CA2364584A1 - Eukaryotic peptide uptake system for transportation of enkephalins - Google Patents

Abstract

An oligopeptide transporter in the yeast Saccharomyces cerevisiae mediates the uptake of tetra- and pentapeptides, including the endogenous opioids leucine enkephalin (Tyr-Gly-Gly-Phe-Leu) and methionine enkephalin (Tyr-Gly-Gly-Phe-Met). The transporter is encoded by the gene OPT1. The system is specific for tetra- and pentapeptides and can be inhibited by the opioid receptor antagonists naloxone and naltrexone. Vectors allowing expression of OPT1 and methods of use are disclosed. Treatment of OPT1p with toxic enkephalins as an antifungal method is also disclosed.

Description

EUKARYOTIC PEPTIDE UPTAKE SYSTEM FOR
TRANSPORTATION OF ENKEPHALINS
RELATED CASES
This application is a conversion of provisional application Serial No.
60/122,312, filed on March l, 1999, entitled: Enkephalins are Transported by a Novel Eucaryotic Peptide Uptake System.
FIELD OF THE INVENTION
The invention relates to plant molecular genetics, and more specifically, to an oligopeptide transporter in the yeast Saccharomyces cerevisiae. The transporter mediates the uptake of tetra- and penta- peptides, including leucine enkephalin and methionine enkephalins.
BACKGROUND OF THE INVENTION
Peptide uptake is the process by which individual cells are able to transport intact peptides across their plasma membranes. The process is a general physiological phenomenon of bacteria, fungi, plant cells and mammalian cells (Becker, J. M.
et al, In: Microorganisms and Nitrogen Sources, Payne, J. W. (ed.), John Wiley and Sons, Inc., pp. 257-279 (1980); Matthews, D. M. et al., Curr. Top. Membr. Transp.
14:331-425 ( 1980)). In every case studied so far, peptide transport is a specific biochemical process in which small peptides (_< 6 amino acids) are transported across a membrane by energy-dependent, saturable carriers.

Three genetically distinct systems of peptide uptake have been identified in gram-negative bacteria. An oligopeptide permease (Opp) system has been identified in bacteria such as E. coli, and S. typhimurium (Andrews, J. C. et al., J.
Bacteriol.
161:484-492 (1985); Hogarth, B. G. et al., J. Bacteriol. 153:1548-1551 (1983)). The Opp system is capable of transporting peptides having up to 5 amino acid residues, regardless of their side chains (Payne, J. W. et al, J. Biol. Chem.
243:3395=3403 ( 1968); Payne, J. W. et al., J. Biol. Chem. 243:6291-6299 ( 1968)). In contrast, tripeptide permease (Tpp) systems, such as that of S. typhimurium, exhibit an apparent affinity for peptides having hydrophobic amino acid residues (Gibson, M. M. et al., J.
Bacteriol. 160:122-130 (1984)). The third system, a dipeptide permease (Dpp) system, has a preference for transporting dipeptides (Abouhamad, W. N., et al., Mol.
Microbiol. 5:1035-1047 (1991)). Functionally similar systems have been described in fungi and yeast (Naider, F. et al., In: Current Topics in Medial Mycology, volume II, McGinnis, M. M. (ed.) (1987)), but have not been well characterized.
The genes that encode the protein components of the oligopeptide transporters ofE. coli (Kashiwagi, K. et al, J. Biol. Chem. 265:8387-8391 (1990)), Salmonella typhimurium (Hiles, I. D. et al., Eur. J. Biochem. 158:561-567 (1986); Hiles, I. D. et al, J. Molec. Biol 195:125-142 (1987)), Bacillus subtilis (Rudner, D. Z. et al., J.
Bacteriol. 173:1388-1398 (1991); Perego, M. et al., Mol. Microbiol. 5:173-185 ( 1991 )), Streptococcus pneumoniae (Alloing, G. et al., Mol. Microbiol. 4:633-1990)), and Lactococcus lactis as well as two dipeptide permeases, one in E.
coli (Abouhamad, W. N., et al., Mol. Microbiol. 5:1035-1047 (1991)), and the other in _3_ Bacillus subtilis (Mathiopoulos, C. et al., Mol. Microbial. 5:1903-1913 ( 1991 )) have been cloned and sequenced.
The ability of bacteria and plant cells to accumulate peptides has been found to be dependent upon peptide transport systems (Becker, J. M. et al., In:
Microorganisms and Nitrogen Sources, Payne, J. W. (ed.), John Wiley and Sons, Inc., pp. 257-(1980); Matthews, D. M. et al., Curr. Top. Membr. Transp. 14:331-425 (1980);
Higgins, C. F. et al., In: Microorganisms and Nitrogen Sources, Payne, J. W.
(ed.), John Wiley and Sons, Inc., pp. 211-256 (1980); Naider, F. et al., In: Current Topics in Medial Mycology, volume II, McGinnis, M. M. (ed.) (1987)). These systems are distinct from the mechanisms that mediate the uptake of amino acids.
The existence of peptide transport systems in plants was demonstrated by showing that plants could accumulate non-hydrolyzable, non-physiological peptide substrates, intact and against a concentration gradient (Higgins, C. F. et al., Planta 134:205-206 (1977); Higgins, C. F. et al., Planta 136:71-76 (1977); Higgins, C. F. et al, Planta 138:211-216 (1978); Higgins, C. F. et al., Planta 142:299-305 (1978);
Sopanen, T. et al., FEBS Lett. 79:4-7 (1977)). The transport system was found to exhibit saturation kinetics and to be inhibited by a range of metabolic inhibitors (Higgins, C. F. et al., Planta 136:71-76 (1977)). The plant peptide transport system can transport both di- and tripeptides (Sopanen, T. et al., FEBS Lett. 79:4-7 (1977);
Higgins, C. F. et al., Planta 142:299-305 (1978)). Plant peptide transport systems are capable of transporting a wide variety of peptides. These systems exhibit broad transport specificity with respect to amino acid side-chains. The presence of D-amino acids, however, reduces the transport rate, thus indicating that the transporters have WO 00/52162 PCT/iJS00/05158 strong stereospecificity. Two proteins, approximately 66 D and 41 D, have been suggested as components of the plant peptide transport system in barley grains (Payne, J. W. et al., Planta 170:263-271 (1987).
The primary function of peptide transport is to supply amino acids for nitrogen nutrition (Payne, J. W. et al., In: Microorganisms and Nitrogen Sources, Payne, J. W.
(ed.), John Wiley and Sons, Inc., pp. 257-279 (1980); Matthews, D. M. et al., Curr.
Top. Membr. Transp. 14:331-425 (1980); Becker, J. M. et al., In:
Microorganisms and Nitrogen Sources, Payne, J. W.:(ed.), John Wiley and Sons, Inc., pp. 257-279 (1980);
Adibi, S. A. et al., Metabolism 36:1001-1011 (1987); Higgins, C. F. et al., Planta 138:211-216 (1978); Sopanen, T. et al., FEBS Lett. 79:4-7 (1977); Higgins, C.
F. et al., Planta 138:217-221 (1978)). In bacteria, peptide transport has, however, also been associated with sporulation (Perego, M. et al., Mol. Microbiol. 5:173-185 (1991);
Mathiopoulos, C. et al., Mol. Microbiol. 5:1903-1913 ( 1991 )); chemotaxis (Manson, M. D. et al., Nature 321:253-256 (1986), and the recycling of cell wall peptides (Goodell, E. W. et al., J. Bacteriol 169:3861-3865 (1987)).
Small peptides containing four to five amino acid residues are transported by a recently identified class of peptide transporters named the OPTl family (Lubkowitz et al. Mol. Microbiol. (1998) 28(4):729-741, incorporated herein by reference).
The amino acid sequence of this family is distinct from that of the PTR family, a ubiquitous group of proton-coupled transporters which selectively transports di- and tripeptides. Phylogenetic analysis suggests that the OPT family is also distinct from the major facilitator superfamily (MFS), a diverse collection of proteins which catalyzes the transport of a wide variety of substrates, including sugars, amino acids, neurotransmitters, and drugs.
Members of the OPT family have been identified and characterized in the yeasts Candida albicans, Schizosaccharomyces pombe, and Saccharomyces cerevisiae. Additional members exist in plants, as indicated by searches of publicly accessible data bases. In mammalian tissues, reports in the literature suggest that the enkephalins, endogenous pentapeptides involved in analgesia in the central nervous system, are transported across the blood-brain barrier by a specific, saturable transport system. The existence of enkephalin transporters has been inferred from data obtained by measuring whole brain flux of the peptides in rodents. To date, no protein has been identified in eukaryotes as the discrete enkephalin carrier.
SUMMARY OF THE INVENTION
It has now been discovered that the endogenous opioids Met-enkephalin and Leu-enkephalin, pentapeptides of amino acid sequence YGGFM and YGGFL, respectively, can be transported by cells expressing the S. cerevisiae ORF
YJL212C.
When expressed under the control of a constitutive promoter in a high copy number vector, this OPT family member is necessary and sufficient to transport Leu-enkephalin into yeast cells. This is the first example of a genetically defined eukaryotic transport protein which can transport enkephalins across the cell membrane. This gene has been named OPTl.
The invention also comprises a method for obtaining mammalian enkephalin transporters by functional complementation of OPT1 deficient yeast.

