AU2416600A - Exocytosis pathway proteins and methods of use - Google Patents

Description

WO 00/43419 PCT/USOO/01431 EXOCYTOSIS PATHWAY PROTEINS AND METHODS OF USE FIELD OF THE INVENTION The invention relates to molecules involved in the exocytosispathway and more particularly to novel polypeptideswhich associatewith exocytoticproteins, n ucleicacids and antibodies. The invention further relates to the use of proteins associated with the exocytosis pathway 5 in methods for identifying candidate agents which modulate exocytosis. BACKGROUND OF THE INVENTION In eukaryotic cells, proteins destined for the plasma membrane or the extracellular space are delivered along the secretory pathway. This comprises a series of sequential, vesicle mediatedtransport steps, each of which requires the specific targeting of transportvesicles 10 to the appropriate acceptor membrane and the subsequent fusion of vesicle and acceptor membranes. In this way, proteins to be secreted by the cell are translocated into the endoplasmic reticulum and then travel through the Golgi complex. The proteins are sorted into secretory vesicles in the trans-Golgi network and these vesicles then fuse with the plasma membrane. This final membrane fusion event is known as exocytosis and results 15 in the discharge of vesicle contents into the extracellular space as well as the incorporation of vesicle membrane lipids and proteins into the plasma membrane. Exocytosis can be divided into two classes: constitutive and regulated. In constitutive exocytosis, secretor vesicles fuse with the plasma membrane immediately after formation; in regulated exocytosis, secretory vesicles accumulate in the cytoplasm and only undergo 20 fusion upon receipt of an appropriate signal. All eukaryotic cells exhibit constitutive WO 00/43419 PCTIUSOO/01431 -2 Although the fundamental purpose of exocytosis is to deliver lipids and proteins to the plasma membrane and to release vesicle contents from the cell, different cell types utilize this mechanism to fulfill their own particular physiological role. Some examples of the various functions of exocytosis in different cell types are listed in Table 1. 5 Table 1. Functions of exocytosis Constitutive All cells Insertion of plasma membrane proteins Liver cells Serum protein secretion Mammary cells Milk protein secretion 10 Fibroblasts Connective tissue protein secretion Regulated Neurons Neurotransmitter release Adrenal chromaffin cells Adrenaline secretion Pancreatic acinar cells (exocrine) Digestive enzyme secretion 15 Pancreatic D-cells (endocrine) Insulin secretion Mast cells Histamine secretion Mammary cells Milk protein secretion Sperm Enzyme secretion Egg Creation of fertilization envelope 20 Adipocytes Insertion of glucose transporters into plasma membrane It should be noted that exocytosis (i.e., the fusion of vesicles with the plasma membrane) is not only the end point of the secretory pathway, but can also involve vesicles which did not originate fromthe endoplasmic reticulum. For instance, transcytosis occurs in polarized cells and involves endocytic vesicle budding from one pole of the cell, transport to the other 25 pole (often via endosomes) and subsequent exocytic fusion. In mammary cells, transcytosis is used in the uptake of antibodies from the blood and their subsequent secretion in milk. Similarly, some vesicles undergo cycles of exo/endocytic fusion via endosomes without returning to the Golgi. Exocytosis of recycling vesicles may be either constitutive (e.g., transferrin receptor-containing vesicles) or regulated (e.g., synaptic vesicles). 30 Since all cells exhibit constitutive exocytosis, it follows that regulated secretory cells must possess two types of secretory vesicle: one constitutive and on regulated. Morphological studies indicate this to be the case, since constitutive secretory vesicles appear small and clear in the electron microscope, whereas regulated secretory vesicles typically appear el nnTITITF PRHFET (RULE 26) WO 00/43419 PCT/US0O/01431 -3 larger and opaque. Furthermore, the two types of vesicle usually contain different substances (an exception is the mammary cell, where casein secretion occurs by both constitutive and regulated exocytosis). It should be noted that cells may contain more than one type of regulated secretory vesicle. 5 The best example of this is seen in neurons, which may possess synaptic vesicles and large dense-core vesicles in addition to constitutive secretory vesicles. Some properties of the two type of neuronal regulated secretory vesicle are listed in Table 2. Large dense core vesicles contain peptide neurotransmitters and these are very similar to regulated secretory vesicles in endocrine cells. Indeed, much of the information on large dense-core 10 vesicle biogenesis and exocytosis has come from studies of adrenal chromaffin cells and their tumor cell derivatives, PC12 cells, both popular neuronal cell models. Synaptic vesicles appear clear in the electron microscope, are much smallerthan large dense-core vesicles and contain fast neurotransmitters. Synaptic vesicles have evolved in animals to allow the extremely rapid point-to-point communication required for brain function. Recently synaptic 15 like vesicles have been found in endocrine cells, such as adrenal chromaffin cells and pancreatic D-cells. These vesicles also appearto contain fast neurotransmitters, although their physiological role is unclear. Table 2. Characteristics of regulated secretory vesicles in neurons Synaptic vesicles Large dense-core vesicles 20 Vesicle size 50 nm 200 nm Speed of 200 ps milliseconds-seconds transmission Fast neurotransmitters Peptides (endorphins, VIP, Neurotransmitter (GABA, glutamate, ACh, etc.) etc.) Cells affected Post-synaptic contact only Cells in surrounding area 25 Duration of effect Short-lived Longer-lived Stimulation Low-frequency High-frequency Ca 2 concentration Hundreds of micromolar Tens of micromolar Vesicle recycling Yes No via endosome? 30 Abbreviations used: GABA, A-aminobutyric acid; ACh, acetylcholine; VIP, vasoactive intestinal peptide. The more information that is gathered regarding exocytosis, the easier it will beto manipulate exocytosis. Moreover, the more information which is gathered, the easier it will be to diagnosis and treat disorders involving exocytosis. For example, inflammatory mediator 35 release from mast cells leads to a variety of disorders, including asthma. Therapy for allergy SUBSTITUTE SHEET (RULE 26) WO 00/43419 PCT/USOO/01431 -4 remains limited to blocking the individual mediators released from mast cells (anti histamines), non-specificanti-inflammatoryagents such as steroidsand mastcell stabilizers which are only marginally effective at limiting the symptomatology of allergies. Similarly, the Chediak-Higashi Syndrome (CHS) is a rare autosomal recessive disease 5 in which neutrophils, monocytes and lymphocytes contain giant cytoplasmic granules. Similar disorders have been described in mice, mink, cattle, cats and killer whales, with the murine homolog of CHS (designated beige or bg) being the best characterized. See Perouetal., J. Biol. Chem. 272(47):29790(1997) and Barbosaetal., Nature 382:262(1996), both of which are hereby incorporated by reference. 10 There is a therefore a need to determine the proteins involved in exocytosis. Some insights into the process of regulated secretion at the molecular level have allowed the definition of G proteins as important regulators. Early experiments showed that non-hydrolyzable analogues of GTP could induce secretionin peritoneal mast cells (Fernandes, et al., Nature 312: 453 (1984)). More recently, a large body of evidence has been accumulating 15 implicating small G proteinsof the rabfamily as regulatorsin the fusion of secretory granules with plasma membranes during exocytosis. The rab GTPases represent a diverse family of homologous proteins that are generally associated with the membrane of organelles in a wide variety of cells, wherethey regulatedefined steps of intracellular membrane traffic (Zerial, M. and Stenmark, H., Curr. Opin. Cell Biol. 5:613 (1993)). An example of this are 20 the rab3 subfamily proteins which have been found to have limited expression in regulated secretion-competent cells, and to be associated with synaptic or secretory granules, suggesting that they are involved in stimulus-secretion coupling (Lledo, et al., Trends Neurobiol. Sci. 17:426 (1994)). Furthermore, overexpression of rab3d or its GTP binding mutant form (N1351) in the rat basophilline RBL leads to significantinhibitionof IgE mediated 25 exocytosis (Roa, J. Immunol., 159:2815 (1997)). Rab3a, Rab3b, Rab3c, and Rab3d constitute a subgroup of the rab family implicated in regulated exocytosis. Rab3a has been detected in regulated secretory cells such as neurons, endocrine cells, and exocrine cells but not in constitutive secretory cells such as hepatocytes and lymphocytes (Fischer von Mollard, Proc. Natl Acad. Sci. 87: 1988-92 30 (1990), Takai, et al., Int. Rev. Cytol. 133: 187-230 (1992)). In neuromuscular synapses, rab3a has been localized at the synaptic vesicles (Mizoguchi, et al., Biochem. Biophys. Res. Commun. 202: 1235-43 (1994)). Furthermore, Rab3a has also been detected at the secretory granules of chromaffin cells and at the zymogen granules in the exocrine cells of pancreatic acini (Padfield, etal., Proc. NatI. Acad. Sci., USA 89:1656-60 (1992)). Rab3a WO 00/43419 PCT/USOO/01431 -5 has been shown to interact with a numberof other proteins including the exchange proteins GDI and GRF (Matsui, et al., Mol. Cell. Biol. 10: 4116-22 (1990), Burstein, E.S. and Macara I.G., Proc. Natl. acad. Sci., USA 89:1154-8 (1992)) as well as GAP (Burstein, J. Biol. Chem. 266: 2689-92 (1991)) and Rabphilin (Shirataki, et al., Mol. Cell Biol. 13: 2061-8 5 (1993)). It is believedthat Rab3a modulatesexocytosisin regulatory secretory cells. Rab3a is cloned and known in the art (see, i.e., Genbank accession number (no.) M28210). Rab3d is thought to be involved in the modulation of regulated secretion in a number of cell types. Rab3d is predominantly expressed in fat tissue butcan also be found expressed, at lower levels, in a wide range of tissue types including lung, spleen, heart, and brain. 10 Baldini, G., et al., (1992) Proc. Natl. Acad. Sci. USA 89: 5049-52. Rab3d has alsp been implicated in the translocation of the Glut4 glucose transporter in adipocytes. Rab3d has been cloned and is known in the art, i.e., Genbank accession no.: AF081353. Thus, rabs, particularly, tissue /cell specific isoforms of rabs and the proteins which they interact with are of great pharmaceutical interest. Rab7 is cloned and known in the art, 15 (see, i.e., Genbank accession no. U44104). Rab9 is localized to the surface of late endosomes where it appears to act to stimulate the transportofmannose6-phosphate receptors between late endosomes and the trans-Golgi network, both in vitro and in vivo. Recent studies suggest that this GTPase is a rate-limiting component for transport between late endosomes and the trans-Golgi network. Rab9 has 20 been cloned and is known in the art, (see i.e., U44103). Rab1 1 was identified by screening a Madin-Darby canine kidney cell cDNA library using degenerate oligonucleotides derived from conserved sequences of the Rab superfamily. Chavrier, P., et al., (1990), Mol. Cell. Biol. 10:6578-85. The predicted amino acid sequences of the canine, human, rat and rabbit Rab1 1 are 100% identical. This high level 25 of conservation between species might reflect a particular importance of this member of the Rab family. Rab1 1 has been localized to both the constitutive and regulated secretory pathway in PC12 cells. Ora3, a homolog of Rab11 (91% identity at the amino acid level) has been found to be associated with cholinergicsynapticvesicles derived from the electric organ of the marine ray. Although the function of Rab1 1 has yet to be definitively 30 determined, a number of lines of evidence suggest that it may play a role in the targeting of transport vesicles of different origin to a common destination, the plasma membrane. Northern blot analysis has shown that Rab1 1 is ubiquitously expressed but is generally WO 00/43419 PCT/USOO/01431 -6 more abundant in tissues with a high level of secretion. Rab1 1 has been cloned and is known in the art, i.e., Genbank accession no. X56740. Rab5 (a, b, and c) make up a subgroup of the Rab protein family. They are located at the cytoplasmic surface of the plasma membrane, on early endosomes and on plasma 5 membrane derived clathrin coated vesicles. Antibodies directed against Rab5a inhibit the fusion of early endosomes in vitro suggesting that its activity is required in this process. In vivo, overexpression of wild type and mutant Rab5a leads to changes in the rate of internalizationof endocyticmarkersand in morphologicalalterations of the early endosomes. These data suggest that Rab5a is a rate-limiting factor that regulates the kinetics of both 10 lateral fusion of early endosomesand fusion of plasma membranederived endocyticvesicles with early endosomes. Some proteins have been identified to associate with Rab5. For example, a 62 kDa coiled-coil protein that specifically interacts with the GTP-bound form of Rab5 has been identified. This protein shares 42% sequence identity with Rabaptin-5, a previously identified effector of Rab5, and has been named it Rabaptin-5beta. Like 15 Rabaptin-5, Rabaptin-5beta displays heptad repeats characteristic of coiled-coil proteins and is recruited on the endosomal membrane by Rab5 in a GTP-dependent manner. However, Rabaptin-5beta has features that distinguish it from Rabaptin-5. The relative expression levels of the two proteins varies in different cell types. Rabaptin-5beta does not heterodimerize with Rabaptin-5, and forms a distinct complex with Rabex-5, the 20 GDP/GTP exchange factor for Rab5. Immunodepletion of the Rabaptin-5beta complex from cytosol only partially inhibits early endosome fusion in vitro, whereas the additional depletion of the Rabaptin-5 complex has a stronger inhibitory effect. Fusion activity can mostly be recovered by addition of the Rabaptin-5 complex alone, but maximal fusion efficiency requires the presence of both Rabaptin-5 and Rabaptin-5beta complexes. 25 Gournier H., et al., (1998), EMBO J. 17(7):1930-1940. Rab5 is cloned and known in the art (see, i.e., Genbank accession no. M28215). Moreover, key proteins that act in Ca 2 -regulated exocytosis in neurons and endocrine cells includethe vesicle proteins synaptotagminVAMP/synaptobrevin, the target membrane protein SYNTAXINs and SNAP-23/25 and, in addition, the soluble N-ethylmaleimide 30 sensitive fusion protein (NSF) and soluble NSF-attachment proteins (a-, 0-, y-SNAPs). Functional evidence for the importance of the membrane proteins has come from their sensitivityto the specific proteolyticactions of clostridial neurotoxinsand / or geneticanalysis in mice and Drosophila. The soluble factors NSF and SNAP were found to interact, in a 20S complex, with the neurotoxin substrates leading to them being designated as SNAP 35 receptors (SNAREs). Many of the proteins that make up the SNARE complexes contain WO 00/43419 PCT/USOO/01431 -7 coiled coil domainswhich are thoughtto intereact primarily through hydrophobicinteractions. These proteins described function at some point in the exocytic pathway either as central membersof the vesiclefusion complexoras accessory proteins involved in some regulatory step in the vesicle fusion cycle. 5 GS27 is associatedwith the Golgiapparatusand is believed to behave like a SNARE. GS27 (for Golgi SNARE of27K), is identicalto membrin, a protein implicated earlier in ER-to-Golgi transport. Regarding SNAREs, these proteins are known to mediate vesicle transport by docking vesicles onto the target membrane. One study has have reported that the cytoplasmic domain of GS27 or antibodies raised against it quantitatively inhibit transport 10 in vitro from the ER to the trans-Golgi/TGN, acting at a stage between the cis/medial- and the trans-Golgi/TGN,indicating that protein movement from medial- to the trans-Golgi/TGN depends on SNARE-mediated vesicular transport and that GS27 plays a functional role (Lowe, S. L., et al., Nature, 389:881-884 (1997)). Therefore, GS27 is implicated in exocytosis by its relation to vesicular transport. 15 SNAP-23 (used interchangeablywith snap-23) was first identified in a human B lymphocyte cDNA library (Ravichandran, V., et al., (1996), J. Biol. Chem. 271:13300-03). Subsequently, others have independently reported the identification of SNAP-23 in several cell types including mast cells. The primary structure of SNAP-23 is 59% identical to SNAP 25; it contains a central cluster of cysteine residues that is a site of palmitoylation in SNAP 20 25 and predicted coiled-coil that are thought to serve in binding other SNAREs, especially syntaxins 1,3, and 4. SNAP-23, like SNAP-25, has been localized mainly to the plasma membrane. However, recent evidence suggests that SNAP-23 translocates to the surface of secretory granules upon cellular activation and forms a complex with SYNTAXIN-3 and VAMP-2 (Guo, Z., et al., (1998), Cell. 94:537-48). This same study suggests that SNAP-23 25 function is crucialfor compound exocytosis in mast cells in that SNAP-23 specificantibodies can completely block secretion in permeabilized cells. SNAP-23 is cloned and known in the art, (see, i.e., Genbank accession no. U55936). NSF and alpha-snap were originally detected as factors required for transport through the Golgi in in vitro assays and yeast homologues of these proteins, sec18 and sec17, are 30 essential for secretion in vivo. In Golgi transport assays and in the formation of a Golgi membrane derived 20S complex, a and p-SNAP appear to be functionally redundant. In contrastmore recent results suggestthat a and -SNAP havedistinctfunctionsin regulated exocytosis based on the ability of alpha-snap to displace synaptotagmin from the SNARE WO 00/43419 PCT/USOO/01431 -8 complex. Sollner, T., et al., Cell, 75: 409-18 (1993). Alpha-snap has been cloned and is known in the art, i.e., Genbank accession number (no.) U39412. Sec1 is a hydrophilic protein that plays an essential role in exocytosis from the yeast Saccharomyces cerevisiae. Syntaxin (a T-SNARE), together with SNAP-25 and 5 synaptobrevinNAMP (a T- and a V-SNARE, respectively), is thought to form the core of the docking-fusion complex in synaptic vesicle exocytosis. Proteins that exhibit similarity to Sec1 were identified in the nervous system of Drosophila melanogaster (Rop) and Caenorhabditiselegans(UNC1 8).Munc-1 8/n-Secl/rbSecl,a brain homologueoftheyeast Secip protein, is thought to participate in regulating the docking and fusion of synaptic 10 vesicles. Munc-1 8/n-Secl/rbSecl expression has been reported to be neural-specific and a number of non-neural isoforms have been identified which are more ubiquitously expressed. Shaywitz, D. A., et al., J. Cell. Biol. 128:769-777 (1995). The role of Muncl8c, previously identified as an n-Sec1/Munc18 homolog in 3T3-L1 adipocytes, in insulin-regulated GLUT4 trafficking has been investigated in 3T3-L1 15 adipocytes. Lowe, S. L., et al, Nature. 389:881-884 (1997). In these cells, Muncl8c predominantly associated with syntaxin4, although it bound both syntaxin2 and syntaxin4 to similar extents in vitro. In addition, SNAP-23, an adipocyte homolog of SNAP-25, associated with both syntaxins 2 and 4 in 3T3-L1 adipocytes. Overexpression of Munc1 8c in 3T3-L1 adipocytes by adenovirus-mediated gene transfer results in inhibition of insulin 20 stimulated glucose transport in a virus dose-dependent manner (maximal effect, approximately 50%) as well as in inhibition of sorbitol-induced glucose transport (by approximately 35%), which is mediated by a pathway different from that used by insulin. In contrast, Muncl8b, which is also expressed in adipocytes but which did not bind to syntaxin4, had no effect on glucose transport. These results suggest that Muncl8c is 25 involved in the insulin-dependent trafficking of GLUT4 from the intracellular storage compartment to the plasma membrane in 3T3-L1 adipocytes by modulating the formation of a SNARE complex that includes syntaxin4. Unc1 8-1 has been cloned and is known in the art, i.e., Genbank accession number (no.) D63851. Tetanus toxin inhibits neurotransmitter release by selectively blocking fusion of synaptic 30 vesicles. Tetanus toxin has been shown to proteolytically degrade synaptobrevin il (also named VAMP-2), a synaptic vesicle-specific protein, in vitro and in nerve terminals. As targetsoftetanustoxin, synaptobrevinsprobablyfunctionin the exocytoticfusion of synaptic vesicles.A synaptobrevinhomologue, cellubrevin (VAMP-3), present in all cells and tissues tested, is a membrane trafficking protein of a constitutively recycling pathway. McMahon WO 00/43419 PCT/USOO/01431 -9 H.T., et al, Nature. 364(6435):346-52 (1993). Like synaptobrevin 11, cellubrevin is proteolysed by tetanus toxin light chain in vitro and after transfection.These resultsindicate that constitutive and regulated vesicular pathways use homologousproteins for membrane trafficking, likely for membrane fusion at the plasma membrane, indicating a greater 5 mechanistic and evolutionary similarity between these pathways than previously thought. The homologueof Vamp3, vamp2 (synaptobrevin), has been localizedto mastcell granules and may play a critical role in mast cell exocytosis (Guo, Z., et al., Cell, 94:537-48 (1998)). Vamp3 has been cloned and is known in the art, i.e., Genbank accession no.: AF26007. Accordingly, the proteins involved in exocytosis, termed Exo proteins herein, particularly 10 those associatedwith GS27, Rab3a, Rab7, Rab9, Rab1 1, Rab3d, Rab5, alpha snap, unc1 8 1, vamp3, and snap-23are of interest, and it is desirable to providesuch proteinsand related molecules. It is a further aspect of the invention to provide recombinant nucleic acids encoding Exo proteins and expression vectors and host cells containing the nucleic acid encoding the Exo protein. A further aspect of the invention is to provide methods for 15 screening for antagonists and agonists of Exo proteins, particularly those which modulate exocytosis, secretion and/or vesicular transport. SUMMARY OF THE INVENTION Accordingly, the present invention provides recombinant nucleic acids encoding an Exo protein that has at least about 85% sequence identity, and more preferably at least about 20 90% sequence identity, and most preferably about 95% sequence identity with an amino acid sequence encoded by a nucleic acid comprising the first 100 nucleic acid residues of a sequence selected from the group consisting of SEQ ID NOS:15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 63, 64, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 25 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134,135,136,137,138,139,140,141, 142,143,147,148, 150,151,152, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 193, 194,195,196,197,198,199,200,201,202,203,204,205,210,211. Preferably, the Exo proteins bind to a protein selected from the group consisting of GS27, rab7, rab9, snap-23, 30 rab3a, rab1 1, rab3d, rab5, alpha-snap, unc1 8-1, and vamp3. Also provided are recombinant nucleic acids which have at least about 75% sequence identity, more preferably, at least 85% sequence identity and most preferably at least about 95% sequence identity with a nucleicacid sequence comprising the first 100 nucleic acid residues of a sequence selected from the group consisting of SEQ ID NOS: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, WO 00/43419 PCT/USOO/01431 -10 39, 41, 43, 45, 47, 49, 51, 53, 63, 64, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103,104,105,106,107,108,109,110,111, 112,113,114,115,116,117, 118, 119,120,121, 122,123,124,125,126,127,128,129,130,131,132,133, 134,135, 136,137,138,139,140,141, 142,143, 147, 148,150,151, 152,155,156, 157,158,159, 5 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 210, 211 and their complements. Recombinant Exo proteins, expression vectors and host cells comprising the nucleic acids are also included. In an additionalaspect, the invention provides recombinant Exo proteinsExo3, Exo4, Exo5, Exo6, Exo7, Exo8, Exo9, Exo10, Exo11, Exo12, Exo13, Exo14, Exo15, Exo16, Exo17a, 10 Exo17b, Exo18, Exo19, Exo20, Exo21, Exo22, Exo23, Exo24, Exo25, Exo26, Exo27, Exo28, Exo29, Exo30, Exo3l, Exo32, Exo33, Exo34, Exo35, Exo36, Exo37, Exo38, Exo39, Exo40, Exo41, Exo42, Exo43, Exo44, Exo45, Exo46, Exo47, Exo48, Exo49, Exo5O, Exo51, Exo52, Exo53, ExoS4, Exo55, Exo56, Exo57, Exo58, Exo59, Exo60, Exo6l, Exo62, Exo63, Exo64, Exo65, Exo66, Exo67, Exo68, Exo69, Exo70, Exo7l, Exo72, Exo73, Exo74, Exo75, Exo76, 15 Exo77, Exo78, Exo79, Exo8O, Exo8l, Exo82, Exo83, Exo84, Exo85, Exo86, Exo87, Exo88, Exo89, Exo9O, Exo9l, Exo92, Exo93, Exo94, Exo95, Exo96, Exo97, Exo98, Exo99, Exol 00, Exol01, Exol02, Exol03, ExolO4, Exol05, ExolO6, Exol07, Exo1O8, Exol09, ExollO, Exo111, Exo112, Exo113, Exo114, Exo115, Exo116, Exo117, and Exo118, and the nucleic acids encoding said Exo proteins. 20 In a further aspect, the invention provides methods of making Exo proteins, comprising providing a cell comprising an Exo nucleic acid and subjecting the cell to conditions which allow the expression of Exo proteins. In a further aspect, the present invention provides methods for screening for a bioactive agent capable of binding to an Exo protein. The method comprises combining an Exo 25 protein and a candidate bioactive agent, and determining the binding of the candidateagent to the Exo protein. In an additional aspect, the present invention provides methods for screening for agents capable of interfering with the binding of an Exo protein and GS27. The methods comprise combining an Exo protein, a candidate bioactiveagentand a GS27 protein, and determining 30 the binding of the Exo protein and the GS27 protein. In anotheraspect, the present invention provides methods for screening for agents capable of interferingwith the binding of an Exo protein and rab7. The methodscomprise combining WO 00/43419 PCTIUSOO/01431 -11 an Exo protein, a candidatebioactive agent and a rab7 protein, and determining the binding of the Exo protein and the rab7 protein. In a furtheraspect, the present invention provides methods for screening for agents capable of interfering with the binding of an Exo protein and rab9. The methodscomprise combining 5 an Exo protein, a candidate bioactiveagentand a rab9 protein, and determining the binding of the Exo protein and the rab9 protein. In yet another aspect, the present invention provides methods for screening for agents capable of interfering with the binding of an Exo protein and snap-23. The methods comprise combining an Exo protein, a candidate bioactive agent and a snap-23 protein, 10 and determining the binding of the Exo protein and the snap-23 protein. In an additional aspect, the present invention provides methods for screening for agents capable of interfering with the binding of an Exo protein and rab3a. The methods comprise combining an Exo protein, a candidatebioactiveagent and a rab3a protein, and determining the binding of the Exo protein and the rab3a protein. 15 In another aspect, the present invention provides methodsfor screening for agents capable of interfering with the binding of an Exo protein and rabl. The methods comprise combiningan Exo protein, a candidate bioactive agent and a rab1 1 protein, and determining the binding of the Exo protein and the rab1 1 protein. In a further aspect, the present invention providesmethodsforscreeningfor agentscapable 20 of interfering with the binding of an Exo protein and rab3d. The methods comprise combiningan Exo protein, a candidatebioactiveagent and a rab3d protein, and determining the binding of the Exo protein and the rab3d protein. In yet an additional aspect, the present invention provides methodsfor screening for agents capable of interfering with the binding of an Exo protein and rab5. The methods comprise 25 combining an Exo protein, a candidate bioactive agent and a rab5 protein, and determining the binding of the Exo protein and the rab5 protein. In a furtheraspect, the presentinvention provides methods for screeningforagentscapable of interfering with the binding of an Exo protein and alpha-snap. The methods comprise combining an Exo protein, a candidate bioactive agent and an alpha-snap protein, and 30 determining the binding of the Exo protein and the alpha-snap protein.

