WO2001036671A2

WO2001036671A2 - A new method for the examination of protein localization in living cells

Info

Publication number: WO2001036671A2
Application number: PCT/EP2000/010943
Authority: WO
Inventors: Fabian KÖHLER
Original assignee: Koehler Fabian
Priority date: 1999-11-04
Filing date: 2000-11-06
Publication date: 2001-05-25
Also published as: WO2001036671A8; AU2354501A; WO2001036671A3

Abstract

A method for the detection of a compound within a cell comprising the steps of (a) introducing into a cell proteinaceous material comprising at least two fusion proteins or derivatives thereof or nucleic acid, encoding upon expression said fusion proteins, wherein said compound or a precursor thereof is present or expressed in said cell before said proteinaceous material is introduced into or expressed in said cell; (b) allowing expression of said nucleic acid, if applicable; and (c) assessing for a signal from said first or second signaling entity that is provided, restored, altered or influenced.

Description

A New Method for the Examination of Protein Localization in Living Cells

The present invention relates to a method for the detection of a compound within a cell comprising the steps of (a) introducing into a cell proteinaceous material comprising at least two fusion proteins or derivatives thereof or nucleic acid, encoding upon expression said fusion proteins wherein one of said fusion proteins or derivatives thereof comprises (aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a first portion of said compound; (ab) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal for a subcellular structure; and (ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein further a second of said fusion proteins or derivatives thereof comprises (ad) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a second portion of said compound wherein said first portion and said second portion are spatially distinct to allow the simultaneous interaction of said amino acid sequence or non-proteinaceous structure (aa) and said amino acid sequence or non-proteinaceous structure (ad) with said compound; (ae) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal specific for the same subcellular structure as said amino acid sequence or non-proteinaceous structure (ab); and (af) an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; wherein said compound or a precursor thereof is present or expressed in said cell before said proteinaceous material is introduced into or expressed in said cell; (b) allowing expression of said nucleic acid, if applicable; and (c) assessing for a signal from said first or second signaling entity that is provided, restored, altered or influenced. The description cites a number of published documents. The disclosure content of these documents is herewith specifically incorporated by reference.

The knowledge of the intracellular localization of a given protein is of fundamental interest for the understanding of its functions in health and disease. Most signals a cell receives lead to an alteration of the localization of proteins involved in the respective signal transduction pathway. Frequently, signal transduction pathways result in the translocation of transcription factors to the nucleus and a change in the transcription pattern.

Current methods for the investigation of protein localization are based on the detection with mono- and/or polyclonal antibodies and a coupled color-/light reaction. The necessary preparation of antibody derivatives as well as the fixation, staining and analysis of single cells under the microscope is laborious and time consuming, thus the number of analyzed cells limited. Furthermore the availability of specific antibodies is a limiting factor, although under certain circumstances this problem can be encountered by genetically fusing an epitope tag to the protein of interest and using an antibody against this epitope tag for detection.

Using fusions of the protein of interest with the green fluorescent protein (GFP) of the jelly-fish Aequorea victoria (Chalfie, M. et al. (1994); Prasher, D.C. et al. (1992); Inouye, S. and Tsuji, F.I. (1994A); Wang, S. and Hazelrigg, T. (1994); Cody, C.W. et al. (1993); Inouye, S. and Tsuji, F.I. (1994B); Heim R. et al. (1994); Ormo, M. et al. (1996); Yang, F. et al. (1996)), no fixation and staining is needed but still single cells must be analyzed for the intracellular localization of the fluorescence under a fluorescence microscope. Since, the GFP portion of the fusion protein with its approximately 240 amino acids is quite large, it cannot be excluded that it changes the behavior and localization of the protein of interest.