The invention also comprises a fungicidal composition comprising a toxic analogue of enkephalin as an active ingredient, and a method of killing fungi comprising applying a toxic analogue of enkephalin to a substrate or organism to be treated.
The invention also comprises a method of preventing or reducing fungal growth on substrates.
The invention also comprises OPT1 transformed plants, methods of such transformation and methods of growing transformed plants.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 shows shows the growth of S. cerevisiae BY4730 expressing OPT family members.
Fig. 2 shows uptake of [3H]Leu-enkephalin by S. cerevisiae BY4730 transformants.
Fig. 3 shows a chromatographic analysis of Leu-enkephalin.
Fig. 4 shows the effects of naloxone and naltrexone on the uptake of [3H]Leu-enkephalin and [3H]leucyl-leucine.
Fig. 5 shows the amino acid sequence of the OPT family member (isp4-like protein) fromArabidopsis thaliana designated emb CAB43855.1 Fig. 6 shows the amino acid sequence of the OPT family member (isp4-like protein) from Arabidopsis thaliana designated emb CAB 10414.1 Fig. 7 shows the amino acid sequence of the OPT family member(isp4 like protein) fromArabidopsis thaliana designated Genbank GB:D14061.
Fig. 8 shows the amino acid sequence of the OPT family member (previously unknown protein) from Arabidopsis thaliana designated emb CAB38285.

_7_ Fig. 9 shows the amino acid sequence of the OPT family member (isp4 like protein) from Arabidopsis thaliana designated Genbank GB:D83992.
Fig. 10 shows the amino acid sequence of the OPT family member (Optlp) from Candida albicans designated Genbank GB:AAB69628.1.
Fig. 11 shows the amino acid sequence of the OPT family member (Optl) from Saccharomyces cerevisiae designated YJL212c.
Fig. 12 shows the amino acid sequence of the OPT family member (YPR194c) from Saccharomyces cerevisiae designated YPR194c.
Fig. 13 shows the amino acid sequence of the OPT family member (previously unknown protein) from Schizosaccharomyces pombe designated emb CAB 16254:'1.
Fig. 14 shows the amino acid sequence of the OPT family member from Schizosaccharomyces pombe designated emb CAA19062.1.
Fig. 15 shows the amino acid sequence of the OPT family member from Schizosaccharomyces pombe designated sp40900 or Gi 729859.
Fig. 16 shows a sequence comparison of Optl family members.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention arises, in part, from the exploitation of the S.
cerevisiae OPTIp. S. cerevisiae strains that carry mutations in OPT1 may be created by knocking out OPTl by any means known in the art, such as homologous recombination with a defective OPTl , by homologous recombination replacing OPTI
with another gene, by mutation and selection, and the like. These knock-out yeasts will be completely deficient for enkephalin transport, which can be determined by _g_ their resistance to toxic derivatives of enkephalin and lack of uptake of radiolabeled Met- and Leu-enkephalin.
In addition to obtaining mammalian homologues to OPTl by functional complementation, functional augmentation may also be used. In all the wild-type yeast strains tested, enkephalin transport was deficient in the absence of a strong promoter such as the ADH promoter. Therefore, yeast vectors carrying exogenous mammalian DNA may be transformed into wild-type yeast and examined for enkephalin transport. Preferably, these vectors comprise a strong promoter to augment expression of a functional enkephalin transport protein.
Thus, one aspect of the present invention is the construction of a stable S.
cerevisiae OPTI mutant. Methods for isolating such mutants are described below, and by Perry, J. R. et al. (In: "Isolation and Characterization of a Saccharomyces cerevisiae Peptide Transport Gene," Molecular and Cellular Biology, volume 14 (1994), herein incorporated by reference in its entirety). Polynucleotides that encode the peptide transport genes of higher plants have been identified and isolated by their capacity to complement the peptide transport deficiency of the stable S.
cerevisiae ptr2 strain. In a like manner, knock-out S. cerevisiae may be used in functional complementation assays using polynucleotides that encode mammalian proteins.
Transformation of knock-out S. cerevisiae with mammalian sequences and subsequent growth on Leu-enkephalin as the source of leucine may reveal mammalian homologues of OPTI. In addition, functional augmentation may be employed to obtain mammalian homologues.