WO 00/43419 PCT/USOO/01431 -12 In yet another aspect, the present invention provides methods for screening for agents capable of interfering with the binding of an Exo protein and unc18-1. The methods comprise combining an Exo protein, a candidate bioactive agent and an unc1 8-1 protein, and determining the binding of the Exo protein and the unc18-1 protein. 5 In an additional aspect, the present invention provides methods for screening for agents capable of interfering with the binding of an Exo protein and vamp3. The methods comprise combining an Exo protein, a candidatebioactiveagent and a vamp3 protein, and determining the binding of the Exo protein and the vamp3 protein. In another aspect, the invention provides methods for screening for an bioactive agent 10 capable of modulating the activity of an Exo protein. The method comprises the steps of adding a candidate bioactiveagentto a cell comprising a recombinant nucleic acid encoding an Exo protein, and determining the effect of the candidate bioactive agent on cellular activity. In a preferred embodiment the cellular activity is exocytosis or vesicular transport. In anotheraspect, the invention provides a method of treating an exocytosis related disorder 15 comprising administering an agent that interferes with specific binding of a protein selected from those shown in the Sequence Listing with a protein selected from the group consisting of GS27, Rab3a, Rab7, Rab9, Rabl1, Rab3d, Rab5, alpha snap, uncl8-1, vamp3, and snap-23, expressed in a tissue such that said disorder is ameolerated. Also provided herein is a method of treating an exocytosis related disorder comprising 20 administering to a patient an agent that binds to a protein encoded by a sequence selected from the group consisting of those set forth in the Sequence Listing, such that exocytosis is altered. Further provided herein is a method of reducing or inhibiting exocytosis in a cell comprising administering an agent that interferes with specific binding of a protein selected from those 25 encoded by a sequence selected from the group consisting of SEQ ID NOS:1-51 (odd numbers) and 53-2 11with a protein selected from the group consisting of GS27, Rab3a, Rab7, Rab9, Rab1 1, Rab3d, Rab5, alpha snap, uncle 8-1, vamp3, and snap-23, expressed in said cell such that exocytosis is inhibited. In yet another aspect, the invention provides a method of neutralizing the effect of a protein 30 encoded by a sequence selected from the group consisting of SEQ ID NOS:1-51 (odd numbers) and 53-211 comprising contacting an agent specific for said protein with said WO 00/43419 PCT/USOO/01431 -13 protein in an amount sufficient to effect neutralization. Other aspects of the invention are set forth as described below. BRIEF DESCRIPTION OF THE DRAWINGS Figures1A and 1 B showthe underlying mechanism of yeasttwo-hybridand yeastone-hybrid 5 systems. Figure 1A shows the two-hybrid system: GAL4A represent GAL4 transcription activation domain. cDNA represents cDNA library inserts. X represents any bait gene. GAL4B represents GAL4 DNA binding domain. HIS/lacZ indicates that the reporter gene is either HIS or lacZ. Figure 2 shows the outline of yeast two-hybrid screening. Solid black dots represent 10 colonies on plates. Transformation steps of both bait plasmid and cDNA library plasmids are indicated. Figure 3 shows the outline of yeast one-hybrid screening. Solid black dots represent colonies on plates. Figures 4A-4D show vectors used in the yeast two-hybrid and one-hybrid screening, 15 respectively. Fig ures4A-4B show the two-hy brid vectors. Bait vectors can be pHybLex/Zeo (Invitrogen), pBD-GAL4 (Stratagene), pAS2-1 (Clontech), pGBT9 (Clontech), or pGilda (Origene, Clontech). Arrows indicate transcription of fusion proteins on either bait or cDNA vector. The binding domain can be either GAL4 or LexA. MCS underlined represents multiple cloning sites, where either bait gene or cDNA fragments should be cloned. 2 p 20 Ori represents yeast 2 micron replicationorig in. cDNA vectors can be pYESTrp2(Invitrogen), pAD-GAL4 (Stratagene), pACT2 (Clontech), pGADGH (Clontech), pGAD424 (Clontech), or pJG4-5 (Origene).Activationdomain can be GAL4, VP16, or other transcription activator. Figures 4C-4D show the one-hybrid reporter vectors. DNA sequences of interest should be inserted into the multiple cloning sites (MCS) underlined. The enzyme used to Linearize 25 reporter vector for integration is shown by solid arrow. The dashed arrow indicates the transcription of either HIS or lacZ gene. DETAILED DESCRIPTION OF THE INVENTION The present invention provides Exo proteins and nucleic acids involved in the exocytotic pathway. In a preferred embodiment, the Exo proteins are from vertebrates and more 30 preferably from mammals, including rodents (rats, mice, hamsters, guinea pigs, etc.), WO 00/43419 PCT/USOO/01431 -14 primates, farm animals (including sheep, goats, pigs, cows, horses, etc) and in the most preferred embodiment, from humans. An Exo protein of the present invention may be identified in several ways. "Protein" in this sense includes proteins, polypeptides, and peptides. The Exo proteins of the invention 5 fall into two general classes: proteins that are completely novel, i.e. are not part of a public database as of the time of discovery, although they may have homology to either known proteins or expressed sequence tags (ESTs). Alternatively, the Exo proteins are known proteins, but that were not known to be involved in exocytosis; i.e. they are identified herein as having a novel biological function. Accordingly, an Exo protein may be initially identified 10 by its association with a protein known to be involved in exocytosis or vesicular transport. In one embodiment provided herein, Exo proteins bind to a protein selected from the group consisting of GS27, rab7, rab9, snap-23, rab3a, rab11, rab3d, rab5, alpha-snap, uncl8-1, and vamp3. Exo proteins may be novel or may have been known in the art to exist, but not known to bind to GS27, rab7, rab9, snap-23, rab3a, rab1 1, rab3d, rab5, alpha-snap, 15 unc1 8-1, or vamp3. Wherein the Exo proteins and nucleic acids are novel, compositions and methodsof use are provided herein. In the case that the Exo proteins and nucleic acids were known but not known to bind to GS27, rab7, rab9, snap-23, rab3a, rab1 1, rab3d, rab5, alpha-snap, unc18-1, or vamp3, methods of use, i.e. functional screens, are provided. In one embodiment, Exo nucleic acids or Exo proteins are initially identified by substantial 20 nucleic acid and/or amino acid sequence identity or similarity to the sequences provided herein. In a preferred embodiment, Exo nucleic acids or Exo proteins have sequence identity or similarityto the sequences provided herein as described below and bind to an exocytosis or vesiculartransport protein. Preferred exocytosis and vesicular transport proteinsinclude GS27, rab7, rab9, snap-23, rab3a, rab1 1, rab3d, rab5, alpha-snap, unc1 8-1, and vamp3 25 (these proteins are known in the art are considered the same whether "-" is used within the name or capitols are used). Such sequence identity or similarity can be based upon the overall nucleic acid or amino acid sequence. SEQ ID NO:1 shows nucleic acid sequence encoding at least a portion of mouse syntaxin4, Genbankaccessionno.: U76832, described in Hay JC, et al., J Cell Biol 1998,141(7):1489 30 1502. SEQ ID NO:3 shows nucleic acid sequence encoding at least a portion of mouse LZIP-1 and LZIP-2, Genbank accession no.:AC003675,described in Burbelo PD, etal. Gene 1994, 139(2):241-245.

WO 00/43419 PCT/USOO/01431 -15 SEQ ID NO:5 shows the nucleic acid sequence encoding at least a portion of mouse IL-3 receptor, Genbank accession no.:M29855, described in Tabira T, et al., Ann N YAcad Sci. 1998, 840: 107-116. 5 SEQ ID NO:7 shows nucleic acid sequence encoding at least a portion of mouse IL-4 receptor, accession no.: M27959, describedin Ryan JJ, et al., J Immunol. 1998,161(4):1811 1821. SEQ ID NO:9 shows a second nucleic acid sequence encoding at least a portion of mouse IL-4 receptor, accession no.:M27959, described in Ryan JJ, et al., J Immunol. 1998, 10 161(4):1811-1821. SEQ ID NO: 11 shows nucleic acid sequence encoding at least a portion of mouse LDL receptor-related protein 6 (Lrp6), accession no.:AF074265, described in Brown,SD, et al., Biochem. Biophys. Res. Commun. 248 (3):879-888 (1998). SEQ ID NO: 13 shows nucleic acid sequence encoding at least a portion of mouse abc2, 15 accession no.:X75927, described in Illing M, et al., J Biol Chem 1997,11;272(15):10303 10310. SEQ ID NOS:1 5,17,19,21,23, and 25 show nucleic acid sequences which encode Exo3-8, respectively. SEQ ID NO:27 shows the nucleic acid sequence encoding Exo9, which may share some 20 characteristics with human syntaxinl6A, accession no.:AF008937, described in Hay JC, et al., J Cell Biol 1998, 141(7):1489-1502. SEQ ID NO:29 shows the nucleic acid sequence encoding Exol 0, which which may share some characteristics with human putative RNA binding protein (RBP56), accession no.:U51334, described in Genomics 38:51-57 (1996). 25 SEQ ID NO:31 showsthe nucleicacid sequenceencoding Exol 1, which has some similarity with GenBank accession no.: AA144083. SEQ ID NO:33 shows the nucleicacid sequenceencoding Exol 2, which has some similarity with GenBank accession no.: AA103185.