The present invention relates in part to the isolation of a novel polynucleotide that is capable of hybridizing to, or recombining with, a plant gene that encodes a peptide transport protein. The polynucleotides of the present invention are "substantially purified," in that they have been purified from undesired yeast genes with which they are associated in nature. The molecules maybe in either a double-stranded or single-stranded form. Such polynucleotides are capable of augmenting the transport capacity of a recipient plant, and thus may be used to facilitate the delivery of desired compounds to the plant. In an alternative embodiment, the polynucleotides of the present invention can be used to disrupt or otherwise inactivate endogenous transport systems. Such disruption renders the plant incapable of transporting toxic peptides, and thus resistant to pathogens that produce such peptides.
The capacity of the polynucleotides of the present invention to hybridize to a plant gene arises out of the extent of homology between the respective sequences of the polynucleotides. As used herein, a polynucleotide of the present invention is said to be able to "hybridize" to a plant gene if the two molecules are capable of forming an anti-parallel, double-stranded nucleic acid structure. The molecules are said to be "minimally complementary" if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional "low-stringency" conditions. Similarly, the molecules are said to be "complementary"
if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional "high-stringency" conditions. Such conventional stringency conditions are described by Sambrook, J., et al., (In:

Molecular Cloning, a Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. ( 1989)), and by Haymes, B. D., et al. (In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985)), both herein incorporated by reference).
Complementary molecules thus need not exhibit "complete complementarity,"
but need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure. Departures from complete complementarity are, therefore, permissible, so long as such departures do not completely preclude the capacity of the molecules to form a double-stranded structure. In contrast, where two nucleic acid molecules exhibit "complete complementarity," every nucleotide of one of the molecules is complementary to a nucleotide of the other; such molecules need not have the same lengths.
The capacity of the polynucleotides of the present invention to recombine with a plant gene is determined by the extent of sequence "homology" between the polynucleotide and the plant gene. Homologous recombination is a well-studied natural cellular process which involves the exchanges of a region of one polynucleotide with a region of another (see, Sedivy, J. M.,Bio-Technol.
6:1192-1196 (1988)). Sufficient homology for recombination requires only minimal homology in regions of the polynucleotide that flank the portion of the polynucleotide that undergoes recombination. The region may be of any length from a single base to a substantial fragment of a chromosome. Generally, a region having a length of about ten nucleotide residues is sufficient. Recombination is catalyzed by enzymes which are naturally present in both prokaryotic and eukaryotic cells.

The polynucleotides of the present invention comprise isolated nucleic acid molecules that can complement or augment a tetra or pentapeptide transport deficiency of S. cerevisiae. The term "polynucleotide" encompasses nucleic acid molecules that encode a complete protein, as well as nucleic acid molecules that encode fragments of a complete protein. The polynucleotides may comprise the wild-type allele (or a portion of such allele) of a functional peptide transport gene, or they may comprise mutated or disrupted (as by the insertion of additional DNA
or RNA) alleles of such genes. As used, herein a "fragment" of a polynucleotide is an oligonucleotide whose nucleotide sequence is identical to that of a region of the polynucleotide, and whose length is greater than about 15 nucleotide residues, and preferably greater than about 20 nucleotide residues.
Functional complementation or augmentation The isolation and cloning of polynucleotides that encode S. cerevisiae enkephalin transport proteins permits the isolation of analogous, complementary polynucleotides from mammalian cells. The functional role of such isolated polynucleotides can be readily determined by transforming them into the above-described stable enkephalin transport-deficient yeast strain, and evaluating whether transformants acquire the capacity to transport intact enkephalin.
Thus, the methods of the present invention permit the isolation of polynucleotides from mammalian cells. Such polynucleotides are the equivalents of the preferred polynucleotides of the present invention.
In one embodiment of the invention, mammalian protein encoding sequences are cloned into suitable yeast expression vectors. Such vectors comprise regulatory sequences such as promoters, termination signals and restriction endonuclease recognition sequences to permit the introduction of heterologous sequences, such as the mammalian protein encoding sequences. Any suitable vector for expression of proteins in yeast known in the art may be used.
Examples of suitable yeast vectors include the yeast 2-micron circle, the expression plasmids YEP13, YCP and,YRP, etc., or their derivatives. Such plasmids are well known in the art (Botstein, D., et al., Miami Wntr. Syrup. 19:265-274 (1982);
Broach, J. R., In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p. 445-(1981); Broach, J. R., Cell 28:203-204 (1982); Sherman, F. et al., In: Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
(1986)).
Yeast transformation may be accomplished by any means known in the art.
Selection of transformed yeast with functional complementation or augmentation of the deleted OPT1 may be on enkephalin containing media as the source of leucine, for example.
Yeast transformants that display functional complementation or augmentation may be further analyzed by isolating the heterologous DNA and determining the nucleic acid sequence. The deduced amino acid sequence may be analyzed for identity and/or similarity by any of the available sequence analysis programs.
A score of high identity or similarity, and predicted structural features of the deduced amino acid sequence (particularly the presence and number of transmembrane domains and the presence of the putative consensus sequence for OPT1 family members) indicates homology of the mammalian sequence to S. cerevisiae OPTlp.

For complementation, OPTI knock-out yeast strains may be generated and used. Alternatively, wild-type yeast may be used in augmentation studies. In either case, yeast cells are transformed with yeast expression vectors carrying heterologous mammalian DNA. Preferably, in augmentation studies, the expression of the heterologous sequence in the yeast expression vector is under the control of a strong promoter, such as the ADH promoter.
Antifungal Compositions Another embodiment of the mention comprises an antifungal composition comprising at least one toxic derivative of enkephalin as an active ingredient.
Another embodiment is a method of using the antifungal compositions comprising applying the antifungal composition to a substrate to reduce or prevent fungal growth.
There is a plethora of toxic moieties that may be employed in the antifungal composition and method using the antifungal composition. Virtually any toxic moiety may be used that satisfies the following criteria: the toxic moiety or moieties must be able to be associated with the enkephalin peptide, and the moiety or moieties must not interfere with the uptake of the peptide into the cells.
Substantial evidence suggests that the uptake of toxic peptides is mediated by peptide transport systems (McCarthy, P. J. et al.,Antimicrob. Agents Chemother.
28:494-499 (1985); McCarthy, P. J. et al., J. Gen. Micro. 131:775-780 (1985);
Moneton, P. et al., J. Gen. Micro. 132:2147-2153 (1986); Yadan, J. C. et al., J.
Bacteriol. 160:884-888 (1984)); Payne, J. W. et al., FEMS Microbiol. Letts.
28:55-60 (1985); Mehta, R. J. et al., Antimicrob. Agents Chemother. 25:373-374 (1984)).
Since the polynucleotides of the present invention define the genetic loci responsible for enkephalin transport in yeast, fungi would be take up toxic enkephalin derivatives by transport, and fungal growth would be prevented or reduced.
In this regard, the present invention provides a method for conjugating an antimicrobial or antifungal agent or a pesticide to a peptide in order to provide a more effective treatment against fungal growth. In a similar manner, toxic peptide derivatives maybe used as herbicides to eliminate fungal growth around plants (particularly crops).
Examples of toxic peptide or peptidyl molecules that may be used include, but are not limited to:
(A) metabolic toxins (such as the antifungal agent FMDP >N³ -(4-methoxyfumaroyl)-L-2,3 diaminopropanoic acid), toxic nucleotides (such as halogenated nucleotides (e.g., 5-fluoroorotic acid), dideoxynucleotides, mutagenic nucleotide or nucleoside analogs, etc. (Kingsbury, W. D. et al., J. Med. Chem.
27:1447-1451 (1984); Andruszkiewicz, R. et al., J. Med. Chem.30:1715-1719 (1987);
Andruszkiewicz, R. et al., J. Med. Chem. 33:132-135 (1990); Andruszkiewicz, R.
et al., J. Med.Chem. 33:2755-2759 (1990); Milewski, S. et al., J. Drugs Expt.
Clin. Res.
14:461-465 (1988));
(B) peptides that contain toxic amino acids (such as oxalysine, fluorophenylalanine, ethionine, unusual D amino acids, etc.)(McCarthy, P. J.
et al., Antimicrob. Agents Chemother. 28:494-499 (1985); Marder, R. et al., J.
Bacteriol.
36:1174-1177(1978); Moneton, P. et al., J. Gen. Micro. 132:2147-2153 (1986);
Mehta, R. J. et al., Antimicrob. Agents Chemother.25:373-374 (1984); Bosrai, M. et al., J. Gen. Microbiol. 138:2353-2362 (1992));

(C) toxic peptides and peptidyl molecules such as bacilysin (Milewski, S. et al., Arch. Microbiol. 135:130-136 (1983);Moneton, P. et al., J. Gen.
Microbiol.
132:2147-2153 (1986); Kenig, M. et al. J. Gen. Microbiol. 94:37-45 (1976)), polyoxins (especially polyoxin D) (Becker, J. M. et al., Antimicrob. Agents Chemother. 23:926-929 (1983)), nikkomycins(especially nikkomycin Z) (Dahn, U.
et al., Arch. Microbiol. 107:143-160 (1976)), and their analogs (Smith, H. A. et al.,Antimicrob. Agents Chemother. 29:33-39 (1986); Naider, F. et al., Antimicrob.
Agents Chemother. 24:787-796 (1983);Krainer, E. et al., J. Med. Chem. 34:174-(1991); Shenbagamurthi. P. et al., J. Med. Chem. 26:1518-1522 (1983);
Shenbagamurthi. P. et al., J. Med. Chem. 29:802-809 (1986); Khare, R. K. et al., J.
Med. Chem. 83:650-656 (1988); Emmer, G. et al., J. Med. Chem. 28:278-281 (1985);
Decker, H. et al., J. Gen Microbiol. 137:1805-1813 (1991); Delzer, J.et al., J.
Antibiot. 37:80-82 (1984); all herein incorporated by reference).
In a preferred embodiment, the peptides of such conjugates will be N-.alpha.-acetylated, since such modification facilitates theuptake of peptide molecules.
In the method of reducing or preventing fungal growth, a composition containing at least one toxic analogue of enkephalin as an active ingredient are applied to a substrate or plant in an amount suitable to prevent or reduce fungal growth.
OPT1 Vectors Vectors to allow the expression of OPT1 in plants comprise nucleic acid molecules comprising the coding sequence of OPT1, regulatory sequences suitable for use and functional in plants (such as promoters, enhancers, termination sequences and the like), and may include selectable marker genes as is well known in the art (such as antibiotic resistance genes, and the like). Such vectors may be used to transform plant cells to provide expression of OPTIp in plants.
In one embodiment of the invention, the polynucleotides will be operably linked to regulatory sequences sufficient to permit the polynucleotide's transcription.
Such polynucleotides may be incorporated into nucleic acid vectors that are sufficient to permit either the propagation or maintenance of the polynucleotide within a host cell. The nature of the regulatory elements will depend upon the host cell, and the desired manner of expressing the polynucleotide. Examples of suitable regulatory elements include constitutive or inducible prokaryotic promoters, such as the .lambda.
pL or pR promoters, or other well-characterized promoters (e.g., lac, gal, trp, ara, hut, etc.). Other promoters which may be employed are the nos, ocs and CaMv promoters.
Efficient plant promoters that may be used are over-producing plant promoters such as the small subunit (ss) of the ribulose 1, 5 biphosphate carboxylase from soybean (Berry-Lowe, et al., J. Molec. App. Gen. 1:483-498 ( 1982)) and the promoter of the chlorophyll a/b binding protein. These two promoters are known to be light induced in eukaryotic plant cells (see Genetic Engineering of Plants, An Agricultural Perspective," Cashmore, A. (ed), Plenum, N.Y., pp. 29-38 (1983); Coruzzi, G.
et al., J. Biol. Chem. 258:1399 (1983); and Dunsmeier, P. et al., J. Molec. App. Gen.
2:285 (1983)). The 35S promoter is particularly preferred.
Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli such as, for example, pBR322,Co1El, pSC101, pACYC 184, .pi.VX. Such plasmids are, for example, disclosed by Maniatis, T., et al. (In:

Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1982)). Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan, T. (In: The Molecular Biology of the Bacilli, Academic Press, N.Y. (1982), pp. 307-329). Suitable Streptomyces plasmids include p1J101 (Kendall, K. J., et al., J. Bacteriol.169:4177-4183 (1987)), and Streptomyces bacteriophages such as .o slashed.C31 (Chater, K. F., et al., In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids are reviewed by John, J. F., et al.
(Rev.
Infect. Dis. 8:693-704 (1986)), and Izaki, K. (Jpn. J. Bacteriol. 33:729-742 (1978)).
As indicated, the invention particularly contemplates providing the polynucleotides of the present invention to plants, especially tobacco, coffee, wheat and other cereals, apple and other non-citrus fruit producers, and citrus fruit crops.
Suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonefla, Vigna, Citrus, Linum, Geranium, Manicot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, A tropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis, Nemesia, Pelargonium, Panicurn, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Lolium, Zea, Triticum, Sorghum, Ipomoea, Passiflora, Cyclamen, Malus, Prunus, Rosa, Rubus, Populus, Santalure, Allium, Lilium, Narcissus, Ananas, Arachis, Phaseolus, Pisum and Datura.
In one embodiment, OPT1 polynucleotide is provided without promoters or other regulatory elements, but under conditions sufficient to permit the polynucleotide to recombine with and replace a region of the endogenous plant peptide transport gene. In an alternative embodiment, the polynucleotides will be administered to the plant operably linked to regulatory elements and/or vector elements.
Any of a variety of methods may be used to introduce the OPT1 polynucleotide of the present invention into a plant cell. The genetic material can be microinjected directly into the plant embryo cells or introduced by electroporation as described in Fromm et al.,"Expression of Genes Transformed into Monocot and Dicot Plant Cells by Electroporation," Proc. Nat'1. Acad. Sci. U.S.A.82:5824-28 (1985) or it can be introduced by direct precipitation using polyethylene glycol as described in Paszkowski et aI.,EMBO J. 3:2717-22 (1984). In the case of monocotyledonous plants, pollen may be transformed with total DNA or an appropriate functional clone providing resistance, and the pollen then used to produce progeny by sexual reproduction.
The Ti plasmid of Agrobacterium tumefaciens provides a means for introducing DNA into plant cells (Caplan, A., et al., Science815-821 (1983);
Schell, J.
et al., Bio/Technology, April 1983, pp. 175-1980; Fraley, R. T., et al., Proc.
Nat'l.
Acad. Sci. U.S.A. 80:4803 (1983); (Hooykass, P. J. J. et al., In: Molecular Form and Function of the Plant Genome, Vlotan-Doltan, L. et al. (eds.), Plenum Press, N.Y., pp.
655-667 (1984); Horsch, R. B. et al., In: Current Communications in Molecular Biology, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., pp. 13-19 (1988);
Horsch et al., Science 233:496-498 (1984); all herein incorporated by reference). As such, it provides a highly preferred method for introducing the polynucleotides of the present invention into plant cells Ti plasmids contain two regions essential for the production of transformed cells. One of these, termed "transfer DNA" (TDNA), induces tumor formation.
The other, termed "virulent region," is essential for the formation but not maintenance of tumors. It is possible to insert the polynucleotides of the present invention into the T
DNA region without affecting its transfer function. By removing the tumor-causing genes so that they no longer interfere, the modified Ti plasmid can then be used as a vector for the transfer of the gene constructs of the invention into an appropriate plant cell. The polynucleotides of the present invention are preferably inserted between the terminal sequences that flank the T-DNA.
A particularly useful Ti plasmid vector is pGV3850, a non-oncogenic derivative of the nopaline Ti plasmid C58 (Caplan, A., et al., Science 815-821 ( 1983)). This vector utilizes the natural transfer properties of the Ti plasmid. The internal T DNA genes that determine the undifferentiated crown gall phenotype have been deleted and are replaced by any commonly used cloning vehicle (such as pBR322). The cloning vehicle sequence contained between T DNA border regions serves as a region of homology for recombination to reintroduce foreign DNA
cloned in a derivative of the same cloning vehicle. Any polynucleotide of the present invention cloned in such plasmid can thus be inserted into pGV3850 by a single recombination of the homologous sequences. Antibiotic resistance markers can be added to the plasmid to select for the recombination event. The presence of thenopaline synthase (nos) gene in pGV3850 facilitates the monitoring of the transformation.