WO 00/43419 PCT/USOO/01431 -16 SEQ ID NO:35 shows the nucleicacid sequenceencoding Exol 3, which has some similarity with GenBank accession no.: AA919222. SEQ ID NO:37 showsthe nucleic acid sequenceencoding Exo14, which has some similarity 5 with GenBank accession no.:AA276016 and human (xs99). SEQ ID NO:39 showsthe nucleicacid sequenceencoding Exol 5, which has some similarity with GenBank accession no.:AA617266, and CREB-RP (creb-rp), Genebank accession no.: U31903. 10 SEQ ID NO:41 shows the nucleicacid sequence encoding Exol 6, which has some similarity with GenBank accession no.:AA221293 and rat lamina-assocated peptide. SEQ ID NO:43 shows the nucleic acid sequence encoding Exo17a, which has some similarity with GenBank accession no.:AA1 66109 and rat syntaxin5, Genebank accession 15 no.:L20822, described in Rowe T, et al., Science 1998 279(5351):696-700. SEQ ID NO:45 shows the nucleic acid sequence encoding Exo17b, which has some similarity with GenBank accession no.:AA1 66109 and rat syntaxin5, Genebank accession no.:L20822, described in Rowe T, et al., Science 1998 279(5351):696-700. SEQ ID NO:47 showsthe nucleicacid sequenceencoding Exol 8, which has some similarity 20 with GenBankaccession no.:AA1 66109and has some similarityto rat syntaxin5, Genebank accession no.:L20822, described in Rowe T, et al., Science 1998 279(5351):696-700. SEQ ID NO:49 showsthe nucleicacid sequenceencoding Exol 9, which has some similarity with GenBank accession no.:U76832 and mouse syntaxin4, described in Hay JC, J Cell 25 Biol 1998, 141(7):1489-1502. SEQ ID NO:51 shows the nucleic acid sequence which encodes Exo20. SEQ ID NO:53 shows the nucleic acid sequence which encodes Exo2l. SEQ ID NO:54 shows the nucleic acid sequence encoding a portion of human axonal transporter of synaptic vesicles, Genbank accession no.: X90840.

WO 00/43419 PCT/USOO/01431 -17 SEQ ID NO:55 shows the nucleic acid sequence encoding a portion of human cargo selection protein TIP47 (TIP47), Genbank accession no.: AF057140. SEQ ID NO:56 shows the nucleic acid sequence encoding a portion of human cargo selection protein TIP47 (TIP47), Genbank accession no.: AF057140. 5 SEQ ID NO:57 shows the nucleic acid sequence encoding a portion of human cargo selection protein TIP47 (TIP47), Genbank accession no.: AF057140. SEQ ID NO:58 shows the nucleic acid sequence encoding a portion of human cargo selection protein TIP47 (TIP47), Genbank accession no.: AF057140. SEQ ID NO:59 shows the nucleicacid sequenceencodinga portion of human tax interaction 10 protein, Genbank accession no.: AF028824. SEQ ID NO:60shows the nucleicacid sequenceencodinga portionof human tax interaction protein, Genbank accession no.: AF028824. SEQ ID NO:61 showsthe nucleic acid sequenceencodinga portionof humantax interaction protein, Genbank accession no.: AF028824. 15 SEQ ID NO:62 shows the nucleicacid sequenceencoding a portion of human human inositol polyphosphate 5-phosphatase, Genbank accession no.: M74161. SEQ ID NO:63 shows the nucleic acid sequence encoding Exo22. SEQ ID NO:64 shows the nucleic acid sequence encoding Exo23. SEQ ID NO:65 shows the nucleic acid sequence encoding a portion of mouse C57BL/6J 20 Sec6l protein complex gamma subunit; Genbank accession no.: U11027. SEQ ID NO:66 shows the nucleic acid sequence encoding a portion of mouse HMG-1; GenBank accession no.: U00431. SEQ ID NO:67 shows the nucleic acid sequence encoding a portion of mouse cyclin B2; GenBank accession no.: X66032.

WO 00/43419 PCTIUSOO/01431 -18 SEQ ID NO:68 shows the nucleic acid sequence encoding a portion of mouse cyclin B2; GenBank accession no.: X66032. SEQ ID NO:69 shows the nucleic acid sequence encoding a portion of mouse pancreatic beta-cell kinesin heavy chain; GenBank accession no.: U86090. 5 SEQ ID NO:70 shows the nucleic acid sequence encoding a portion of mouse pancreatic beta-cell kinesin heavy chain; GenBank accession no.: U86090. SEQ ID NO:71 shows the nucleic acid sequence encoding a portion of mouse syntaxin4; GenBank accession no.: U76832. SEQ ID NO:72 shows the nucleic acid sequence encoding a portion of mouse syntaxin4; 10 GenBank accession no.:U76832. SEQ ID NO:73 shows the nucleic acid sequence encoding a portion of mouse syntaxin4; GenBank accession no.:U76832. SEQ ID NO:74 shows the nucleicacid sequenceencoding a portion of mouse stearoyl-CoA desaturase (SCD2); GenBank accession no.: M26270. 15 SEQ ID NO:75 shows the nucleic acid sequence encoding a portion of mouse spermidine aminopropyltransferase (Mspmsy); GenBank accession no.: AF031486. SEQ ID NO:76 shows the nucleic acid sequence encoding a portion of mouse prothymosin alpha; GenBank accession no.: X56135. SEQ ID NO:77 shows the nucleic acid sequence encoding a portion of mouse protein 20 cofactor; GenBank accession no.: U74079. SEQ ID NO:78 shows the nucleic acid sequence encoding a portion of mouse outer dense fiber protein 2 (Odf2); GenBank accession no.: AF000968. SEQ ID NO:79 shows the nucleic acid sequence encoding a portion of mouse protein expressed in E12 brain (clone C2); GenBank accession no.: X83589.

WO 00/43419 PCT/USOO/01431 -19 SEQ ID NO:80 shows the nucleic acid sequence encoding a portion of mouse hnRNP K homologue;GenBank accession no.: L29769. SEQ ID NO:81 shows the nucleic acid sequence encoding a portion of mouse NRF1 (NFE2-related factor 1); GenBank accession no.: X78709. 5 SEQ ID NO:82 shows the nucleic acid sequenceencodinga portion of mouse RNA-binding protein; GenBank accession no.: L17076. SEQ ID NO:83 shows the nucleic acid sequence encoding a portion of mouse dynactin1; GenBank accession no.: U60312. SEQ ID NO:84 shows the nucleic acid sequence encoding a portion of mouse 10 hormone-sensitive lipase; GenBank accession no.: U08188. SEQ ID NO:85 shows the nucleic acid sequence encoding a portion of mouse mtprda (human TPRD homologue); GenBank accession no.: AB008516. SEQ ID NO:86 shows the nucleic acid sequence encoding Exo24, having some similarity with GenBank accession no.: AA268561. 15 SEQ ID NO:87 shows the nucleic acid sequence encoding Exo25, having some similarity with GenBank accession no.: AA097037. SEQ ID NO:88 shows the nucleic acid sequence encoding Exo26, having some similarity with GenBank accession no.: AA097037. SEQ ID NO:89 shows the nucleic acid sequence encoding Exo27, having some similarity 20 with GenBank accession no.: AA259474. SEQ ID NO:90 shows the nucleic acid sequence encoding Exo28, having some similarity with GenBank accession no.: AA555886. SEQ ID NO:91 shows the nucleic acid sequence encoding Exo29, having some similarity with GenBank accession no.: AA770839.

WO 00/43419 PCT/USOO/01431 -20 SEQ ID NO:92 shows the nucleic acid sequence encoding Exo30, having some similarity with GenBank accession no.: AA770839. SEQ ID NO:93 shows the nucleic acid sequence encoding Exo3l, having some similarity with GenBank accession no.: AA415504. 5 SEQ ID NO:94 shows the nucleic acid sequence encoding Exo32, having some similarity with GenBank accession no.: AA415504. SEQ ID NO:95 shows the nucleic acid sequence encoding Exo33, having some similarity with GenBank accession no.: AA415504. SEQ ID NO:96 shows the nucleic acid sequence encoding Exo34, having some similarity 10 with GenBank accession no.: AA415504. SEQ ID NO:97 shows the nucleic acid sequence encoding Exo35, having some similarity with GenBank accession no.: AA415504. SEQ ID NO:98 shows the nucleic acid sequence encoding Exo36, having some similarity with GenBank accession no.: AA415504. 15 SEQ ID NO:99 shows the nucleic acid sequence encoding Exo37, having some similarity with GenBank accession no.: AA415504. SEQ ID NO: 100 shows the nucleic acid sequence encoding Exo38, having some similarity with GenBank accession no.: AA415504. SEQ ID NO: 101 shows the nucleic acid sequence encoding Exo39, having some similarity 20 with GenBank accession no.: AA415504. SEQ ID NO: 102 shows the nucleic acid sequence encoding Exo40, having some similarity with GenBank accession no.: AA415504. SEQ ID NO: 103 shows the nucleic acid sequence encoding Exo4l, having some similarity with GenBank accession no.: AA172925.

WO 00/43419 PCT/USOO/01431 -21 SEQ ID NO:104 shows the nucleic acid sequence encoding Exo42, having some similarity with GenBank accession no.: AA288130. SEQ ID NO:105 shows the nucleic acid sequence encoding Exo43, having some similarity with GenBank accession no.: Al181639. 5 SEQ ID NO:106 shows the nucleic acid sequence encoding Exo44, having some similarity with GenBank accession no.: AA184709. SEQ ID NO:107 shows the nucleic acid sequence encoding Exo45, having some similarity with GenBank accession no.: AA266406. SEQ ID NO:108 shows the nucleic acid sequence encoding Exo46, having some similarity 10 with GenBank accession no.: AA563185. SEQ ID NO:109 shows the nucleic acid sequence encoding Exo47, having some similarity with GenBank accession no.: AA519170. SEQ ID NO: 110 shows the nucleic acid sequence encoding Exo48, having some similarity with GenBank accession no.: AA519170. 15 SEQ ID NO: 111 shows the nucleic acid sequence encoding Exo49, having some similarity with yeast ORMI, GenBank accession no.: AA175198. SEQ ID NO: 112 shows the nucleic acid sequence encoding Exo50, having some similarity with rat mt-GrpE no.1 precursor, GenBank accession no.: AA060861. SEQ ID NO: 113 shows the nucleic acid sequence encoding Exo51, having some similarity 20 with human CENP-F kinetochore protein, GenBank accession no.: A1034171. SEQ ID NO: 114 shows the nucleic acid sequence encoding Exo52, having some similarity with human arfaptin 2 (putative target of ADP-ribosylation factor), GenBank accession no.:AA543955. SEQ ID NO: 115 shows the nucleic acid sequence encoding Exo53, having some similarity 25 with human brain and reproductive organ-expressed protein (BRE); GenBank accession no.: AA200608.

WO 00/43419 PCT/USOO/01431 -22 SEQ ID NO: 116 shows the nucleic acid sequence encoding Exo54, having some similarity with human brain and reproductive organ-expressed protein (BRE); GenBank accession no.: AA200608. SEQ ID NO:117 shows the nucleic acid sequence encoding Exo55, having some similarity 5 with human cell cycle progression 2 protein (CPR2); GenBank accession no.: W87077. SEQ ID NO:118 shows the nucleic acid sequence encoding Exo56, having some similarity with human spliceosomeassociatedprotein (SAP145); GenBank accession no.: Al 119401. SEQ ID NO: 119 shows the nucleic acid sequence encoding Exo57, having some similarity with mesocricetus auratus stearyl-CoA desaturase (FAR-17c); GenBank accession no.: 10 AA387696. SEQ ID NO:120 shows the nucleic acid sequence encoding Exo58, having some similarity with rat inositol trisphosphate receptor subtype 3 (IP3R-3); GenBank accession no.: AA823026. SEQ ID NO:121 shows the nucleic acid sequence encoding Exo59, having some similarity 15 with L-Asparaginase; GenBank accession no.: Al 118730. SEQ ID NO: 122 shows the nucleic acid sequence encoding Exo60, having some similarity with L-Asparaginase; GenBank accession no.: A1118730. SEQ ID NO:123 shows the nucleic acid sequence encoding Exo6l, having some similarity with human RB-binding protein 2 (RBBP-2); GenBank accession no.: AA755315. 20 SEQ ID NO:124 shows the nucleic acid sequence encoding Exo62, having some similarity with human secreted apoptosis related protein 3 (SARP3); GenBank accession no.: AU018890. SEQ ID NO: 125 shows the nucleic acid sequence encoding Exo63, having some similarity with myosin heavy chain; GenBank accession no.: AA237764. 25 SEQ ID NO:126 shows the nucleic acid sequence encoding Exo64, having some similarity with myosin heavy chain; GenBank accession no.: AA237764.

WO 00/43419 PCT/USOO/01431 -23 SEQ ID NO:127 shows the nucleic acid sequence encoding Exo65, having some similarity with myosin heavy chain; GenBank accession no.: AA237764. SEQ ID NO: 128 shows the nucleic acid sequence encoding Exo66, having some similarity with myosin heavy chain; GenBank accession no.: AA237764. 5 SEQ ID NO: 129 shows the nucleic acid sequence encoding Exo67, having some similarity with rat tomosyn; GenBank accession no.: AA437465. SEQ ID NO: 130 shows the nucleic acid sequence encoding Exo68, having some similarity with rat tomosyn; GenBank accession no.: AA437465. SEQ ID NO:131 shows the nucleic acid sequence encoding Exo69, having some similarity 10 with rat tomosyn; GenBank accession no.: AA437465. SEQ ID NO:132 shows the nucleic acid sequence encoding Exo70, having some similarity with rat tomosyn; GenBank accession no.: AA437465. SEQ ID NO:133 shows the nucleic acid sequence encoding Exo7l, having some similarity with human mcag29 CTG repeat region; GenBank accession no.: AA089340. 15 SEQ ID NO:134 shows the nucleic acid sequence encoding Exo72, having some similarity with rat G protein gamma-5 subunit; GenBank accession no.: AA021879. SEQ ID NO:135 shows the nucleic acid sequence encoding Exo73, having some similarity with rat G protein gamma-5 subunit; GenBank accession no.: AA021879. SEQ ID NO:136 shows the nucleic acid sequence encoding Exo74, having some similarity 20 with rat G protein gamma-5 subunit; GenBank accession no.: AA021879. SEQ ID NO:137 shows the nucleic acid sequence encoding Exo75, having some similarity with rat G protein gamma-5 subunit; GenBank accession no.: AA021879. SEQ ID NO:138 shows the nucleic acid sequence encoding Exo76, having some similarity with rat G protein gamma-5 subunit; GenBank accession no.: AA021879.

WO 00/43419 PCT/USOO/01431 -24 SEQ ID NO: 139 shows the nucleic acid sequence encoding Exo77, having some similarity with rat G protein gamma-5 subunit; GenBank accession no.: AA021879. SEQ ID NO:140 shows the nucleic acid sequence encoding Exo78. SEQ ID NO:141 shows the nucleic acid sequence encoding Exo79. 5 SEQ ID NO:142 shows the nucleic acid sequence encoding Exo80. SEQ ID NO:143 shows the nucleic acid sequence encoding Exo8l. SEQ ID NO:144 shows the nucleic acid sequence encoding a portion of human HLA-B-associated transcript 3; GenBank accession no.: M33519. SEQ ID NO:145 shows the nucleic acid sequence encoding a portion of human 10 HLA-B-associated transcript 3; GenBank accession no.: M33519. SEQ ID NO:146 shows the nucleic acid sequence encoding a portion of human T cell leukemia/lymphoma 1; GenBank accession no.: X82240. SEQ ID NO:147 shows the nucleic acid sequence encoding Exo82. 15 SEQ ID NO:148 shows the nucleic acid sequence encoding Exo83; may have some homology with rat rabin3. SEQ ID NO:149 shows the nucleic acid sequence encoding at least a portion of human KIAA0665; GenBank accession no.: AB014565. SEQ ID NO:150 shows the nucleic acid sequence encoding Exo84 which may have some 20 similarity with Rabin 3, GenBank accession no.: AA846576. SEQ ID NO:151 shows the nucleic acid sequence encoding Exo85 which may have some similarity with Rabin 3, GenBank accession no.: AA846576. SEQ ID NO:152 shows the nucleic acid sequence encoding Exo86 which may have some 25 similarity with human KIAA0665, GenBank accession no.: AA757034.

WO 00/43419 PCT/USOO/01431 -25 SEQ ID NO:153 shows the nucleic acid sequence encoding a portion of mouse protein cofactor; GenBank accession no.: U74079. SEQ ID NO:154 shows the nucleic acid sequence encoding a portion of mouse protein cofactor; GenBank accession no.: U74079. 5 SEQ ID NO:155 shows the nucleic acid sequence encoding Exo87, may have some similarity with calcium-dependent protein kinase, Genbank accession no.: AA770736. SEQ ID NO:156 shows the nucleic acid sequence encoding Exo88, may have some similarity with calcium-dependent protein kinase, Genbank accession no.: AA770736. 10 SEQ ID NO:157 shows the nucleic acid sequence encoding Exo89, may have some similarity with calcium-dependent protein kinase, Genbank accession no.: AA770736. SEQ ID NO:158 shows the nucleic acid sequence encoding Exo90, may have some similarity with calcium-dependent protein kinase, Genbank accession no.: AA770736. SEQ ID NO:159 shows the nucleic acid sequence encoding Exo9l, may have some 15 similarity with human mcag29 CTG repeat region, Genbank accession no.: AA473325. SEQ ID NO:160 shows the nucleic acid sequence encoding Exo92, may have some similarity with human mcag29 CTG repeat region, Genbank accession no.: AA473325. SEQ ID NO:161 shows the nucleic acid sequence encoding Exo93, may have some similarity with Genbank accession no.: AA1 38122. 20 SEQ ID NO:162 shows the nucleic acid sequence encoding Exo94, may have some similarity with Genbank accession no.: AA1 38122. SEQ ID NO:163 shows the nucleic acid sequence encoding Exo95, may have some similarity with Genbank accession no.: AA1 38122. SEQ ID NO:164 shows the nucleic acid sequence encoding Exo96, may have some 25 similarity with Genbank accession no.: AA1 38122.

WO 00/43419 PCT/USOO/01431 -26 SEQ ID NO:165 shows the nucleic acid sequence encoding Exo97, may have some similarity with Genbank accession no.: AA1 38122. SEQ ID NO:166 shows the nucleic acid sequence encoding Exo98, may have some similarity with Genbank accession no.: AA060976. 5 SEQ ID NO:167 shows the nucleic acid sequence encoding Exo99, may have some similarity with Genbank accession no.: AA277208. SEQ ID NO:168 shows the nucleic acid sequence encoding Exo100, may have some similarity with Genbank accession no.: AA277208. SEQ ID NO:169 shows the nucleic acid sequence encoding Exo101, may have some 10 similarity with Genbank accession no.: AA467477. SEQ ID NO:170 shows the nucleic acid sequence encoding Exo102, may have some similarity with Genbank accession no.: AA467477. SEQ ID NO:171 shows the nucleic acid sequence encoding Exo103, may have some similarity with Genbank accession no.: AA833213. 15 SEQ ID NO:172 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403. SEQ ID NO:173 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403. SEQ ID NO:174 shows the nucleic acid sequence encoding a portion of Rab2; GenBank 20 accession no.: X95403. SEQ ID NO:175 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403. SEQ ID NO:176 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403.

WO 00/43419 PCT/USOO/01431 -27 SEQ ID NO:177 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403. SEQ ID NO:178 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403. 5 SEQ ID NO: 179 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403. SEQ ID NO: 180 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403. SEQ ID NO:181 shows the nucleic acid sequence encoding a portion of Rab2; GenBank 10 accession no.: X95403. SEQ ID NO:182 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403. SEQ ID NO:183 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403. 15 SEQ ID NO: 184 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403. SEQ ID NO: 185 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403. SEQ ID NO: 186 shows the nucleic acid sequence encoding a portion of Rab2; GenBank 20 accession no.: X95403. SEQ ID NO:187 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403. SEQ ID NO:188 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403.

WO 00/43419 PCT/USOO/01431 -28 SEQ ID NO:189 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403. SEQ ID NO:190 shows the nucleic acid sequence encoding a portion of Rab5C; GenBank accession no.: AA230407. 5 SEQ ID NO:191 shows the nucleic acid sequence encoding a portion of Rab5C; GenBank accession no.: AA230407. SEQ ID NO: 192 shows the nucleic acid sequence encoding a portion of Rab5C; GenBank accession no.: AA230407. 10 SEQ ID NO:193 shows the nucleic acid sequence encoding Exol04. SEQ ID NO: 194 shows the nucleic acid sequence encoding Exol 05, a human gene similar to mouse testis-specific protein PBSI3; may have some similarity to GenBank Accession no.: AA184365. 15 SEQ ID NO:195 shows the nucleic acid sequence encoding Exol06; may have some similarity to GenBank Accession no.: AA504490. SEQ ID NO:196 shows the nucleic acid sequence encoding Exol07; may have some similarity to GenBank Accession no.: AA504490. 20 SEQ ID NO:197 shows the nucleic acid sequence encoding Exol08; may have some similarity to GenBank Accession no.: Al181750. SEQ ID NO:198 shows the nucleic acid sequence encoding Exo109; may have some similarity to GenBank Accession no.: Al181750. 25 SEQ ID NO:199 shows the nucleic acid sequence encoding Exo110; may have some similarity to GenBank Accession no.: AU0431 11. SEQ ID NO:200 shows the nucleic acid sequence encoding Exol 11, a human gene similar to rat alpha-soluble NSF attachment protein (SNAP).

WO 00/43419 PCT/USOO/01431 -29 SEQ ID NO:201 shows the nucleic acid sequence encoding Exol 12. shows the nucleic acid sequence encoding Exol 05, a human gene similar to mouse testis-specific protein PBS1 3; may have some similarity to GenBank Accession no.: AA1 84365. 5 SEQ ID NO:202 shows the nucleic acid sequence encoding Exol 13 which may be similar to a mouse zinc finger protein. SEQ ID NO:203 shows the nucleic acid sequence encoding Exol 14 which may be similar to a mouse zinc finger protein. SEQ ID NO:204 shows the nucleic acid sequence encoding Exol 15 which may be similar 10 to chicken c-hairy 1; may have some similarity to GenBank Accession no.: AA116067. SEQ ID NO:205 shows the nucleic acid sequence encoding Exol 16 which may be similar to chicken c-hairy 1; may have some similarity to GenBank Accession no.: AA116067. SEQ ID NO:206 shows the nucleic acid sequence encoding a portion of mouse syntaxin4; 15 GenBank accession no.: U76832. SEQ ID NO:207 shows the nucleic acid sequence encoding a portion of mouse interleukin (IL) -3 receptor; GenBank accession no.: M29855. SEQ ID NO:208 shows the nucleic acid sequence encoding a portion of mouse interleukin (IL) -3 receptor; GenBank accession no.: M29855. 20 SEQ ID NO:209 shows the nucleic acid sequence encoding a portion of mouse low density lipoprotein (LDL) receptor-related protein; GenBank accession no.: AF074265. SEQ ID NO:210 shows the nucleic acid sequence encoding Exol17, similar to a human ANF126 zinc protein. 25 SEQ ID NO:211 shows the nucleic acid sequence encoding Exo118, similar to a rat isoprenylated 67 kDa protein. As indicated above, Exo3-Exol 18 are novel. The Exo proteinsencoded by SEQ ID NOS:1 51 (odd numbers) and Sequence ID NOS:53-211 are each novel in the aspect that they are shown herein to bind to an exocytosisorvesiculartransport protein, or fragment thereof, WO 00/43419 PCTIUSOO/01431 -30 for the first time. In preferred embodiments, the proteins encoded by SEQ ID NOS:1-51 (odd numbers) bind to GS27; the proteins encoded by SEQ ID NO:53 bind to rab7; the proteins encoded by SEQ ID NOS:54-64 bind to rab9; the proteins encoded by SEQ ID NOS:65-143bind to snap-23; the proteins encoded by SEQ ID NOS: 144-148 bind to rab3a; 5 the proteins encoded by SEQ ID NOS:149-152 bind to rab1 1; the proteins encoded by SEQ ID NOS:153-171 bind to rab3d; the proteins encoded by SEQ ID NOS:172-193 bind to rab5; the proteins encoded by SEQ ID NOS: 194-201 bind to alpha-snap; the proteins encoded by SEQ ID NOS:202-205 bind to unc1 8-1; and, the proteins encoded by SEQ ID NOS:206 211 bind to vamp3. 10 In a preferred embodiment, a protein is a "Exo protein" if the overall sequence identity of the protein sequence to any one of the amino acid sequences encoded by SEQ ID NOS: 1 51, odd numbers, and SEQ ID NOS:53-21 1, preferably those sequences encoding Exo3 118, is preferably greater than about 75%, more preferably greater than about 80%, even more preferably greater than about 85% and most preferably greater than 90%. In some 15 embodiments the sequence identity will be as high as about 93 to 95 or 98%. As is known in the art, a number of different programs can be used to identify whether a nucleic acid has sequence identity or similarity to a known gene or expression sequence tag (EST). Sequence identity will be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Apple. 20 Math. 2:482 (1981), by the sequence identity alignmentalgorithmofNeedleman& Wunsch, J. Mol. Biool.48:443(1970), by the search for similarity method of Pearson& Lipman, PNAS USA 85:2444(1988), by computerizedimplementationsof these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the Best Fit sequence program described by 25 Devereux et al., Nucl. Acid Res. 12:387-395 (1984), preferably using the default settings, or by inspection. Preferably, percent identity is calculated by FastDB based upon the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis," MacromoleculeSequencingand Synthesis, Selected Methods and Applications, pp 127-149 30 (1988), Alan R. Liss, Inc. An exampleofa useful algorithm is PILEUP. PILEUPcreatesa multiplesequencealignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplificationof the progressivealig nmentmethod of Feng & Doolittle, J. Mol. Evol. 35:351 35 360 (1987); the method is similar to that described by Higgins & Sharp CABIOS 5:151-153 WO 00/43419 PCT/USOO/01431 -31 (1989). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps. Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin et al., PNAS USA 90:5873-5787 (1993). A 5 particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., Methods in Enzymology, 266: 460-480 (1996); http://blast.wustl/edu/blast/ README.html]. WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span =1, overlap fraction = 0.125, word threshold (T) = 11. The HSP S and HSP S2 10 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. A % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of 15 the "longer" sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored). In a similar manner, "percent (%) nucleic acid sequence identity" with respect to the coding sequence of the polypeptides identified herein is defined as the percentage of nucleotide 20 residuesin a candidatesequencethat are identicalwith the nucleotideresiduesin the coding sequence of the Exo protein. A preferred method utilizes the BLASTN module of WU BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively. The alignment may include the introduction of gaps in the sequences to be aligned. In 25 addition, for sequences which contain either more or fewer amino acids than the protein encoded by the sequences in the Sequence Listing, it is understood that the percentage of sequence identity will be determined based on the number of identical amino acids in relation to the total number of amino acids. Thus, for example, sequence identity of sequences shorter than that shown in the Sequence Listing, as discussed below, will be 30 determined using the number of amino acids in the shorter sequence. As an example, SEQ ID NOS: 1-51 (odd numbers)and SEQ ID NOS:53-211 were identified as follows. A basic Blast search has been performed using program "Blastn" and database "nr". Blastn is a NCBI BLAST family of program used to compared a nucleotide query WO 00/43419 PCT/USOO/01431 -32 sequence against a nucleotide sequence database, and nr is a nucleotide sequence database that includes all non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF. Two numbers, Score (bits) and E values, will be returned after search querry is submitted. In general sequences considered known had 5 a Score > 100 and E < 0.001. Using the same Blast search, the nucleic acid sequences encoding Exo3-118 had a Score < 100 or E > 0.001. These nucleic acid sequences encoding Exo3-118 were then further searched using program "Blastn" and database "dbest". The dbest database is a nucleotidesequence database that includes non-redundant Database of GenBank+EMBL+DDBJEST Divisions. Using this criteria, some of the nucleic 10 acid sequences had a Score> 100 and E <0.001, thus, these sequences are considered novel, yet have "some similarity"to a known sequenceas indicated by the accession number provided. The sequences of the accession numbers provided herein are readily available to the skilled artisan. As will be appreciated by those in the art, the nucleic acid sequences of the invention can 15 be used to generate protein sequences. There are a variety of ways to do this, including cloning the entire gene and verifying its frame and amino acid sequence, or by comparing it to known sequences to search for homology to provide a frame, assuming the novel Exo protein has homology to some protein in the database being used. Generally, the nucleic acid sequences are input into a program that will search all three frames for homology. 20 This is done in a preferred embodiment using the following NCBI Advanced BLAST parameters. The program is blastx or blastn. The database is nr. The input data is as "Sequence in FASTA format". The organism list is "none". The "expect" is 10; the filter is default. The "descriptions" is 500, the "alignments" is 500, and the "alignment view" is pairwise. The "Query Genetic Codes" is standard (1). The matrix is BLOSUM62; gap 25 existence cost is 11, per residue gap cost is 1; and the lambda ratio is .85 default. This results in the generation of a putative protein sequence. While this program can be used to generatea preferred protein sequence, it is understood thatthe present invention provides polypeptides encoded by each of the three frames of each nucleic acid provided herein. Thus, when a protein encoded by the nucleic acid herein is described, the skilled artisan 30 understands that the protein begins with the first amino acid encoded by the first codon of the coding region, which is not necessarily the first nucleotide in the sequence listing. As will be appreciated by those skilled in the art, the sequences of the present invention may contain sequencing errors. That is, there may be incorrect nucleosides, frameshifts, unknown nucleosidesorothertypesof sequencingerrorsin any of the sequences; however, 35 the correct sequences will fall within the homology and stringency definitions herein. In WO 00/43419 PCT/USOO/01431 -33 addition, as will be appreciated by those in the art, in general, the first 200 bases or so of sequence contains the fewest errors. In a preferred embodiment, the Exo proteins are encoded by a nucleic acid comprising the first 100 nucleotides of the sequences set forth in the Sequence Listing and bind to an exocytosis or vesicular transport protein or fragment 5 thereof. Exo proteins of the present invention may be shorter or longer than the amino acid sequences encoded by the nucleic acids shown in the Sequence Listing. Thus, in one embodiment, Exo proteins can be portions or fragments of the amino acid sequences encoded by the nucleic acid sequences provided herein. In one embodiment herein, 10 fragmentsofExo proteinsare considered Exo proteins if a) they share at leastone antigenic epitope; b) have at least the indicated sequence identity; c) and preferably have Exo biologicalactivity, including binding to an exocytosis or vesicular transport protein. In some cases, where the sequence is used diagnostically, that is, when the presence or absence of Exo protein nucleic acid is determined, only the indicated sequence identity is required. 15 The nucleic acids of the present invention may also be shorteror longerthan the sequences in the Sequence Listing. The nucleic acid fragmentsincludeany portion of the nucleic acids provided herein which have a sequence not exactly previously identified; fragments having sequences with the indicated sequence identity to that portion not previously identified are provided in an embodiment herein. 20 In addition, as is more fully outlined below, Exo proteins can be made that are longer than those depicted in the Sequence Listings; for example, by the addition of epitope or purification tags, the addition of other fusion sequences, or the elucidation of additional coding and non-coding sequences. As described below, the fusion of an Exo peptide to a fluorescent peptide, such as Green Fluorescent Peptide (GFP), is particularly preferred. 25 Exo proteins may also be identified as encoded by Exo nucleic acids which hybridize to any one of the sequences depicted in SEQ ID NOS:1-51, odd numbers, and SEQ ID NOS:53-211, preferably those encoding Exo3-118. Hybridization conditions are further described below. In a preferred embodiment, when an Exo protein is to be used to generate antibodies, an 30 Exo protein must share at least one epitope or determinant with the full length protein. By "epitope" or "determinant" herein is meant a portion of a protein which will generate and/or bind an antibody. Thus, in most instances, antibodies made to a smaller Exo protein will be able to bind to the full length protein. In a preferred embodiment, the epitope is unique; WO 00/43419 PCT/USOO/01431 -34 that is, antibodies generated to a unique epitope show little or no cross-reactivity. The term "antibody" includes antibody fragments, as are known in the art, including Fab Fab,, single chain antibodies (Fv for example), chimeric antibodies, etc., either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA 5 technologies. In a preferred embodiment, the antibodies to Exo are capable of reducing or eliminating the biological function of Exo, as is described below. That is, the addition of anti-Exo antibodies(eitherpolyclonal or preferably monoclonal) to Exo (or cells containing Exo)may reduce or eliminate the Exo activity. Generally, at least a 25% decrease in activity is 10 preferred, with at least about 50% being particularly preferred and about a 95-100% decrease being especially preferred. The Exo antibodies of the invention specifically bind to Exo proteins. In a preferred embodiment, the antibodies specifically bind to Exo proteins. By "specifically bind" herein is meant that the antibodies bind to the protein with a binding constant in the range of at 15 least 1 0 4- 10-6 M-, with a preferred range being 10 - 10 M 1 . Antibodies are further described below. In the case of the nucleic acid, the overall sequence identity of the nucleic acid sequence is commensurate with amino acid sequence identity but takes into account the degeneracy in the genetic code and codon bias of different organisms. Accordingly, the nucleic acid 20 sequence identity may be either lower or higher than that of the protein sequence. Thus the sequence identity of the nucleic acid sequence as compared to the nucleic acid sequences of the Sequence Listing, is preferablygreaterthan 75%, more preferablygreater than about80%, particularly greaterthan about 85% and most preferablygreaterthan 90%. In some embodiments the sequence identity will be as high as about 93 to 95 or 98%. 25 In a preferred embodiment, an Exo nucleic acid encodes an Exo protein. As will be appreciated by those in the art, due to the degeneracy of the genetic code, an extremely large number of nucleic acids may be made, all of which encode the Exo proteins of the present invention. Thus, having identified a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids, by simply modifying the 30 sequence of one or more codons in a way which does not change the amino acid sequence of the Exo.