The introduction of the Ti plasmid is typically accomplished by infecting a wounded leaf of the plant with Agrobacterium tumefaciens bacteria that contains the plasmid. Under appropriate growth conditions, a ring of calli forms around the wound(Hooykass, P. J. J. et al., In: Molecular Form and Function of the Plant Genome, Vlotan-Doltan, L. et al. (eds.), Plenum Press, N.Y., pp. 655-667 (1984)).
The calli are then transferred to growth medium, allowed to form shoots, roots and develop further into plants.
The procedure can alternatively be performed in tissue culture. All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the introduced polynucleotide. There is an increasing body of evidence that practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major cereal crop species, sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables (Hooykass, P. J. J. et al., In: Molecular Form and Function of the Plant Genome, Vlotan-Doltan, L. et al. (eds.), Plenum Press, N.Y., pp. 655-(1984); Horsch, R. B. et al., In: Current Communications in Molecular Biology, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., pp. 13-19 (1988)). Methods for regenerating plants from cultural protoplasts are described by Evans et al.
(Handbook of Plant Cell Culture 1:124-176; by Davey, M. R., In: Protoplasts1983--Lecture Proceedings, pp. 19-29, Birkhauser, Basel (1983)); Dale, P. J. (In:
Protoplasts 1983--Lecture Proceedings, pp. 31-41, Birkhauser, Basel (1983)); Binding, H.
(In:
Plant Protoplasts, CRC Press, Boca Raton, pp. 21-37 (1985)) and Cooking, E. C.
In:

Molecular Form and Function of the Plant Genome, Vlotan-Doltan, L. et al.
(eds.), Plenum Press, N.Y., pp.27-32 ( 1984)).
Regeneration efficiency varies from species to species of plants, but generally a suspension of transformed protoplasts containing the introduced gene sequence is formed. Embryo formation can then be induced from the protoplast suspensions, to the stage of ripening and germination as natural embryos. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Shoots and roots normally develop simultaneously.
Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable.
Other systems, such as cauliflower mosaic virus, CaMV (Hohn, B., et al., In "Molecular Biology of Plant Tumors," Academic Press, New York, pp. 549-560;
and Howell, U.S. Pat. No. 4,407,956) can also be used to introduce the OPT1 polynucleotide of the present invention into plant cells. In accordance with such methods, the entire CaMV viral DNA genome is inserted into apparent bacterial plasmid thus creating a recombinant DNA molecule which can be propagated in bacteria. After cloning, the recombinant plasmid is cleaved with restriction enzymes either at random or at unique sites in the viral portion of the recombinant plasmid for insertion of the polynucleotides of the present invention. The modified viral portion of the recombinant plasmid is then excised from the parent bacterial plasmid, and used to inoculate the plant cells or plants.

After transformation of the plant cell or plant, the same may be selected by aid of an appropriate marker, such as antibiotic resistance, and then assessed to determine whether it contains the desired polynucleotide of the invention. The mature plants, grown from the transformed plant cells, can be selfed to produce an inbred plant whose seeds will contain the introduced polynucleotides of the present invention.
These seeds can be grown to produce plants that exhibit any of a set of desired properties.
In one embodiment of the present invention, the exhibited property will be an increased facility to transport peptides, particularly, enkephalin. In this embodiment, the OPT1 polynucleotide of the invention are provided to the plant or plant cells along with transcriptional regulatory sequences, such that an overexpression of the plant's peptide transport gene occurs. Such plants are desirable in that their enhanced peptide transport system can be used to facilitate the up take of peptide-associated molecules.
The invention, therefore, also contemplates a method for growing plants, particularly crops such as those mentioned above, by providing said crops with the OPTIp encoding polynucleotide to allow for expression of OPTIp in the plant and providing the plant with a growth enhancement amount of an enkephalin to promote growth of the plant.
In all the embodiments of the invention, the polynucleotide of OPT1 that is suitable is at least the nucleotide sequence encoding the protein of SEQ ID
N0:2.
EXPERIMENTS
Growth on Leu-Enkephalin-- An experiment was designed to determine whether members of the OPT family could transport leucine enkephalin (Leu-enkephalin;

YGGFL) to satisfy an auxotrophic requirement for leucine. For this study, a strain of S. cerevisiae auxotrophic for methionine and leucine (BY4730) along with the prototrophic parental strain (BY4700) were selected for use. Fig. 1 shows the growth of S. cerevisiae BY4730 expressing OPT family members. Cells were transformed with pDB20 (empty vector), pCaOPTI (C. albicans OPT1) under its endogenous promoter, pADH 194C (S. cerevisiae ORF 194C under the S. cerevisiae ADH
promoter), and pADHOPTl (S. cerevisiae OPT1 under the S. cerevisiae ADH
promoter). Cells were spotted onto proline medium supplemented with various sources of leucine, as indicated on the figure, to meet auxotrophic requirements and were grown for 72 h at 30 °C. S. cerevisiae BY4730 transformed with the vector (pDB20) and transformants expressing three members of the OPT family were able to use either leucine or leucyl-leucine for growth as shown in Fig. 1. In contrast, only cells transformed with YJL212C, expressing OPT1 (pADHOPTl), could grow on Leu-enkephalin as a sole source of leucine. The parental strain BY4700 transformed with an empty vector (pDB20) or three members of the OPT family (pCaOPTI, pADH 194C, or pADHOPTl ) grew well in the presence of Leu-enkephalin at all concentrations (10-1000 ~,M), indicating this peptide was not toxic. Growth on Leu-enkephalin in cells expressing OPT 1 was concentration-dependent, with the most robust growth seen at the highest concentrations. In a similar experiment, it was determined that cells expressing OPT1 could grow on methionine enkephalin (Met-enkephalin) as a sole source of methionine.