WO 00/43419 PCT/USOO/01431 -35 In one embodiment, the nucleic acid is determined through hybridization studies. Thus, for example, nucleic acids which hybridize under high stringency to the nucleic acid sequences shown in the sequence listing, or its complement is considered an Exo gene. High stringency conditions are known in the art; see for example Maniatis et al., Molecular 5 Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al., both of which are hereby incorporatedby reference. Stringentconditions are sequence-dependentand will be differentin differentcircumstances. Longersequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology- 10 Hybridization with Nucleic Acid Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-1 0'C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target 15 hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30*C for short probes (e.g. 10 to 50 nucleotides) and at least about 60'C 20 for long probes (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. In another embodiment, less stringent hybridization conditions are used; for example, moderate or low stringency conditions may be used, as are known in the art; see Maniatis and Ausubel, supra, and Tijssen, supra. 25 The Exo proteins and nucleic acids of the present invention are preferably recombinant. As used herein, "nucleic acid" may refer to either DNA or RNA, or molecules which contain both deoxy- and ribonucleotides. The nucleic acids include genomic DNA, cDNA and oligonucleotides including sense and anti-sense nucleic acids. Such nucleic acids may also contain modifications in the ribose-phosphate backbone to increase stability and half 30 life of such molecules in physiological environments. The nucleicacid may be double stranded, single stranded, or contain portionsof both double stranded or single stranded sequence. As will be appreciated by those in the art, the depiction of a single strand ("Watson") also defines the sequence of the other strand ("Crick"); thus the sequences depicted in the Sequence Listing also includethe complement WO 00/43419 PCT/USOO/01431 -36 of the sequence. By the term "recombinant nucleic acid" herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid by endonucleases, in a form not normally found in nature. Thus an isolated Exo nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally 5 joined, are both considered recombinant for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e. using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered 10 recombinant for the purposes of the invention. Similarly, a "recombinant protein" is a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid as depicted above. A recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics. For example, the protein may be isolated or purified away from some or 15 all of the proteins and compounds with which it is normally associated in its wild type host, and thus may be substantially pure. For example, an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight of the total protein in a given sample. A substantially pure protein comprises at least about 20 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred. The definition includes the production of an Exo protein from one organism in a different organism or host cell. Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of a inducible promoter or high expression promoter, such that the protein is made at 25 increased concentration levels. Alternatively, the protein may be in a form not normally found in nature, as in the addition of an epitope tag or amino acid substitutions, insertions and deletions, as discussed below. Also included within the definition of Exo proteins of the present invention are amino acid sequence variants. These variants fall into one or more of three classes: substitutional, 30 insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding an Exo protein, using cassette or PCR mutagenesisorothertechniqueswell known in theart, to produce DNA encodingthe variant, and thereafterexpressingthe DNA in recombinant cell culture as outlined above. However, variant Exo protein fragments having up to about 100-150 residues may be prepared by 35 in vitro synthesis using established techniques. Amino acid sequence variants are WO 00/43419 PCT/USOO/01431 -37 characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the Exo protein amino acid sequence. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified 5 characteristics as will be more fully outlined below. While the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the targetcodon or region and the expressed Exo variantsscreenedfor the optimal combination 10 of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M1 3 primer mutagenesis and PCR mutagenesis. Screening of the mutants is done using assays of Exo protein activities. Amino acid substitutions are typically of single residues; insertions usually will be on the 15 order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger. Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize 20 the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of the Exo protein are desired, substitutions are generally made in accordance with the following chart: Chart I Original Residue Exemplary Substitutions 25 Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser 30 GIn Asn Glu Asp Gly Pro His Asn, GIn WO 00/43419 PCT/USOO/01431 -38 lie Leu, Val Leu lie, Val Lys Arg, Gin, Glu Met Leu, Ile 5 Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe 10 Val lie, Leu Substantialchanges in functionor immunological identity are made by selectingsubstitutions that are less conservative than those shown in Chart I. For example, substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or 5 hydrophobicity of the molecule at the target site; or the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue 10 having an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine. The variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected 15 to modify the characteristics of the Exo proteins as needed. Alternatively, the variant may be designed such that the biological activity of the Exo protein is altered. For example, glycosylationsites may be altered or removed. Similarly, mutationswithin the kinasedomain and/or the cell death domain may be made. Covalent modifications of Exo polypeptides are included within the scope of this invention. 20 One type of covalent modification includes reacting targeted amino acid residues of an Exo polypeptide with an organic derivatizing agent that is capable of reacting with selected side chainsorthe N-or C-terminalresidues of an Exo polypeptide. Derivatizationwith bifunctional agents is useful, for instance, for crosslinking Exo to a water-insoluble support matrix or surface for use in the method for purifying anti-Exo antibodies or screening assays, as is WO 00/43419 PCT/USOO/01431 -39 more fully described below. Commonly used crosslinkingagents include, e.g., 1,1-bis(diazo acetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimideesters, forexample, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N 5 maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the 10 "-amino groups of lysine, arginine, and histidine side chains [T.E. Creighton, Proteins: Structureand MolecularProperties, W. H. Freeman& Co., San Francisco, pp. 79-86(1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group. Another type of covalent modification of the Exo polypeptide included within the scope of 15 this invention comprisesaltering the nativeglycosylationpattern of the polypeptide. "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence Exo polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence Exo polypeptide. 20 Addition of glycosylation sites to Exo polypeptides may be accomplished by altering the amino acid sequence thereof. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence Exo polypeptide (for 0-linked glycosylation sites). The Exo aminoacid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding 25 the Exo polypeptide at preselected bases such that codons are generatedthatwill translate into the desired amino acids. Another means of increasing the number of carbohydrate moieties on the Exo polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 September 1987, and in Aplin and 30 Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981). Removal of carbohydrate moieties present on the Exo polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. 35 Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic WO 00/43419 PCT/USOO/01431 -40 cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo-and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987). Another type of covalent modification of Exo comprises linking the Exo polypeptide to one 5 of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. Exo polypeptides of the present invention may also be modified in a way to form chimeric molecules comprising an Exo polypeptide fused to another, heterologous polypeptide or 10 amino acid sequence. In one embodiment, such a chimeric molecule comprises a fusion of an Exo polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino-orcarboxyl terminus of the Exo polypeptide. The presence of such epitope-tagged forms of an Exo polypeptide can be detected using an antibody against the tag polypeptide. Also, provision 15 of the epitope tag enables the Exo polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. In an alternative embodiment, the chimeric molecule may comprise a fusion of an Exo polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule, such a fusion could be to the Fc region of an IgG 20 molecule as discussed further below. Varioustag polypeptidesand theirrespectiveantibodiesarewell known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., 25 Molecular and Cellular Bioloqy, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky etal., Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al., BioTechnoloqy, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the 30 T7 gene 10 protein peptidetag [Lutz-Freyermuthet al., Proc. Natl. Acad. Sci. USA, 87:6393 6397 (1990)]. In an embodiment herein, Exo proteins of the Exo family and Exo proteins from other organisms are cloned and expressed as outlined below. Thus, probe or degenerate WO 00/43419 PCTIUSOO/01431 -41 polymerase chain reaction (PCR) primer sequences may be used to find other related Exo proteins from humans or other organisms. As will be appreciated by those in the art, particularly useful probe and/or PCR primer sequences include the unique areas of the Exo nucleic acid sequence. As is generally known in the art, preferred PCR primers are 5 from about 15 to about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosine as needed. The conditions for the PCR reaction are well known in the art. It is thereforealso understood that provided along with the sequences in the sequences listed herein are portions of those sequences, wherein unique portions of 15 nucleotides or more are particularly preferred. The skilled artisan can routinely 10 synthesize or cut a nucleotide sequence to the desired length. Once the Exo nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombinedto form the entire Exo nucleic acid. Once isolated from its natural source, e.g., contained within a plasmid or other vector or excised therefrom as a linear nucleic acid segment, the recombinant Exo nucleic acid can be further-used as a probe to identify 15 and isolate other Exo nucleic acids. It can also be used as a "precursor" nucleic acid to make modified or variant Exo nucleic acids and proteins. The skilled artisan understands that wherein two or more nucleic acids overlap, the overlapping portion(s) of one of the overlapping nucleic acids can be omitted and the nucleic acids combined for example, by ligation, to form a longer linear Exo nucleic acid so as to, for example, encode the full length 20 or mature peptide. The same applies to the amino acid sequences of Exo polypeptides in that they can be combined so as to form one contiguous peptide. Using the nucleic acids of the present invention which encode an Exo protein, a variety of expression vectors are made. The expression vectors may be either self-replicating extrachromosomalvectors or vectors which integrate into a hostgenome. Generallythese 25 expressionvectorsincludetranscriptional and translational regulatorynucleicacid operably linked to the nucleic acid encoding the Exo protein. The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding 30 site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleicacid sequence. Forexample, DNAfora presequenceorsecretory leaderis operably 35 linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the WO 00/43419 PCTIUSOO/01431 -42 secretionof the polypeptide;a promoterorenhanceris operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of 5 a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. The transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the Exo protein; for example, 10 transcriptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the Exo protein in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells. In general, the transcriptional and translational regulatory sequences may include, but are 15 not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. In a preferredembodiment, the regulatory sequences include a promoterand transcriptional start and stop sequences. Promoter sequences encode either constitutive or inducible promoters. The promoters 20 may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention. In addition, the expression vector may comprise additional elements. For example, the expression vector may have two replication systems, thus allowing it to be maintained in 25 two organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification. Furthermore, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct. The integrating vector may be directed to a specific locus in the host cell by selecting the 30 appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.

WO 00/43419 PCT/USOO/01431 -43 In addition, in a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used. A preferred expression vector system is a retroviral vector system such as is generally 5 described in PCT/US97/01019 and PCT/US97/01048, both of which are hereby expressly incorporated by reference. Exo proteins of the present invention are produced by culturing a host cell transformed with an expressionvectorcontaining nucleicacid encodingan Exo protein, underthe appropriate conditions to induce or cause expression of the Exo protein. The conditions appropriate 10 for Exo protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation. For example, the use of constitutive promoters in the expression vector will require optimizingthe growth and proliferationof the hostcell, while the use of an inducible promoter requires the appropriate growth conditionsfor induction. In addition, in someembodiments, 15 the timing of the harvest is important. For example, the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield. Appropriate host cells include yeast, bacteria, archebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melangaster cells, 20 Saccharomyces cerevisiae and otheryeasts, E. coli, Bacillus subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells, fibroblasts, Schwanoma cell lines, immortalized mammalian myeloid and lymphoid cell lines, Jurkatcells, livercells, mammary cells, sperm, egg, adipocytes, granulocytes,adrenalchromaffin cells, mast cells, basophils, endocrine and exocrine cells, muscle cells, eosinophils and neuronal cells. 25 In a preferredembodiment,the Exo proteinsare expressed in mammaliancells. Mammalian expressionsystemsare also known in the art, and include retroviralsystems. A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequencefor Exo protein into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to 30 the 5' end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase I to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element (enhancer element), typically located within 100 WO 00/43419 PCT/US0O/01431 -44 to 200 base pairs upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include 5 the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter. Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3 to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3' terminus of the 10 mature mRNA is formed by site-specific post-translational cleavage and polyadenylation. Examples of transcription terminator and polyadenlytion signals include those derived form SV40. The methods of introducing exogenous nucleic acid into mammalianhosts, as well as other hosts, is well known in the art, and will vary with the host cell used. Techniques include 15 dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei. In a preferred embodiment, Exo proteins are expressed in bacterial systems. Bacterial expression systems are well known in the art. 20 A suitable bacterialpromoteris any nucleicacid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3) transcription of the coding sequence of Exo protein into mRNA. A bacterial promoter has a transcription initiation regionwhich is usually placed proximal to the 5 end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. 25 Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactoseand maltose, and sequencesderived from biosynthetic enzymes such as tryptophan. Promoters from bacteriophage may also be used and are known in the art. In addition, synthetic promoters and hybrid promoters are also useful; 30 for example, the tac promoter is a hybrid of the trp and lac promoter sequences. Furthermore,a bacterialpromoter can include naturally occurring promotersof non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.

WO 00/43419 PCT/USOO/01431 -45 In addition to a functioning promoter sequence, an efficient ribosome binding site is desirable. In E. coli, the ribosome binding site is called the Shine-Delgarno (SD) sequence and includes an initiation codon and a sequence 3-9 nucleotides in length located 3 - 11 nucleotides upstream of the initiation codon. 5 The expression vector may also include a signal peptide sequence that provides for secretion of the Exo protein in bacteria. The signal sequence typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell, as is well known in the art. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer 10 membrane of the cell (gram-negative bacteria). The bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol,erythromycin, kanamycin, neomycinand tetracycline. Selectablemarkers 15 also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways. These components are assembled into expressionvectors. Expressionvectorsfor bacteria are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others. 20 The bacterialexpression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others. In one embodiment, Exo proteins are produced in insect cells. Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art. 25 In a preferred embodiment, Exo protein is produced in yeast cells. Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragiis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica. Preferred promoter sequences for expression in yeast include the 30 inducible GAL1,10 promoter, the promoters from alcohol dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase, WO 00/43419 PCT/USOO/01431 -46 hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and the acid phosphatase gene. Yeast selectable markers include ADE2, HIS4, LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; the neomycin phosphotransferase gene, which confers resistance to G418; and the CUP1 gene, which allows yeast to grow in the 5 presence of copper ions. The Exo protein may also be made as a fusion protein, using techniques well known in the art. Thus, for example, for the creation of monoclonal antibodies, if the desired epitope is small, the Exo protein may be fused to a carrier protein to form an immunogen. Alternatively, the Exo protein may be made as a fusion protein to increase expression, or 10 for other reasons. For example, when the Exo protein is an Exo peptide, the nucleic acid encoding the peptide may be linked to othernucleicacid forexpression purposes. Similarly, Exo proteins of the invention can be linked to protein labels, such as green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), etc. 15 In one embodiment, the Exo nucleic acids, proteins and antibodies of the invention are labeled. By "labeled" herein is meant that a compound has at least one element, isotope or chemical compound attached to enablethe detectionof the compound. In general, labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes. 20 The labels may be incorporated into the compound at any position. In a preferred embodiment, the Exo protein is purified or isolated after expression. Exo proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods includeelectrophoretic, molecular, immunological and chromatographic techniques, 25 including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing. For example, the Exo protein may be purified using a standard anti-Exo antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification, Springer-Verlag, NY (1982). The degree 30 of purification necessary will vary depending on the use of the Exo protein. In some instances no purification will be necessary. Once expressed and purified if necessary, the Exo proteins and nucleic acids are useful in a number of applications.

WO 00/43419 PCT/USOO/01431 -47 The nucleotide sequences (or their complement) encoding Exo proteins have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA. Exo protein nucleic acid will also be useful for the preparation of Exo protein polypeptides by 5 the recombinant techniques described herein. The full-length native sequence Exo protein gene, or portions thereof, may be used as hybridizationprobesfora cDNAlibraryto isolate the full-length Exo proteingeneorto isolate still other genes (for instance, those encoding naturally-occurring variants of Exo protein or Exo proteinfrom other species) which have a desired sequence identityto the Exo protein 10 coding sequence. Optionally, the length of the probes will be about 20 to about 50 bases. The hybridization probes may be derived from the nucleotide sequences herein or from genomic sequences including promoters, enhancer elements and introns of native sequences as provided herein. By way of example, a screening method will comprise isolating the coding region of the Exo protein gene using the known DNA sequence to 15 synthesize a selected probe of about 40 bases. Hybridization probes may be labeled by a variety of labels, including radionucleotides such as 1 2 P or 3S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the Exo protein gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to 20 determine which members of such libraries the probe hybridizes. The probes may also be employed in PCR techniques to generate a pool of sequences for identification of closely related Exo protein coding sequences. Nucleotide sequences encoding a Exo protein can also be used to construct hybridization probes for mapping the gene which encodes that Exo protein and for the genetic analysis 25 of individuals with genetic disorders. The nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries. Nucleic acids which encode Exo protein or its modified forms can also be used to generate 30 eithertransgenicanimalsor"knockout" animalswhich, in turn, are useful in the development and screening of therapeutically useful reagents. A transgenic animal (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A WO 00/43419 PCT/USOO/01431 -48 transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, cDNA encoding an Exo protein can be used to clone genomic DNA encoding an Exo protein in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express 5 the desired DNA. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009. Typically, particular cells would be targeted for the Exo protein transgene incorporation with tissue-specific enhancers. Transgenic animals that include a copy of a transgene encoding an Exo protein introduced into the 10 germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of the desired nucleic acid. Such animals can be used as tester animalsfor reagentsthought to confer protection from, for example, pathologicalconditions associated with its overexpression. In accordance with this facetof the invention, an animal is treated with the reagent and a reduced incidence of the pathologicalcondition, compared 15 to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition. Alternatively, non-human homologues of the Exo protein can be used to construct a Exo protein "knock out" animal which has a defective or altered gene encoding an Exo protein 20 as a result of homologous recombination between the endogenous gene encoding an Exo protein and altered genomic DNA encoding an Exo protein introduced into an embryonic cell of the animal. For example, cDNA encoding an Exo protein can be used to clone genomic DNA encodingan Exo protein in accordancewith established techniques. A portion of the genomic DNA encoding an Exo protein can be deleted or replaced with another gene, 25 such as a gene encoding a selectable marker which can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see e.g., Thomasand CapecchiCell, 51:503 (1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously 30 recombined with the endogenous DNA are selected [see e.g., Li et al., Cell, 6:915 (1992)]. The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see e.g., Bradley, in TeratocarcinomasandEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryocan then be implantedintoa suitablepseudopregnantfemalefosteranimal 35 and the embryo brought to term to create a "knock out" animal. Progeny harboring the homologously recombined DNA in their germ cells can be identifiedby standard techniques and used to breed animals in which all cells of the animal contain the homologously WO 00/43419 PCT/USOO/01431 -49 recombined DNA. Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the Exo protein polypeptide. It is understood that cell based knock-out or "knock-in" systems can also be made and utilized in accordance with the 5 present disclosure. It is understood that the models described herein can be varied. For example, "knock-in" models can be formed, or the models can be cell-based rather than animal models. Nucleic acid encoding the Exo polypeptides, antagonists or agonists may also be used in gene therapy. In gene therapy applications, genes are introduced into cells in order to 10 achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene. "Gene therapy" includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can be used as 15 therapeutic agents for blocking the expression of certain genes in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane. (Zamecnik etal., Proc. Nat. Acad. Sci. USA 83, 4143-4146 [1986]). The oligonucleotides can be modified to enhance their uptake, e.g. by substituting their 20 negatively charged phosphodiester groups by uncharged groups. There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, 25 microinjection, cell fusion, DEAE-dextran, the calcium phosphateprecipitationmethod, etc. The currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]). In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an 30 antibody specific for a cell surface membrane protein or the targetcell, a ligand fora receptor on the target cell, etc. Where liposomes are employed, proteins which bind to a cell surface membrane protein associatedwith endocytosismay be used fortargetingand/orto facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular WO 00/43419 PCT/USOO/01431 -50 localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu etal., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and gene therapy protocols see Anderson et al., Science 256, 808-813 (1992). 5 In a preferred embodiment, the Exo proteins, nucleic acids, modified proteins and cells containing the native or modified Exo proteins are used in screening assays. Identification of this important exocytosis protein permits the design of drug screening assays for compounds that modulate Exo activity. Screens may be designed to first find candidate agents that can bind to Exo proteins, and 10 then these agents may be used in assays that evaluate the ability of the candidate agent to modulate Exo activity. Thus, aswill be appreciatedby those in the art, there are a number of different assays which may be run; binding assays and activity assays. Thus, in a preferred embodiment, the methods comprise combining an Exo protein and a candidate bioactive agent, and determining the binding of the candidate agent to the Exo 15 protein. Preferred embodiments utilize the human Exo protein, although other mammalian proteins may also be used, including rodents (mice, rats, hamsters, guinea pigs, etc.), farm animals (cows, sheep, pigs, horses, etc.) and primates. These latter embodiments may be preferredin the developmentof animal modelsof human disease. In some embodiments, as outlined herein, variant or derivative Exo proteins may be used, including deletion Exo 20 proteins as outlined above. The term "candidatebioactiveagent" or "exogeneous compound" as used herein describes any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., with the capability of directly or indirectly altering the bioactivity of Exo. Generallya pluralityof assay mixtures are run in parallel with different agent concentrations 25 to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection. Candidateagents encompass numerouschemicalclasses, though typicallythey are organic 30 molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Candidate agents comprise functional groups necessaryfor structuralinteractionwith proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of WO 00/43419 PCT/USOO/01431 -51 the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functionalgroups. Candidateagents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, 5 structural analogs or combinations thereof. Particularly preferred are peptides. Candidate agents are obtained from a wide varietyof sources including librariesofsynthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form 10 of bacterial, fungal, plant and animal extractsare availableor readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjectedto directedor random chemical modifications,such as acylation, alkylation, esterification, amidification to produce structural analogs. 15 In a preferred embodiment,the candidate bioactive agents are proteins. By "protein" herein is meantat leasttwo covalentlyattached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The protein may be made up of naturallyoccurringamino acids and peptide bonds, or synthetic peptidomimetic structures. Thus "amino acid", or "peptide residue", as used herein means both naturally occurring and synthetic amino acids. For 20 example, homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chains may be in either the (R) or the (S) configuration. In the preferredembodiment, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, 25 for example to prevent or retard in vivo degradations. In a preferred embodiment, the candidate bioactive agents are naturally occurring proteins or fragments of naturally occuring proteins. Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be used. In this way libraries of procaryotic and eukaryotic proteins may be made for screening 30 againstExo. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.

WO 00/43419 PCTIUSOO/01431 -52 In a preferred embodiment, the candidate bioactive agents are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. The peptides may be digests of naturally occuring proteins as is outlined above, random peptides, or "biased" random 5 peptides. By "randomized" or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to 10 allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceousagents. In one embodiment, the library is fully randomized, with no sequence preferences or constants at any position. In a preferred embodiment, the library is biased. That is, some positions within the sequenceareeitherheld constant,orare selectedfrom a limited number 15 of possibilities. For example, in a preferred embodiment, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc. 20 In a preferred embodiment, the candidate bioactive agents are nucleic acids. By "nucleic acid" or"oligonucleotide"or grammatical equivalents herein means at least two nucleotides covalently linked together. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide 25 (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Patent No. 30 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321 (1989), 0 methylphophoroamiditelinkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566(1993); Carlssonetal., Nature 380:207 (1996), all of which 35 are incorporated by reference). Other analog nucleic acids include those with positive WO 00/43419 PCT/USOO/01431 -53 backbones(Denpcy et al., Proc. Nat. Acad. Sci. USA 92:6097 (1995); non-ionicbackbones (U.S. Patent Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 5 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y.S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, includingthose described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate 10 Modifications in Antisense Research", Ed. Y.S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp169-176). Several nucleic acid analogs are described in Rawls, C & E News June 2,1997 page 35. All of these references are hereby expressly incorporated by reference. These modifications of the ribose 15 phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments. In addition, mixtures of naturally occurring nucleic acids and analogs can be made. Alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occuring nucleicacidsand analogsmay be made. The nucleicacids may be single stranded 20 or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid,wherethe nucleicacid contains any combination of deoxyribo-and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc. 25 As described above generally for proteins, nucleic acid candidate bioactive agents may be naturally occuring nucleicacids, random nucleicacids, or biased" random nucleic acids. For example, digestsof procaryoticoreucaryoticgenomes may be used as is outlinedabove for proteins. In a preferred embodiment, the candidate bioactive agents are organic chemical moieties, 30 a wide variety of which are available in the literature. The assays provided utilize Exo proteins as defined herein. In one embodiment, portions of Exo proteins are utilized; in a preferred embodiment, portions having Exo activity are used. Exo activity is described further below and includes binding activity to GS27, rab7, rab9, snap-23, rab3a, rabl1, rab3d, rab5, alpha-snap, uncl8-1, vamp3 or Exo protein WO 00/43419 PCT/USOO/01431 -54 modulatorsas furtherdescribed below. In addition, the assays described herein may utilize either isolated Exo proteins or cells comprising the Exo proteins. Generally, in a preferred embodiment of the methods herein, the Exo protein or the candidate agent is non-diffusibly bound to an insoluble support having isolated sample 5 receiving areas (e.g. a microtiterplate, an array, etc.). The insolublesupports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. 10 These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose, teflon TM, etc. Microtiter platesand arraysare especiallyconvenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. In some cases magnetic beads and the like are included. The particular manner of binding of the composition is not crucial so long as it is compatible 15 with the reagents and overall methods of the invention, maintains the activity of the compositionand is nondiffusable. Preferred methods of binding includethe use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to "sticky" or ionic supports, chemical crosslinking,the synthesisof the protein or agent on the surface, etc. In some embodiments, 20 GS27 can be used. Other embodiments include using, rab7, rab3a, rab3d, snap23, rab9, rab5, alpha snap, rab1 1, unc18-1 or vamp3. Following binding of the protein or agent, excess unbound material is removed by washing. The sample receiving areas may then be blockedthrough incubationwith bovine serum albumin (BSA), casein or other innocuous protein or other moiety. Also included in this invention are screening assays wherein solid 25 supports are not used. In a preferred embodiment, the Exo protein is bound to the support, and a candidate bioactive agent is added to the assay. Alternatively, the candidate agent is bound to the support and the Exo protein is added. Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. 30 Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.

WO 00/43419 PCT/USOO/01431 -55 The determination of the binding of the candidate bioactive agent to the Exo protein may be done in a number of ways. In a preferred embodiment, the candidate bioactive agent is labelled, and binding determined directly. For example, this may be done by attaching all or a portion of the Exo protein to a solid support, adding a labelled candidate agent (for 5 example a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps may be utilized as is known in the art. By "labeled" herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g. radioisotope, fluorescers, enzyme, 10 antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc. Forthe specific binding members, the complementarymember would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above. The label can directly or indirectly provide a 15 detectable signal. In some embodiments, only one of the components is labeled. For example, the proteins (or proteinaceous candidate agents) may be labeled at tyrosine positions using 1251, or with fluorophores. Alternatively, more than one component may be labeled with different labels; using 1 for the proteins, for example, and a fluorophor for the candidate agents. 20 In a preferred embodiment, the binding of the candidate bioactive agent is determined through the use of competitive binding assays. In this embodiment, the competitor is a binding moiety known to bind to the targetmolecule(i.e. Exo), such as an antibody, peptide, binding partner, ligand, etc. In a preferred embodiment, the competitoris GS27, rab7, rab9, snap-23, rab3a, rab11, rab3d, rab5, alpha-snap, uncl8-1, or vamp3. Under certain 25 circumstances, there may be competitive binding as between the bioactive agent and the binding moiety, with the binding moiety displacing the bioactive agent. This assay can be used to determine candidateagents which interfere with binding between Exo proteins and GS27, rab7, rab9, snap-23, rab3a, rab11, rab3d, rab5, alpha-snap, uncl8-1, or vamp3. In one embodiment, the candidatebioactiveagent is labeled. Either the candidate bioactive 30 agent, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present. Incubations may be performed at any temperature which facilitates optimalactivity, typically between 4 and 40*C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening. Typically WO 00/43419 PCTIUSOO/01431 -56 between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding. In a preferred embodiment, the competitoris added first, followed by the candidate bioactive 5 agent. Displacement of the competitor is an indication that the candidate bioactive agent is binding to the Exo protein and thus is capable of binding to, and potentially modulating, the activity of the Exo protein. In this embodiment, either component can be labeled. Thus, forexample, if the competitor is labeled, the presence of label in the wash solution indicates displacement by the agent. Alternatively, if the candidate bioactive agent is labeled, the 10 presence of the label on the support indicates displacement. In an alternative embodiment, the candidate bioactive agent is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor may indicate that the bioactive agent is bound to the Exo protein with a higher affinity. Thus, if the candidate bioactive agent is labeled, the presenceof the label on the support, coupled 15 with a lackof competitorbinding, may indicatethat the candidate agent is capable of binding to the Exo protein. In a preferredembodiment,the methods comprisedifferential screening to identity bioactive agents that are capable of modulating the activity of the Exo proteins. In this embodiment, the methods comprise combining an Exo protein and a competitor in a first sample. A 20 second sample comprises a candidate bioactive agent, an Exo protein and a competitor. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the Exo protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable 25 of binding to the Exo protein. Alternatively, a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native Exo protein, but cannot bind to modified Exo proteins. The structure of the Exo protein may be modeled, and used in rational drug design to synthesize agents that interact with that site. Drug candidates that affect Exo bioactivity 30 are also identified by screening drugs for the ability to either enhance or reduce the activity of the protein.

WO 00/43419 PCTUSOO/01431 -57 Positive controls and negative controls may be used in the assays. Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, all samples are washed free of non-specifically bound material and 5 the amount of bound, generally labeled agentdetermined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counterto determinethe amount of bound compound. A variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to 10 facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbialagents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding. The components provided herein for the assays provided herein may also be combined 15 to form kits. The kits can be based on the use of the protein and/orthe nucleicacid encoding the Exo proteins. Assays regarding the use of nucleic acids are further described below. Screening for agents that modulate the activity of Exo may also be done. In a preferred embodiment, methods for screening for a bioactive agent capableof modulatingthe activity of Exo comprise the steps of adding a candidate bioactive agent to a sample of Exo, as 20 above, and determiningan alterationin the biologicalactivity of Exo. "Modulating the activity of Exo" includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present. Thus, in this embodiment, the candidate agent should both bind to Exo (although this may not be necessary), and alter its biological or biochemical activity as defined herein. The methods include both in vitro screening methods, as are generally 25 outlined above, and in vivo screening of cells for alterations in the presence, distribution, activity or amount of Exo. Thus, in this embodiment, the methods comprise combiningan Exo sampleand a candidate bioactive agent, and evaluating the effect on exocytosis. By "Exo activity" or grammatical equivalents herein is meant one of Exo's biological activities, including, but not limited to, 30 its ability to affect exocytosis, secretion and/or vesicular transport. Included within exocytosis, secretion and/or vesicular transport activitiesinclude regulating or involvement in steps therein such as docking, fusion and targeting activities of proteins involved in the entire pathway of exocytosis, secretion and/or vesicular transport. In one embodiment, WO 00/43419 PCT/USOO/01431 -58 vesicular refers to any vesicle including synaptic or secretory granules and vesicles those involved in exocytosis, endocytosis or the trans-golgi network. In one embodiment, exo activity includes GTPase activity or regulation thereof. One exo activity herein is binding to at least one protein selected from the group consisting of GS27, rab7, rab9, snap-23, 5 rab3a, rab1 1, rab3d, rab5, alpha-snap, unc1 8-1 and vamp3. Other exo activities include the activities and regulation thereof of GS27, rab7, rab9, snap-23, rab3a, rab1 1, rab3d, rab5, alpha-snap, unc18-1 and vamp3. In a preferred embodiment, the activity of the Exo protein is increased; in another preferred embodiment, the activity of the Exo protein is decreased. Thus, bioactive agents that are 10 antagonists are preferred in some embodiments, and bioactive agents that are agonists may be preferred in other embodiments. In a preferred embodiment, the invention provides methods for screeningfor bioactiveagents capable of modulating the activity of an Exo protein. The methods comprise adding a candidate bioactive agent, as defined above, to a cell comprising Exo proteins. Preferred 15 cell types include almost any cell. The cells containa recombinantnucleicacid thatencodes an Exo protein. In a preferred embodiment, a library of candidate agents are tested on a plurality of cells. In some embodiments, the assays include exposing the cells to an exocytosis agent that will induce exocytosis in control cells, i.e. cells of the same type but that do not contain the 20 exogeneous nucleic acid encoding an Exo. Suitable exocytosis agents are known in the art and include but are not limited to ionomycin and Ca*, such as, but not limited to the Ca**ionophore A23187. Alternatively, the cells may be exposed to conditionsthat normally result in exocytosis, and changes in the normal exocytosis progression are determined. Alternatively, the cells into which the Exo nucleic acids are introduced normally under 25 exocytosis, and thus changes (for example, inhibition of exocytosis) are determined. Optionally, the cells normally do not undergo exocytosis, and the introduction of a candidate agent causes exocytosis. Thus, the effect of the candidate agent on exocytosis is then evaluated. Detection of exocytosis may be done as will be appreciated by those in the art. In one 30 embodiment, indicators of exocytosis are used. Suitable exocytosis labels include, but are not limited to, annexin. Accordingly, these agents can be used as an affinity ligand, and attached to a solid support such as a bead, a surface, etc. and used to pull out cells WO 00/43419 PCTIUSOO/01431 -59 that are undergoing exocytosis. Similarly, these agents can be coupled to a fluorescent dye such as PerCP, and then used as the basis of a fluorescent-activated cell sorting (FACS) separation. Moreover, FACS or other optical methods can be used to detect exocytosis activity and the modulation thereof based on light scattering, light absorption, 5 dye uptake and release, granule enzyme activity and quantification of granule specific proteins. In this way, bioactive agents are identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of the Exo protein. The compounds having the desired pharmacological activity may be administered in a physiologically acceptable 10 carrier to a host, as previously described. The agents may be administered in a variety of ways, orally, parenterally e.g., subcutaneously, intraperitoneally, intravascularly, etc. Depending upon the manner of introduction, the compounds may be formulatedin a variety of ways. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt.%. 15 The pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds. Diluents known to the art include aqueous media, vegetable and animal oils 20 and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents. Without being bound by theory, it appears that Exo is an important protein in exocytosis. Accordingly, disorders based on mutant or variant Exo genes may be determined. In one 25 embodiment, the invention provides methods for identifying cells containing variant Exo genes comprising determining all or part of the sequence of at least one endogeneous Exo genes in a cell. As will be appreciated by those in the art, this may be done using any number of sequencing techniques. In a preferred embodiment, the invention provides methods of identifying the Exo genotype of an individual comprising determining all or part 30 of the sequence of at least one Exo gene of the individual. This is generally done in at least one tissue of the individual, and may include the evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced Exo gene to a known Exo gene, i.e. a wild-type gene.

WO 00/43419 PCT/USOO/01431 -60 The sequence of all or part of the Exo gene can then be compared to the sequence of a known Exo gene to determine if any differences exist. This can be done using any number of known sequence identity programs, such as Bestfit, etc. In a preferred embodiment, the presenceof a difference in the sequence between the Exo gene of the patientand the known 5 Exo gene is indicative of a disease state or a propensity for a disease state, as outlined herein. The present discovery relating to the role of Exo in exocytosis thus provides methods for inducing or preventing exocytosis in cells. In a preferred embodiment, the Exo proteins, and particularly Exo fragments, are useful in the study or treatment of conditions which 10 are mediated by exocytosis, i.e. to diagnose, treat or preventexocytosis-mediateddisorders. Thus, "exocytosis mediated disorders" or"disease state" include conditions involving both insufficient or excessive exocytosis, vesicular transport, and/or secretion via the secretory pathway, including inflammatory mediator release from mast cells including asthma, allergies, and Chediak-Higashi Syndrome (CHS). Additionally, control of neurotransmitter 15 release can be used to treat Alzheimer's disease, Parkinson's and Huntington's disease states as well as some skitzophrenia, thus these can also be included in exocytosis mediated disorders in some cases. In other cases, fertilization and lactation disorders can be included as disease states which can be treated with the compositions provided and/or identified herein. Additionally, some diabetes, digestion and wound healing disorders can 20 be exocytosis mediated disorders. Thus, in one embodiment, methods of modulating exocytosis in cells or organisms are provided. In one embodiment, the methods comprise administering to a cell an anti-Exo antibody that reduces or eliminates the biological activity of the endogeneous Exo protein. Alternatively, the methods comprise administering to a cell or organism a recombinant 25 nucleic acid encoding an Exo protein. As will be appreciated by those in the art, this may be accomplished in any number of ways. In a preferred embodiment, the activity of Exo is increased by increasing the amount of Exo in the cell, for example by overexpressing the endogeneous Exo or by administering a gene encoding an Exo, using known gene therapy techniques, for example. In a preferred embodiment, the gene therapy techniques 30 include the incorporation of the exogeneous gene using enhanced homologous recombination (EHR), for example as described in PCT/US93/03868, hereby incorporated by reference in its entireity. In one embodiment, the invention provides methods for diagnosing an exocytosis related condition in an individual. The methods comprise measuring the activity of Exo in a tissue WO 00/43419 PCT/USO0/01431 -61 from the individual or patient, which may include a measurement of the amount or specific activity of Exo. This activity is compared to the activity of Exo from either a unaffected second individual or from an unaffectedtissuefrom the first individual. When these activities are different, the first individual may be at risk for an exocytosis mediated disorder. 5 The proteins and nucleic acids provided herein can also be used for screening purposes wherein the protein-protein interactions of the Exo proteins can be identified. Genetic systems have been described to detect protein-protein interactions. The first work was done in yeast systems, namely the "yeast two-hybrid" system. The basic system requires a protein-protein interaction in order to turn on transcriptionof a reportergene. Subsequent 10 work was done in mammalian cells. See Fields et al., Nature 340:245 (1989); Vasavada et al., PNAS USA 88:10686 (1991); Fearon et al., PNAS USA 89:7958 (1992); Dang et al., Mol. Cell. Biol. 11:954 (1991); Chien et al., PNAS USA 88:9578 (1991); and U.S. Patent Nos. 5,283,173, 5,667,973, 5,468,614, 5,525,490, and 5,637,463. a preferred system is described in Serial No. 09/050,863, filed March 30, 1998, entitled "Mammalian Protein 15 Interaction Cloning System". For use in conjunctionwith these systems, a particularlyuseful shuttle vector is described in Serial No. 09/133,944, filed August 14, 1998, entitled "Shuttle Vectors". In general, two nucleic acids are transformed into a cell, where one is a "bait" such as the gene encoding GS27, rab7, rab9, snap-23, rab3a, rab1 1, rab3d, rab5, alpha-snap, unc1 8-1, 20 vamp3 or a portion thereof, and the other encodes a test candidate. Only if the two expression products bind to one another will an indicator, such as a fluorescent protein, be expressed. Expression of the indicator indicates when a test candidate binds to the GS27, rab7, rab9, snap-23, rab3a, rab11, rab3d, rab5, alpha-snap, uncl8-1 orvamp3 and can be identified as an Exo protein. Using the same system and the identified Exo proteins 25 the reverse can be performed. Namely, the Exo proteins provided herein can be used to identify new baits, or agents which interact with Exo proteins. Additionally, the two-hybrid system can be used wherein a test candidate is added in addition to the bait and the Exo protein encoding nucleic acids to determine agents which interfere with the bait, such as GS27, rab7, rab9, snap-23, rab3a, rab1 1, rab3d, rab5, alpha-snap, unc1 8-1 orvamp3, and 30 the Exo protein. In one embodiment, a mammalian two-hybrid system is preferred. Mammalian systems provide post-translational modifications of proteins which may contribute significantly to their ability to interact. In addition, a mammalian two-hybrid system can be used in a wide 35 varietyofmammaliancell typesto mimicthe regulation, induction, processing,etc. of specific WO 00/43419 PCT/USOO/01431 -62 proteins within a particular cell type. Forexample, proteins involved in a disease state such as those describedabove could be tested in the relevant disease cells. Similarly, for testing of random proteins, assaying them underthe relevant cellular conditionswill give the highest positive results. Furthermore, the mammalian cells can be tested under a variety of 5 experimental conditions that may affect intracellular protein-protein interactions, such as in the presence of hormones, drugs, growth factors and cytokines, cellular and chemical stimuli, etc., that may contribute to conditions which can effect protein-protein interactions, particularlythose involved in exocytosis, the secretory pathway, and/or vesicular transport. Expression in various cell types, and assays for Exo activity are described above. The 10 activityassays, such as having an effect on exocytosis, secretion and/or vesiculartransport can be performed to confirm the activity of Exo proteins which have already been identified by their sequence identity/similarity or binding to GS27, rab7, rab9, snap-23, rab3a, rabl 1, rab3d, rab5, alpha-snap, unc1 8-1 or vamp3 as well as to further confirm the activity of lead compounds identified as modulators of exocytosis, secretion and/or vesicular transport. 15 Assays involving binding such as the two-hybrid system may take into accountnon-specific binding proteins (NSB). In one embodiment, the Exo proteins of the present invention may be used to generate polyclonal and monoclonalantibodiesto Exo proteins, which are useful as described herein. 20 Similarly, the Exo proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify Exo antibodies. In a preferred embodiment, the antibodies are generated to epitopes unique to the Exo protein; that is, the antibodies show little or no cross-reactivity to other proteins. These antibodies find use in a number of applications. For example, the Exo antibodies may be 25 coupled to standard affinity chromatography columns and used to purify Exo proteins as further described below. The antibodies may also be used as blocking polypeptides, as outlined above, since they will specifically bind to the Exo protein. The anti-Exo protein antibodiesmay comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised 30 in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the Exo protein polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized.