Transport of Radiolabeled Leu-Enkephalin-- To further explore the possibility that Leu-enkephalin transport was carrier-mediated, transport was measured directly using radiolabeled Leu-enkephalin ([3H]YGGFL). Fig. 2 shows uptake of [3H]Leu-enkephalin by S. cerevisiae BY4730 transformants. Part A shows uptake versus time at 30 °C (~) or 4 °C (o) for cells transformed with pADHOPTI and at 30°C for the empty vector pDB20 (X). The inset iri part A shows Leu-enkephalin uptake at 30°C
versus concentration of Leu-enkephalin for cells transformed with pADHOPT 1.
Part B shows uptake versus pH for cells transformed with pADHOPT 1.
Leu-enkephalin was transported into cells expressing OPT1 (Fig. 2A) in a time- and temperature-dependent manner. In contrast, cells transformed with the vector pDB20 did not accumulate enkephalin. The uptake of Leu-enkephalin was pH-dependent. Transport of the substrate was highest at pH 5.5 and declined sharply as the proton concentration was raised or lowered (Fig. 2B). This pH optimum is similar to those reported for the eukaryotic di- and tripeptide transport systems, as well as that for peptide transport in the prokaryote Lactococcus lactis. Treatment of cells with the metabolic uncouplers 2,4-dinitrophenol, CCCP, or sodium azide, all of which deplete intracellular ATP and collapse the proton gradient, or treatment with the sulfhydryl reagent pCMBS substantially reduced enkephalin uptake (Table I). These data are consistent with a carrier-mediated uptake system for Leu-enkephalin encoded by OPT 1.

Table I
Leu-enkephalin uptake in the presence of various compounds The uptake ofLeu-enkephalin (250 ~M) was measured over a 12-min time course in the presence of the compounds indicated. Each measurement was completed a minimum of four times. The results were normalized to uptake after 12 min of incubation measured in the absence of any other compound (none, 100%) and are reported as mean ~ standard deviation.
Compound Percent of control None 100%

Leucine Enkephalin (YGGFL)a 12 ~

Methionine Enkephalin (YGGFM)a 25 ~
4%

Tyrosine a 95 ~
12%

Leu-Leu a 97 ~
12%

Gly-Gly-Phe a 99 ~
5%

Gly-Gly-Phe-Leu a 41 ~
8%

Lys-Leu-Gly-Leu a 31 ~
14%

MIF-1 (PLG-NHZ)a 95 ~
7%

Tyr-MIF-1 (YPLG-NHZ)a 7g ~
9%

Tyr-Gly-Gly-Phe-Leu-NHz a 71 ~
5%

DPDPE (Y-D-Pen-GF-D-Pen)a 69 ~

DADLE (Y-D-AGF-D-L)a 58 ~
5%

Sodium Azideb 21 ~
2%

2,4-Dinitrophenolb 17 ~
2%

CCCPb 3 8 ~
6%

pCMBSb 55 ~ 5%
a All competitors were at a final concentration of 2.5 mM and added simultaneously with [3H]Leu-enkephalin in the uptake medium.
b Cells were pre-incubated with sodium azide ( 1 mM), CCCP (0.1 mM) 2,4-dinitrophenol ( 1 mM), or pCMBS (0.2 mM) for 30 min prior to addition of the uptake medium.

Table I shows Leu-enkephalin uptake in the presence of various compounds. The uptake of Leu-enkephalin (250 ~M) was measured over a 12-min time course in the presence of the compounds indicated. Each measurement was completed a minimum of four times. The results were normalized to uptake after 12 min of incubation measured in the absence of any other compound (none, 100%) and are reported as mean ~
standard deviation.
Fig. 3 shows a chromatographic analysis of Leu-enkephalin. Arrows indicate the RF values for tyrosine and intact Leu-enkephalin. Part A shows an analysis of uptake assay medium after 2-min incubation with BY4730 transformed with pADHOPTl.
Similar analysis of medium prior to incubation with cells produced identical results. Part B shows an analysis of material extracted from cells after 12-min incubation interval.
As shown in Figure 3, the rate of Leu-enkephalin uptake remained relatively constant over a 12-min time course, suggesting that the opioid does not remain intact upon entering the cell. Chromatographic analysis of radiolabeled material extracted from cells indicated that the enkephalin was degraded, with virtually all radioactivity associated with free tyrosine. In contrast, analysis of an aliquot of medium from which cells were removed after 12 min of incubation at 30 °C revealed that no extracellular hydrolysis of the peptide had occurred. All radioactivity was still associated with intact Leu-enkephalin. If it is assumed that translocation of the substrate, rather than its hydrolysis, is rate-limiting, then an apparent Km for transport can be determined by measuring the rate of transport as a function of substrate concentration.
Transformation of these data give an apparent Km of 310 qM for the uptake of Leu-enkephalin by transporter (Fig. 2A, inset) From Table I, the transport protein encoded by OPT 1 has a strong preference for both Leu-enkephalin and Met-enkephalin and does not appear to transport amino acids or di- or tripeptides. Accumulation of Leu-enkephalin was not affected by the presence of tyrosine or the di- and tripeptides tested, suggesting that the OPTI
protein does not recognize these compounds. The uptake of radiolabeled Leu-enkephalin decreased by 75-88% in the presence of a 10-fold molar excess of Met-enkephalin or Leu-enkephalin, respectively. The tetrapeptide Lys-Leu-Gly-Leu (KLGL), a known substrate for other oligopeptide transporters was also an effective competitor. The amidated form of Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu-NHZ) showed weak inhibition of enkephalin uptake in yeast.
Inhibition of Uptake by Enkephalin Analogs-- The nonmetabolized pentapeptide enkephalin analogues DADLE and DPDPE were somewhat effective competitors, blocking 30-40% of the uptake (Table I). The amidated tetrapeptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NHZ), a substrate for the previously described PTS-1 whole brain Met-enkephalin transport system was a poor competitor, reducing Leu-enkephalin uptake by only 20%. The tripeptide MIF-1 did not cause a significant reduction in the uptake of Leu-enkephalin, further emphasizing the preference of the OPT1 system for tetra- and pentapeptides.
Fig. 4 shows the effects of naloxone and naltrexone on the uptake of [3H]Leu-enkephalin and [3H]leucyl-leucine. Part A shows the uptake of Leu-enkephalin (250 ~M) as measured over a 12-min time course in the presence of naloxone (black bars) or naltrexone (shaded bars) at the concentrations indicated. The results were normalized to uptake of Leu-enkephalin (control, open bar) measured in the absence of either compound and are reported as mean ~ standard deviation. Part B shows the uptake of leucyl-leucine ( 160 ~M) as measured over a 12-min time course in the presence and absence of naloxone (black bar) or naltrexone (shaded bar) at the concentrations indicated. Results were normalized to control and reported as described for part A.
Naloxone and naltrexone antagonize the binding of enkephalin to the opioid receptor. It was found that these compounds also inhibit the transport of Leu-enkephalin across Optlp (Fig. 4A). In a similar experiment, the presence of these compounds did not inhibit the transport of leucyl-leucine, a substrate for the di- and tripeptide transport system Ptr2p (Fig. 4B).
We have discovered a function of the previously unknown open reading frame YJL212C in the yeast S. cerevisiae. This gene is OPTI (SEQ ID NO. 1). The protein encoded by OPTI (SEQ ID NO. 2) consists of 799 amino acids, and based on the amino acid sequence the predicted protein structure suggests an integral membrane protein containing 12-14 putative membrane-spanning domains. In addition, the protein contains several motifs unique to the OPT family, the largest of which consists of 10 invariable residues (SPYXEVRXXVXX~~DDP) located before the first hydrophobic domain.
OPT1, like other members of the OPT family, encodes a functional oligopeptide transporter.
Because Optlp exhibited all the molecular characteristics of an OPT family member, it was hypothesized that this protein was an oligopeptide transporter, even though it was known that S. cerevisiae could not utilize any tetra- or pentapeptides tested to date to satisfy auxotrophic requirements under routine growth conditions.
To see activity of Optl, it was necessary to express OPTl under the control of the ADH