WO 00/43419 PCTIUSOO/01431 -63 Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovinethyroglobulinand soybeantrypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant(monophosphorylLipid a, synthetic trehalose dicorynomycolate). The immunization 5 protocol may be selected by one skilled in the art without undue experimentation. The anti-Exo protein antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit 10 lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. The immunizing agent will typically include the Exo protein polypeptide or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian 15 sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103]. Immortalized cell lines are usually transformedmammalian cells, particularlymyeloma cells of rodent, bovineand human origin. Usually, rator mouse myeloma cell lines are employed. 20 The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survivalof the unfused, immortalizedcells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase(HGPRTor HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"),which substances preventthe 25 growth of HGPRT-deficient cells. Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalizedcell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, 30 San Diego, California and the American Type Culture Collection, Rockville, Maryland. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].

WO 00/43419 PCTIUSOO/01431 -64 The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against Exo protein. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or 5 enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107220 (1980). After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution proceduresand grown by standard methods[Goding, supral. Suitableculture media 10 for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal. The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein a-Sepharose, hydroxylapatite chromatography, gel 15 electrophoresis, dialysis, or affinity chromatography. The monoclonalantibodiesmay also be made by recombinantDNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding 20 the heavy and lightchains ofmurineantibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. 25 The DNAalso may be modified, forexample, by substituting the coding sequencefor human heavy and lightchain constant domains in placeof the homologous murine sequences[U.S. Patent No. 4,816,567; Morrison et al., supral or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of 30 an antibody of the invention, or can be substituted for the variable domains of one antigen combining site of an antibody of the invention to create a chimeric bivalent antibody. The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant WO 00/43419 PCT/USOO/01431 -65 expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternativelythe relevantcysteineresiduesare substituted with another amino acid residue or are deleted so as to prevent crosslinking. 5 In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. Theanti-Exo protein antibodiesof the invention may furthercomprise humanizedantibodies or human antibodies. Humanizedforms of non-human(e.g., murine)antibodiesare chimeric 10 immunoglobulinsimmunoglobulin chains orfragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipientantibody)in which residues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species 15 (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanizedantibodiesmay also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all 20 of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 25 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)]. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as 30 "import" residues, which are typicallytaken from an "import"variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" WO 00/43419 PCT/USOO/01431 -66 antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted 5 by residues from analogous sites in rodent antibodies. Human antibodiescan also be produced using varioustechniquesknown in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal 10 Antibodies and CancerTherapy, Alan R. Liss, p. 77 (1985) and Boerneret al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in 15 all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technoloqy 10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); 20 Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995). Bispecific antibodies are monoclonal, preferably human or humanized, antibodiesthat have binding specificitiesforat leasttwo differentantigens. In the present case, one of the binding specificities is for the Exo protein, the other one is for any other antigen, and preferably 25 for a cell-surface protein or receptor or receptor subunit. Methodsfor making bispecificantibodiesare known in the art. Traditionallythe recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305:537-539 (1983)]. Because of the random assortment 30 of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatographysteps. Similarproceduresare disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

WO 00/43419 PCT/US00/01431 -67 Antibody variable domains with the desired binding specificities(antibody-antigencombining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region 5 (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymoloqy, 121:210 (1986). 10 Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalenty joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 15 92/200373; EP 03089]. It is contemplated that the antibodies may be preparedin vitro using known methods in syntheticprotein chemistry, includingthose involving crosslinkingagents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolateand methyl-4-mercaptobutyrimidateand those disclosed, for example, in U.S. 20 Patent No. 4,676,980. The anti-Exo protein antibodies of the invention have various utilities. For example, anti-Exo protein antibodies may be used in diagnostic assays for an Exo protein, e.g., detecting its expression in specificcells, tissues, or serum. Various diagnostic assay techniques known 25 in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in eitherheterogeneousor homogeneous phases [Zola, Monoclonal Antibodies: a Manual of Techniques, CRC Press, Inc. (1987) pp. 147-158]. The antibodiesused in the diagnostic assays can be labeled with a detectable moiety. The detectable moiety should be capable of producing, either directly or indirectly, 30 a detectable signal. For example, the detectable moiety may be a radioisotope, such as 3 H, 1 4 C, 3 2 P, 35 S, or 1251, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta galactosidase or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described 35 by Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth. 40:219 (1981); and Nygren, J. Histochem.and Cytochem. 30:407 (1982).

WO 00/43419 PCT/USOO/01431 -68 Anti-Exo protein antibodies also are useful for the affinity purification of Exo protein from recombinant cell culture or natural sources. In this process, the antibodies against Exo protein are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody then is contacted with a sample 5 containingthe Exo proteinto be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the Exo protein, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the Exo protein from the antibody. The anti-Exo protein antibodies may also be used in treatment. In one embodiment, the 10 genes encoding the antibodies are provided, such that the antibodies bind to and modulate the Exo protein within the cell. In one embodimenta therapeuticallyeffective dose of an Exo protein, agonist orantagonist is administered to a patient. -By "therapeutically effective dose" herein is meant a dose that producestheeffectsforwhich it is administered. The exact dose will depend on the purpose 15 of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art, adjustments for Exo degradation, systemicversus localizeddelivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the 20 art. A "patient" for the purposes of the present invention includes both humans and other animals, particularly mammals, and organisms. Thus the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is human. 25 The administration of the Exo protein, agonist or antagonist of the present invention can be done in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously,intranasally,transdermally,intraperitoneallyintramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. In some instances, for example, in the treatment of wounds and inflammation, the Exo may be directly applied as a solution or spray. 30 The pharmaceuticalcompositionsof the present invention comprise an Exo protein, agonist or antagonist in a form suitable for administration to a patient. In the preferred embodiment, the pharmaceutical compositions are in a water soluble form, such as being present as WO 00/43419 PCT/USOO/01431 -69 pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. "Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, 5 sulfuric acid, nitric acid, phosphoricacid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. "Pharmaceuticallyacceptablebase addition salts" includethose derived from inorganic 10 bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularlypreferredare the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptableorganicnon-toxicbases include salts of primary, secondary, and tertiaryamines, substitutedamines includingnaturallyoccurring substitutedamines, cyclicaminesand basic 15 ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. The pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring 20 agents; and polyethylene glycol. Additives are well known in the art, and are used in a variety of formulations. All references cited herein are incorporated by reference in their entireity. The following examples are merely for illustration. EXAMPLES 25 The yeast two-hybrid cDNA cloning technology is a powerful in vivo protein-protein interaction assay first introduced in Fields S, Song 0 (1989) Nature 340:245-247 (Chevray PM, Nathans D (1992) Proc Natl Acad Sci USA 89:5789-5793; Chien CT, Bartel PL, Sternglanz R, Fields S (1991) Proc Natl Acad Sci USA 88:9578-9582; Durfee T, Becherer K, Chen PL, Yeh SH, Yang Y, Kilburn AE, Lee WH, Elledge S (1993) Genes Dev 7:555-569; 30 Fields S, Song 0 (1989) Nature 340:245-247; Mendelsohn AR, Brent R (1994) Biotechnology 5:482-486; Zervos A, Gyuris J, Brent R (1993) Cell 72:223-232). It is based on co-expression of two proteins, X and Y, fused to GAL4 DNA binding domain (GAL4B) and GAL4 transcription activation domain (GAL4A) respectively (Figure IA). If the protein WO 00/43419 PCTUSOO/01431 -70 X interacts with the protein Y, the GAL4 transcription activation domain will be brought to the promoter containing the GAL4 DNA binding sites and will activate the transcription of reporter gene HIS3 or lacZ. The two-hybrid system can be used to clone cDNA encoding a novel protein that interacts with a known protein (bait) in yeast. It can also be used to 5 study protein-proteininteractionsbetween two known proteins. The yeasttwo-hybrid system has several advantages over other conventional methods to study protein-protein interactions: Protein-protein interactions are studied in eukaryotic cells (yeast); cDNA clones are immediately available after screening. Screening is very fast and convenient. No protein purification is necessary. Both growth 10 selection and lacZ colorselection are very sensitive. No radioisotope is needed. However, this technology also presents a challenge to new users due to certain technical difficulties: Depending on the bait protein used in screening, false positive rates vary significantly. Yeast transformation efficiency is difficult to control. High quality cDNA libraries can be very difficult to construct. The major difference between 15 the yeast one-hybrid system and the yeast-two hybrid system is that the one-hybrid system is used to clone cDNA encoding DNA-bound proteins, rather than protein-bound proteins (Figure 1 B). DNA sequences of interest are inserted upstream of the minimal promoter controlling the expression of either HIS or lacZ gene. cDNA fragments are fused to the C terminal of GAL4 transcription activation domain (GAL4A) to construct cDNA library. If the 20 protein encoded by the cDNA can bind to the specific DNA sequences of interest, the transcription of HIS/lacZis activated.The yeast one-hybrid system is widely used in cloning new transcription factors (Lehming N, Thanos D, Brickman JM, Ma J, Maniatis T, Ptashne M (1994) Nature 371:175-179; Li JJ, Herskowitz 1 (1993) Science 262:1870-1873; Luo Y, Stile J, Zhu L (1996) BioTechniques 20:564-568; Shang J, Luo Y, Clayton D (1997) 25 Developmental Dynamics 209:242-253; Strubin M., Newell JW, Matthias P (1995) Cell 80:497-506; Wilson TE, Fahmer TJ, Johnston M, Milbrandt J (1991) Science 252:1296 1300; Wang MM, Reed RR (1993)Cell 74:205-214). Experimental protocols betweenthese two methods are very similar except one notable exception. The one-hybrid system yeast reporterstrains need to be constructed by individual researcher. The expressionof reporter 30 genes (HIS/lacZ) should be underthe control of specific DNA sequences of interest, rather than the GAL4 DNA-binding sites in the yeast two-hybrid system. cDNA libraries used for two-hybrid screening can also be used for one-hybrid screening. The experimental flow-chart of a yeast two-hybrid cDNA screening experiment is outlined in Figure 2.

WO 00/43419 PCT/USOO/01431 -71 The experimental flow-chart of a yeast one-hybrid cDNA screening experiment is outlined in Figure 3. MATERIALS Medium and Yeast Strains 5 All yeast culture mediums, including YPD, YPD Agar, DOB, DOBA, CSM-TRP, CSM-LEU, CSM-HIS, CSM-URA, CSM-LYS, CSM-LEU-TRP, CSM-LEU-HIS, and CSM-LEU-TRP-HIS, are available from Bio101, Inc. 3AT (3-amino-1,2,4-triazol) is available from Sigma (Cat no.: A-8056, St. Louis, MO, USA). Yeast two-hybrid system reporter strain Y1 90 (MATa, ura3-52, his3-200, lys2-801, 10 ade2-101, trpl-901, leu2-3, 112, gal4A, gaI80A, cyhr2, LYS 2 ::GAL1UAS-HIS 3

TATA

HIS3, URA 3 ::GAL1UAS-GALTATA-lacZ) and yeast one-hybrid system reporter strain YM4271 (MATa, ura3-52, his3-200, lys2-801, ade2-101, trpl-903, leu2-3, 112, tyr1-501) are available from Clontech Laboratories, Inc. (Cat no.K1603-1, Clontech, Palo Alto, CA, USA). 15 Plasmids and cDNA Libraries pAS2 and pACT2 series were originally constructed by Elledge lab (Durfee et al. 1993) and are available from Clontech laboratories (Cat no.K1604-A, K1604-B). Other GAL4 based two-hybrid vectors, such as pGBT9 and pGAD424 series, were originally published by Fields lab and are available from both Stratagene, Inc. (Cat 20 no.235700,235722) and Clontech Laboratories, Inc. (Cat no.K1605-A, K1605-B). LexA based two-hybrid vectors are available from Origene Technologies, Inc. (Cat no.DPL-100, DPL-1 02). cDNA libraries for two-hybrid and one-hybrid screening are available from Origene, Stratagene, Clontech, and Invitrogen. Rigel also makes its own two-hybrid cDNA libraries from various tissues. All of these two 25 hybrid vectors share basic structures as shown in Figure 4A. The cDNA library is to be amplified before screening. It is recommended that at least 200 15cm-plates should be used to grow up 10 million independent cDNA clones. High quality plasmid can be obtained with Qiagene DNA preparation kits. Single-strand carrier DNA for yeast transformation is available from Origene or 30 Clontech. Carrier DNA can also be made according to protocol by Ito et al. (Ito H, Fukada Y, Murata K, Kimura A (1993) Journal of Bacteriology 153:163-168). Special Buffers Z buffer pH7.0 (per liter) WO 00/43419 PCT/USOO/01431 -72 Na 2

HPO

4 .7H 2 0 16.1 g NaH 2

PO

4

.H

2 0 5.50 g MgSO 4 .7H 2 0 0.246 g Kci 0.75 g 5 Z buffer + X-Gal Z buffer 1 ml 20mg/ml X-Gal 40 p1 P-mercaptoethanol(optional) 2 pl PEG/LiAc (10 ml) 10 50% PEG(3350) 8 ml 1OX TE (pH7.5) 1 ml 1M LiAc 1 ml Equipment and Others 300C incubators and liquid nitrogen containers are required. Nylon membrane 15 and Whatman filter for lacZ color assay are available from Fisher Scientific. X Gal is from either Promega (Cat no.V3941, Madison, WI, USA) or Denville Scientific (Cat no.CX-3000-3, Metuchen, NJ, USA). All plastic wares are from Fisher Scientific or VWR. PROCEDURE: Yeast Two-Hybrid System Screening 20 Grow up yeast reporter strains on YPD plates from frozen stock. Since no antibiotics are added into the yeast medium, very stringent sterilization procedures are required during inoculation. It is also recommended that yeast reporter strain be streaked on SD-W, SD-L, SD-H, SD-U, and SD-K plates to test other markers of the yeast before cDNA library screening. Reporter strain such as 25 Y1 90 should be able to grow up on SD-K, SD-U and SD-H plates, but not on SD W, and SD-L plates. Growth on SD-H plate is due to leaky expression of HIS reporter gene. There are many reporter strains available from different resources. In General, Y1 90 consistently showed higher sensitivity than other yeast strains such as HF7c. 30 Yeast reporter strains with both lacZ reporter gene and HIS3 reporter gene are strongly recommended. HIS selection will ensure that only interaction positive clones will grow, which makes colony picking much easier later. Determine optimal 3AT concentration.

WO 00/43419 PCT/USOO/01431 -73 3AT can be used to suppress background expression from HIS reporter gene of Y190. 3AT concentration varies among different reporter strains and ranges from 0 mM (HF7c) to 15 mM (Y190). To test the optimal concentration of 3AT, one yeast colony should be re-suspended in 10 ml of TE. 100 pl of the re-suspended yeast is 5 spread on SD-H+OmM3AT, SD-H+5mM3AT, SD-H+1OmM3AT, SD-H+15mM3AT, SD-H+25mM3AT, and SD-H+40mM3AT plates. Although 15 mM 3AT is sufficient to suppress background HIS expression of Y190, higher concentrations of 3AT (30-40 mM) are routinely used in our cDNA library screening. Construct bait plasmid. 10 pAS2/pACT2 series plasmids showed higher level of sensitivity than pGAD424 /pGBT9 series plasmids (Estojak J, Brent R, Golemis EA (1995) Molecular and Cellular Biology 15:5820-5829; Legrain P, Dokhelar MC, Transy C (1994) Nucleic Acids Research 22:3241-3242). The disadvantage of using pAS2 is the large size of this plasmid (8 kb), which may present a challenge to cloning large 15 cDNA fragments into the plasmid. cDNA fragments should fused to the C-terminal of Gal4 binding domain in frame (Figure 4A). The junction sequence between GAL4 and cDNA should have a GGG amino acid sequence to avoid any interruption of domain structure. Either full-length cDNA or partial fragments can used to generate bait plasmid. 20 Transform bait into yeast: 1st round. 1 pg of bait plasmid is transformed into Y1 90 with small-scale yeast transformation protocol (see SUBPROTOCOL section). Transformants should be plated on SD W, SD-WH, and SD-WH+3AT(5-4OmM) plates. LacZ color assay can also be done after colonies grow to a diameter of 1 mm. If colonies grow up on SD 25 WH+40mM3AT plates after 3 days incubation and/or LacZ color assay of these colonies show positive result after only 30 minutes incubation with X-Gal, the bait gene should be determined not suitable for two-hybrid screening without further modification. The bait gene itself may be able to activate transcription of reporter genes HIS/lacZ. 30 Although co-transformation of bait plasmid and cDNA library can be done in a single step, co-transformation efficiency is at least 10 fold lower than single plasmid transformation. Mating approach may also be used to introduce cDNA library into yeast cells containing the bait vector. Please refer to protocol published by Finley and Brent (Finley R, Brent R (1994) Proc Natl Acad Sci USA 91:12980 35 12984). Transform cDNA library: 2nd round.

WO 00/43419 PCT/USOO/01431 -74 Y1 90 containing bait plasmid is grown up for second round of transformation by cDNA library plasmid (see SUBPROTOCOL section). Incubation time after transformation varies significantly from 4 days to 11 days. Identify positive clones. 5 Identification of positive clones needs experience. It should also be pointed out that background colonies at lightly populated area of the plate tend to grow bigger, occasionally reaching the size of a positive colony in a dense area on the same plate. The size of the positive colony should at least 4 times bigger than the neighboring background colonies. Positive colonies may also turn red faster. 10 Perform lacZ color assay. Positive colonies should be re-streaked to another SD-LWH+3AT plate to isolated single colonies for color assay and plasmid retrieval. See SUBPROTOCOL section for the lacZ color assay protocol. If a colony does not turn blue after a 4-hour incubation, strong protein-protein interaction is highly unlikely. It is not 15 recommended to pick positive clones after 12 hours incubation, except that you know the protein-protein interaction you are studying is very weak. Retrieve plasmids. There are several methods to retrieve plasmids from yeast, ranging from lyticase lysis to glass beads. The glass beads method is listed in SUBPROTOCOL section. 20 Electroporation method is by far the most efficient method to transform plasmids from yeast miniprep into E. coli. Bait and cDNA plasmid may carry different antibiotic selection markers to facilitate separation in E. coli. For example, Rigel's bait plasmid carries Kanr gene and the cDNA plasmid carries Ampr gene. Verify positive clones. 25 cDNA clones recovered from positive HIS/lacZ positive colonies should be re transformed into yeast with other non-specific bait control to rule non-specific binding. In vitro protein binding assays and function assays should also be done to rule out false positive clones. PROCEDURE: Yeast One-Hybrid System Screening 30 Construct HIS and lacZ reporter plasmids. Selection of a very-well-defined DNA sequence is the most important step for one hybrid screening. Many DNA sequences lead to significant elevation of the basal expression levels of the reporter genes in yeast even in the absence of the cDNA library. Multiple copies (-3) of the DNA sequences of interest should be inserted 35 into the multiple cloning sites of both HIS reporter plasmid pHISi-1 and pLacZi (Clontech Cat no.K1603-1, Figure 4B).

WO 00/43419 PCTIUSOO/01431 -75 Grow up yeast reporter strains on YPD plates from frozen stock. It is also recommended that the yeast reporter strain be streaked on SD-W, SD-L, SD-H, SD-U, and SD-K plates to test other markers of the yeast before cDNA library screening. Reporter strain such as YM4271 should be able to grow up on 5 SD-K, SD-U SD-H, SD-W, and SD-L plates. Integrate HIS reporter into yeast. To facilitate integration of pHISi reporter into yeast chromosome, pHISi-1 should be linearized at Xho I site. Since pHISi-1 has no yeast replication origin and can not survive in yeast without integration, no gel purification of digested plasmid is 10 required. Transform 1 pg of digested plasmid into YM4271 using the small-scale yeast transformation protocol (see SUBPROTOCOL section). Use more plasmids if integration efficiency is low. Transformants should be plated on SD-H, and SD-H plates with different concentration of 3AT(5-40 mM) and incubated at 30 0 C for at least 4 days. 15 Determine optimal 3AT concentration. If more than 40 mM 3AT is needed to suppress transformants growth, the DNA sequences inserted into pHISi are not suitable for one-hybrid screening. Note: Integration efficiency of pHISi is very low. 20-100 colonies are expected on SD-H plate. 20 Integrate LacZ reporter plasmid into yeast. Pick a colony from a SD-H plate from step 3 and freeze as single HIS reporter strain YM4271/H. Linearize pLacZi at Nco I site. Transform 1 pg of linearized plasmid into yeast YM4271/H and plate transformants on SD-U plates. This step of integration is very efficient. Several hundred to thousand colonies are expected to 25 grow on each SD-U plates. Pick colonies and freeze as YM4271/HB. Screen cDNA library for DNA binding protein. Transform 100-200 pg of cDNA library into YM4271/HB with large-scale yeast transformation protocol (see SUBPROTOCOL section). Transformants should be plated on SD-LH+3AT ( concentration determined at step 4). 30 Identifv positive clones. Same as step 6 in two-hybrid screening procedure. Perform lacZ color assays. Same as step 7 in two-hybrid screening procedure. Retrieve cDNA plasmid. 35 Same as step 8 in two-hybrid screening procedure. Verify positive clones.

WO 00/43419 PCT/USOO/01431 -76 DNA gel retardation assay and other function assays are required to verify one hybrid screening results. SUBPROTOCOL Small Scale Yeast Transformation (10' transformants/pg DNA) 5 Inoculate one colony of yeast in 100 ml YPD (without plasmid) or corresponding selection medium (SD-W for Y190 with pAS2) at 240 rpm in a 30 0 C shaker overnight. Check OD 600 the next day. If ODc 0 o is between 0.6 and 1.0, the yeast can be used to prepare competent cells. Otherwise, dilute to OD 60 =0.4 and grow another 3 to 4 10 hours. Spin down cells in two 50 ml plastic tubes at 3000 rpm at room temperature for 5 minutes. Remove medium. Add 30 ml TE pH7.5 and re-suspend the cell pellet on vortex at high speed. Combine cell pellet. 15 Spin down cells again in at 3000 rpm at room temperature for 5 minutes. Remove TE. Estimate the size of the cell pellet and add TE up to total volume of 0.9 ml. Re-suspend cell completely by pipetting up and down. Add 100 p 1 M LiAc and mix well by pipetting. Competent cells are ready. 20 Note: Competent cells can be kept on at room temperature for several hours without significant reduction of transformation efficiency, or at 4 0 C overnight with a slight reduction of transformation efficiency. In a clean eppendorf, mix 1 pg of plasmid with 10 pl 10 mg/ml carrier DNA. Add 100 pl competent cells from step 8 to the eppendorf and mix well with the DNA. 25 Add 600 pl PEG/LiAc and mix well. Note: PEG/LiAc should be freshly made. Pre-mixed PEG/LiAc of up to 2 weeks old can also be used if transformation efficiency is not critical. Incubate at 30 0 C for 30 minutes with or without shaking. Add 70 pl DMSO and mix well. 30 Incubate in 421C water bath for 15 minutes. Put on ice for 2 minutes. Spin down cells in an eppendorf centrifuge at 8000 rpm for 1 minute. Remove supernatant. Add 150 pl of TE to re-suspend cell pellet. 35 Plate on selection medium plate. (e.g. SD-W for Y190 transformed by pAS2). Incubate in a 301C incubator for 2 to 3 days.

WO 00/43419 PCT/USOO/01431 -77 Large scale cDNA library transformation (1-10X10 6 transformants/100 pg cDNA) Inoculate one colony of yeast in 200 ml YPD (one-hybrid screening) or corresponding selection medium (SD-W for two-hybrid screening) at 240 rpm in a 300C shaker overnight. 5 1. Check OD 600 the next day. If OD600 is between 0.8 and 1.0, the yeast can be used to prepare competent cells. Otherwise, dilute to OD00=0.6 and grow another 3 to 4 hours. 2. Spin down cells in a 250 ml bottles at 3000 rpm at room temperature for 5 minutes. 3. Remove medium. 10 4. Add 50 ml TE pH7.5 and re-suspend the cell pellet on vortex at high speed. 5. Spin down cells again in at 3000 rpm at room temperature for 5 minutes. 6. Remove TE. 7. Repeat steps 4 to 7 one more time. Estimate the size of cell pellet and add TE up to total volume of 1.8 ml. Re 15 suspend cell pellet completely by vortex. Add 200 pl 1 M LiAc and mix well by vortex. In a clean eppendorf, mix 100-200pg of plasmid with 200 pl 10 mg/ml carrier DNA. Add DNA to competent cells drop by drop on a vortex at 5000 rpm. To ensure sufficient mixture, vortex at the highest speed for 30 seconds. 20 Add 12 ml PEG/LiAc and mix well. Note: PEG/LiAc should be freshly made. Pre-mixed PEG/LiAc of up to 2 weeks old can also be used if transformation efficiency is not critical. Incubate at 30OC for 30 minutes with shaking. Either an orbital shaker or rotator can be used. 25 Add 140 pl DMSO and mix well. Incubate in 420C water bath for 15 minutes. Invert several times during incubation. Put on ice for 5 minutes to chill. Spin down cells at 3000 rpm in a bench-top centrifuge for 1 minute. Remove supernatant. 30 Add 20 ml TE to re-suspend cell pellet by vortex. Plate 400 pl on each 15 cm selection medium plates (50 plates total). SD-LWH+40 mM3AT plates are used for Y190 strain two-hybrid screening; SD-LH+3AT plates are used for one-hybrid screening. Plate 1 pl on a 10 cm plate of SD-LW for transformation efficiency control. 35 Incubate at 300C for up to 8 days until big colonies appear.

WO 00/43419 PCT/USOO/01431 -78 LacZ color assay. Grow up fresh yeast colonies to a 1 mm diameter. Fill a container (e.g. ice bucket) with liquid nitrogen. Use a nylon membrane to lift colonies up from the plate. No special replica-plating 5 device is needed. Simply press the nylon membrane to the plate. Immerse the nylon membrane (Cat no. N04HY08250, N04HY1 3250, Fisher Scientific, PA, USA) colony side face down into the liquid nitrogen. Wait for 20 seconds, remove the nylon membrane and allow to dry on a paper towel for 5 minutes. 10 In a 10 ml tube, add 40 pl X-Gal to each ml of Z buffer. Add 1.5 ml Z buffer/X-Gal solution to a clean 10 cm petri-dish. For 15 cm diameter petri-dish, add 4 ml Z buffer/X-Gal solution. Add a Whatman circle to the petri-dish, ensuring it is evenly soaked any air bubbles are squeezed out. 15 Use forceps to transfer the dried nylon membrane with colony side facing up to lie over the soaked Whatman circle (Cat no.09-805C, Fisher Scientific, PA, USA). Make sure no air bubble in between the membrane and the circle. Incubate the petri-dish with lid on in 37 0 C until blue color is visible. Yeast plasmid mini-isolation. 20 Inoculate 3 ml of selection medium ( e.g. SD-L for cDNA library plasmid pACT ) with a yeast colony. Incubate in a 30 0 C shaker or rotator overnight or until confluent. Spin down yeast in a bench-top centrifuge at 3000 rpm at room temperature. Remove medium and re-suspend pellet in 200 pl lysis buffer. Transfer to an eppendorf. 25 Add 200 pl volume glass beads. Note: The lid of eppendorf can be used as scoop to collect 200 pl glass beads. Add 200 pl phenol/chloroform/isoamyl alcohol (25:24:1). Vortex at the highest speed for 3 minutes. Spin in micro-centrifuge at 14000 rpm for 10 minutes. 30 Transfer top water layer to another eppendorf, add 20 pI 3M NaAc and 500 pl ethanol. Precipitate should be visible immediately. Put the eppendorf into a dry ice bath for 15 minutes or until frozen. Spin in a micro-centrifuge at 14000 rpm for 10 minutes. Remove supernatant and dry pellet. 35 Wash pellet by 100 pl of 80% ethanol, and dry the pellet in air. Re-suspend pellet in 30 pl H 2 0 and use 1 ml for electroporation to transform E. coli.

WO 00/43419 PCT/USOO/01431 -79 TWO-HYBRID SCREENING RESULTS Bait peptide was cloned into pAS2-1 to screen for proteins that can bind thereto. Results are listed below. cDNA Library Human Lymphocyte 5 Bait Vector pAS2-1 Bait Protein Protein involved in exocytosis Yeast Strain Y190 Number of Transformants 15 million HIS*/lacZ* clones 2 10 Clone Identity PCNA (2) PCNA is a published binding protein of the bait peptide. Yeast one-hybrid screening results were previously published ( Luo Y, Stile J, Zhu L (1996) BioTechniques 20:564 568).

Claims (25)

1. A recombinant nucleic acid encoding an Exo protein comprising a nucleic acid that hybridizes under high stringency conditions to a sequence set forth in SEQ ID NO:86, 5 SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID 10 NO:112, SEQ ID NO:113, SEQ ID NO:1 14, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID 15 NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143 and each complement thereof, respectively, wherein said Exo protein binds to SNAP-23.

2. A recombinant nucleic acid comprising a nucleic acid that is at least about 90% identical to a nucleic acid sequence selected forth in SEQ ID NO:86, SEQ ID NO:87, 20 SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID 25 NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID WO 00/43419 PCT/USOO/01431 -81 NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143 and complements, respectively, wherein said Exo protein binds to SNAP-23. 5

3. An expression vector comprising the recombinant nucleic acid according to claims 1 or 2 operably linked to regulatory sequences recognized by a host cell transformed with the nucleic acid.

4. A host cell comprising the recombinant nucleic acid according to claim 3.

5. A process for producing an Exo protein comprising culturing the host cell of claim 4 10 under conditions suitable for expression of an Exo protein.

6. A process according to claim 5 further comprising recovering said Exo protein.

7. A recombinant Exo protein encoded by the nucleic acid of claim 1 or 2.

8. A recombinant polypeptide comprising an amino acid sequence encoded by the first 100 nucleic acid residues of a sequence selected from the group consisting of the 15 sequences set forth in SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID 20 NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID WO 00/43419 PCT/USOO/01431 -82 NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143 and each complement 5 thereof, wherein said polypeptide binds to SNAP-23.

9. A recombinant polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence that will hybridize under high stringency to a nucleic acid selected from the group consisting of the sequences set forth SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID 10 NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID 15 NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID 20 NO:142, SEQ ID NO:143 and each complement thereof.

10. An isolated polypeptide which specifically binds to an Exo protein according to claim 9.

11. A polypeptide according to claim 10 that is an antibody. WO 00/43419 PCT/USOO/01431 -83

12. A polypeptide according to claim 11 wherein said antibody is a monoclonal antibody.

13. The monoclonal antibody of claim 12 wherein said antibody reduces or eliminates the biological function of said Exo protein. 5

14. A method for screening for a bioactive agent capable of binding to an Exo protein, said method comprising combining an Exo protein and a candidate bioactive agent, and determining the binding of said candidate agent to said Exo protein.

15. A method for screening for agents capable of interfering with the binding of Exo and SNAP-23 comprising: 10 a) combining an Exo protein, a candidate bioactive agent and an SNAP-23 protein; and b) determining the binding of said Exo protein and said SNAP-23 protein.

16. A method according to claim 15 wherein said Exo protein and said SNAP-23 protein are combined first. 15

17. A method for screening for an bioactive agent capable of modulating the activity of an Exo protein, said method comprising the steps of: a) adding a candidate bioactive agent to a cell comprising a recombinant nucleic acid encoding an Exo protein; b) determining the effect of the candidate bioactive agent on said cell. 20

18. A method according to claim 17 wherein a library of candidate bioactive agents are added to a plurality of cells comprising a recombinant nucleic acid encoding an Exo protein. WO 00/43419 PCT/USOO/01431 -84

19. A method according to claim 17 or 18 further comprising adding a labeling agent that will label exocytosing cells.

20. A method according to claim 19 further comprising separating the exocytosing cells from the non-exocytosing cells. 5

21. A method according to claim 20 wherein said separation is done by FACS.

22. A method of treating an exocytosis related disorder comprising administering an agent that interferes with specific binding of a protein selected from a protein encoded by the group consisting of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID 10 NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID 15 NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID 20 NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO:143, with SNAP23 expressed in a tissue such that said disorder is 25 ameolerated. WO 00/43419 PCT/USOO/01431 -85

23. A method of treating an exocytosis related disorder comprising administering to a patient an agent that binds to a protein encoded by a sequence selected from the group consisting of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ 5 ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID 10 NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID 15 NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO:143 such that exocytosis is altered. 20

24. A method of reducing or inhibiting exocytosis in a cell comprising administering an agent that interferes with specific binding of a protein encoded by a sequence selected from the group consisting of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID 25 NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID WO 00/43419 PCTIUSOO/01431 -86 NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID 5 NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID 10 NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO:143, with SNAP23 expressed in said cell such that exocytosis is inhibited.

25. A method of neutralizing the effect of a protein encoded by a sequence selected from the group consisting of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID 15 NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID 20 NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID 25 NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID WO 00/43419 PCT/USOO/01431 -87 NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO:143, comprising contacting an agent specific for said protein with said protein in an amount sufficient to effect neutralization.