promoter, a strong, constitutive promoter which would presumably result in high expression of the gene product. Northern blot analysis confirmed that OPT 1 was not expressed at detectable levels under routine conditions of logarithmic growth.
These results were independently confirmed by serial analysis of gene expression (SAGE) which revealed that OPT1 is only expressed at a low level (~1 copy per cell) following nocodazole arrest in the G2/M phase of the cell cycle. Additional analysis of sporulating yeast cells by DNA microarray analysis indicated that OPT1 was expressed during the late stages of sporulation. In light of these observations, OPTlgene expression must be ectopically induced under the control of a heterologous promoter to enable study of Optlp function in log phase cells.
The product of OPT1 is the oligopeptide transporter Optlp, which translocates pentapeptides, including both Met- and Leu-enkephalin. In BY4730, a strain of S.
cerevisiae auxotrophic for leucine and methionine, only cells expressing OPT 1 could grow on Leu-enkephalin in the absence of exogenous leucine. This indicates that enkephalins are transported intact into the cell and then hydrolyzed. If oligopeptides were hydrolyzed by an extracellular protease prior to transport, then the isogenic control strain (BY4730 transformed with the empty vector pDB20), as well as yeast cells transformed with plasmids encoding other OPT family members (CaOPT l, YPR194C) should be able to utilize the hydrolysis products for growth. This was not the case.
Chromatographic analysis supports conclusion. No evidence for degraded forms of Leu-enkephalin could be found in the extracellular medium. In addition, a large body of work exists which demonstrates that di- and tripeptides enter the cell intact and are then rapidly hydrolyzed by intracellular peptidases.

Transport of Leu-enkephalin is pH- and temperature-dependent, suggesting that this is a proton-coupled, energy-dependent process. These observations are supported by the sensitivity of the transporter to agents which disrupt the proton gradient or deplete intracellular ATP. Utilization of the transmembrane proton gradient to energize active transport has been demonstrated for the PTR family of di- and tripeptide transporters.
Uptake of radiolabeled Leu-enkephalin was inhibited in the presence of excess unlabeled Met- or Leu-enkephalin; amidated Leu-enkephalin was an ineffective competitor.
Tyr-MIF-1 is an amidated tetrapeptide with opiate and anti-opiate activity. This peptide is a substrate for the previously described blood-brain barrier PTS-1 enkephalin transport activity but, like the amidated form of authentic Leu-enkephalin, was not an effective competitor for yeast Optlp. This observation is consistent with the need for a free carboxyl terminus for substrate recognition by Optlp. Tetrapeptides were effective inhibitors, with Lys-Leu-Gly-Leu and des-Tyrl Leu-enkephalin (Gly-Gly-Phe-Leu) eliminating over 50% ofradiolabeled enkephalin accumulation, suggesting that an amino-terminal tyrosine is not essential for substrate recognition. Neither the tripeptide enkephalin fragment Gly-Gly-Phe nor the dipeptide Leu-Leu could inhibit uptake, indicating that this system is distinct from Ptr2p and is selective for tetra-and pentapeptides. These data show that intact oligopeptides are gaining access to the cell via a carrier-mediated process, and the discrete carrier is the gene product of OPTI. If enkephalins were entering by a nonspecific mechanism such as simple diffusion or endocytosis, then all strains, not just those expressing OPT1, should be able to utilize this substrate.

Several enkephalin antagonists were assayed in this study for their effect on enkephalin transport across Optlp. DADLE and DPDPE are enzymatically stable delta opioid receptor antagonists that are pentapeptide mimetics. Previous reports indicated that DPDPE gained access to the brain by a saturable, carrier-mediated mechanism in the blood-brain barrier, which has yet to be defined. Interestingly, transport of DPDPE was not inhibited by Leu-enkephalin in those studies, suggesting either the existence of separate transport systems or a common system with different affinities for these two substrates. A recent report suggests that DPDPE crosses the blood-brain barrier by a phenylarsine oxide-sensitive pathway, suggesting a role for a saturable endocytic mechanism in the in vitro and in situ models studied. It was found that DPDPE
and DADLE were weak competitors for Leu-enkephalin transport, indicating that Optlp interacts with the stable antagonists with differential affinities compared with authentic Leu-enkephalin.
Naloxone and naltrexone are synthetic opioid receptor antagonists classically used to reverse the effects of opiate overdose. Naltrexone is also used clinically in the treatment of alcoholism. Despite the fact that these compounds are similar in structure to morphine, rather than resembling a peptide, they were effective competitors for Leu-enkephalin transport. The effect appears to be specific for the Optlp transporter because the presence of the morphine analogs did not influence the activity of the unrelated di-and tripeptide transporter Ptr2p. The nature of the inhibition of Leu-enkephalin transport by naloxone and naltrexone is currently under investigation. Specifically, it would be of interest to determine whether these compounds are substrates for transport or are nonsubstrate competitors for Optlp.

There is increasing evidence that opioids and their analogues enter the central nervous system by carrier-mediated transport across the blood-brain barrier.
Evidence also exists to suggest that the clearance of the enkephalin analogue DPDPE
occurs by saturable efflux from the brain and systemic elimination of intact DPDPE via biliary excretion. Furthermore, it is possible that neuronal re-uptake systems exist for enkephalin similar to the well studied transport systems for neurotransmitters such as serotonin and y-aminobutyric acid. Previously, none of the putative transporters for enkephalin have been cloned or characterized at a molecular level. The present invention presents the first evidence for a genetically defined eukaryotic transport protein, Optlp, which recognizes and translocates both Met- and Leu-enkephalin into an intact eukaryotic cell.
The identification of this transporter in Saccharomyces may facilitate the discovery of mammalian homologues, thus providing greater insight into the process of pain and its mediation. These mammalian homologues may aid in transporting opiates across the blood-brain barrier, and mediation of the homologues could allow pain mediation.
Similarly, the homologues may be helpful in substance abuse treatment or in fording competitors for opiate transport mechanism to aid such treatment.
EXAMPLES
Strains, Media, and Vectors-- BY4700 (Mata ura300) and BY4730 (Mata 1eu200 met1500 ura300) were grown routinely on YEPD medium (1% yeast extract, 2%
peptone, 2% glucose, 2% agar). Strains transformed with a plasmid were cultured on minimal medium lacking uracil (0.67% Difco yeast nitrogen base with ammonium sulfate, without amino acids, 2% glucose, 0.2% casamino acids). For growth assays, cells were inoculated into medium lacking uracil and ammonium sulfate (0.67% Difco yeast nitrogen base without amino acids and ammonium sulfate, 2% glucose) supplemented with 0.1% proline as anitrogen source, 228 ~M leucine, and 191 pM methionine (proline medium). The plasmids pADH212C and pADH 194C were created by polymerase chain reaction amplification of the appropriate ORFs (YJL212C and YPR194C, respectively) and cloning the resultant products into the URA3/2p-based vector pDB20 such that the genes were under the control of an ADH promoter. The plasmid pCaOPTI consists of a 3.8-kilobase genomic fragment from C. albicans which contains the CaOPT 1 gene cloned into pRS202, a URA3/2 ~-based plasmid. Plasmids were transformed into yeast by the method of Geitz, and transformants were selected by growth on minimal medium lacking uracil.
Growth and Uptake Assa,~-- Transformed cells were grown overnight to mid-exponential phase in proline medium. For growth assays, cells were harvested, washed, and adjusted to a final concentration of 2 X 10' cells/ml in water. Five microliters (= 1 X
105 cells) of each sample was spotted onto proline medium plus 2% agar, supplemented with amino acids or peptides, as indicated in the text and Fig. 1. Plates were incubated at 30 °C for 72 h and observed for growth. For uptake assays, cells were harvested and washed with 2% glucose and adjusted to a final concentration of 2 X 108 cells/ml. The uptake assay was initiated by combining equal volumes of pre-warmed (30 °C) cells and 2X uptake assay mixture (2% glucose, 20 mM sodium citrate/potassium phosphate, pH 5.5, 500 pM
Leu-enkephalin (Sigma), 1 gCi/ml [3H]leucine enkephalin (50 Ci/mmol, American Radiolabeled Chemicals), and incubating at 30 °C. For determination of leucyl-leucine accumulation, 320 pM L-leucyl-L-[3H]leucine ( 16 mM,10 mCi/mmol) was used in place of Leu-enkephalin. L-Leucyl-L-[3H]leucine was synthesized by standard solution-phase techniques. For assays done in the presence of competitors, the 2 X uptake assay mixture was supplemented with competitor (2X final concentration) prior to combining with the cells. A concentrated stock of carbomyl cyanide 3-chlorophenylhydrazone (CCCP) (Sigma) was prepared in Me2S0; naloxone and naltrexone (Sigma) were dissolved in methanol. The compounds were diluted such that the solvent was present at a final concentration of 5% in the uptake medium. All other compounds were dissolved in either water or sodium citrate/potassium phosphate buffer (pH 5.5). At the appropriate time, aliquots (90 pl) were removed and washed by vacuum filtration with 4 X 1 ml ice cold water onto a membrane filter (HAWP, Millipore). The membranes were counted by liquid scintillation spectrometry, and results were reported as nmol/mg dry weight. Data points reflect the mean and standard deviation of a minimum of four independent measurements.
ChromatographX-- Cells were incubated with uptake medium for 12 min, harvested, and washed four times with ice-cold water. The cell pellet was extracted by boiling in 50%
methanol. The methanol extracts, along with control samples, were spotted onto silica plates and developed by ascending chromatography using butanol:glacial acetic acid:water solvent system (9:1:2.5). The chromatograms were sprayed with ninhydrin (0.1% in 95% ethanol) to visualize the nonradioactive standards. Lanes containing radioactive samples were scraped in 0.8-cm intervals and counted for retained radioactivity.

Claims (18)

We claim:

1. A method for obtaining mammalian enkephalin transport proteins comprising deleting the OPT1 gene from a strain ofyeast, transforming said strain with a library of mammalian genes cloned into a suitable yeast expression plasmid, thereby forming transformed yeast, growing said transformed yeast on a medium with an appropriate amount of an enkephalin, selecting transformed yeast based on an ability of said transformed yeast to transport said enkephalin.

2. The method of claim 1 wherein said yeast strain is selected from the group consisting of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

3. The method of claim 1 wherein said enkephalin in Leu-enkephalin.

4. An antifungal composition comprising a toxic derivative of enkephalin as an active ingredient in a sufficient amount to prevent or reduce fungal growth.

5. The antifungal composition of claim 4 wherein said toxic derivative of enkephalin comprises a toxic molecule conjugated to an enkephalin.

6. The antifungal composition of claim 5 wherein said toxic molecule is selected from the group consisting of N3-(4-methoxyfumaroyl)-L-2,3 diaminopropanoic acid, 5-fluororotic acid, dideoxynucleotides, mutagenic nucleotide analogues, mutagenic nucleoside analogues, and toxic amino acids.

7. The antifungal composition of claim 6 wherein said toxic amino acids are selected from the group consisting of oxalysine, fluorophenylalanine, ethionine and unusual D-amino acids.

8. A method of reducing or preventing fungal growth comprising applying an effective amount of an antifungal composition to a substrate wherein said antifungal compound comprises a toxic derivative of enkephalin as an active ingredient in a sufficient amount to prevent or reduce fungal growth.

9. The method of claim 8 wherein said substrate is a plant.

10. A vector for transformation of plant cells comprising, operably joined, a promoter functional in plants, regulatory sequences for transcription and translation functional in plants, and a nucleic acid molecule encoding the protein of SEQ ID NO:2.

11. Transformed plant cells comprising plant cells harboring the vector of claim 10.

12. A method for cultivating plant material comprising transforming plant material with the vector of claim 10, and providing a sufficient amount of an enkephalin to said plant material to enhance plant and plant part growth.

13. The method of claim 12 wherein said plant material is a crop plant selected from the genera selected from the group consisting of Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonefla, Vigna, Citrus, Linum, Geranium, Manicot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, A tropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis, Nemesia, Pelargonium, Panicurn, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Lolium, Zea, Triticum, Sorghum, Ipomoea, Passiflora, Cyclamen, Malus, Prunus, Rosa, Rubus, Populus, Santalure, Allium, Lilium, Narcissus, Ananas, Arachis, Phaseolus, Pisum and Datura..

14. The method of claim 12 wherein said plant part growth comprises growth of fruit of said plant.

15. A method for obtaining mammalian enkephalin transport proteins comprising transforming a strain of yeast with a library of mammalian genes cloned into a suitable yeast expression plasmid, thereby forming transformed yeast, growing said transformed yeast on a medium with an appropriate amount of an enkephalin, selecting transformed yeast based on an ability of said transformed yeast to transport said enkephalin.

16. The method of claim 15 wherein said yeast strain is selected from the group consisting of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

17. The method of claim 15 wherein said enkephalin is Met- or Leu-enkephalin.

18. The method of claim 15 wherein said yeast expression plasmid comprises a strong promoter for expression in yeast that drives expression of said mammalian gene cloned into said plasmid.