Consequently, there is a need to improve the prior art methodology for generally detecting and localizing cellular compounds within a cell. The solution to said technical problem is achieved by providing the embodiments characterized in the claims. Accordingly, the present invention relates to a method for the detection of a compound within a cell comprising the steps of (a) introducing into a cell material comprising at least two fusion proteins or derivatives thereof or nucleic acid encoding said fusion proteins wherein one of said fusion proteins or derivatives thereof comprises (aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a first portion of said compound; (ab) an amino acid sequence or a non- proteinaceous structure representing or comprising a targeting signal for a subcellular structure; and (ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein further a second of said fusion proteins or derivatives thereof comprises (ad) an amino acid sequence or a non- proteinaceous structure capable of specifically interacting with a second portion of said compound, wherein said first portion and said second portion are spatially distinct to allow the simultaneous interaction of said amino acid sequence or non-proteinaceous structure (aa) and said amino acid sequence or non-proteinaceous structure (ad) with said compound; (ae) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal specific for the same subcellular structure as said amino acid sequence or non-proteinaceous structure (ab); and (af) an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; wherein said compound or a precursor thereof is present or expressed in said cell before said proteinaceous material is introduced into or expressed in said cell or wherein said compound or precursor is introduced into said cell simultaneously with or after the introduction of said material or wherein said compound is present in said cell per se; (b) allowing expression of said nucleic acid, if applicable; and (c) assessing for a signal from said first or second signaling entity that is provided, restored, altered or influenced.

In connection with the present invention, the term "detection within a cell" not only refers to the option that the presence of a compound within a cell can be detected per se, e.g. when it is of interest whether said compound is quickly degraded by catabolic activities. Said term also bears the meaning of detecting the localization of a compound within a cell or a subcellular structure. Thus, the method of the invention allows the convenient testing for the localization of a compound (e.g. a protein, lipid, sugar etc.), the synthesis of which is, for example, induced in said cell. The first and second portion of said compound specifically interacting with said amino acid sequences or non-proteinaceous structures (aa) and (ad) would in this embodiment be naturally occurring portions of said compound. In this embodiment it would also be required that the spatial relationship of said first and second portion within the protein was known to allow the measuring of a signal from said second signaling entity. Alternatively, a compound may be introduced into a cell and its path within a cell may be followed by using the method of the present invention. Again, the first and second portions of the compound may be naturally occurring portions. They may also be portions that do not naturally occur on said compound. In the later case, said compound may itself be a fusion protein which comprises besides a naturally occurring protein, the location of which is of interest, said first and second portion in close spatial relationship. In a further embodiment, the compound may be an artificial or synthetic compound that has no natural counterpart. Such a compound may be of pharmaceutical interest. The method of the invention may be used to follow its path through the cell or localize the subcellular structure(s) where it is deposited.

It is crucial to note that the order of the various compounds in the fusion proteins or derivatives etc. may be as given above from N-terminal to C-terminal, but may also be of any other order.

The term "derivative of a fusion protein" is intended to mean that said fusion protein may comprise non-proteinaceous or proteinaceous portions not expressed from the same cistron of genetic information. Thus, proteinaceous portions may be connected to the "genuine" fusion protein by chemical linkage. The same holds true for the connection of non-proteinaceous structures that may be of organic or inorganic origin. If of organic origin, said structures may be sugars, lipids, nucleic acids or derivatives thereof such as peptide nucleic acids. Such structures may also be present on said fusion proteins as a result of a post-translational modification. Structures of inorganic origin comprise metal ions, chelating agents etc. The non-proteinaceous structures as well as the proteinaceous portions not forming a part of the genuine fusion protein advantageously represent or are comprised in a functional moiety of said fusion proteins or derivatives thereof, usually the portions that interact specifically with said first and/or said second portion of the compound.

The terms "fusion protein" and "detection protein", in connection with the present invention, refer to the same subject-matter (except when the compound to be detected is a fusion protein) and are thus interchangeably used. "Detection constructs" thus encode "detection proteins'V'fusion proteins". A schematic representation of fusion proteins that may be used in the method of the present invention is shown in Figure 1.

The term "specifically interacting with a portion of a compound" means that the interacting structure does not interact with any other portion of said compound so that the^' desired signal can be measured. Advantageously, said structure does not specifically interact with any other structure within said cell. Accordingly, it is a prerequisite for the method of the invention to properly work that suitable specifically interacting structures are selected. Such a selection does not represent an undue burden for the person skilled in the art. An example of a useful specific interaction is the binding of an antibody or a functional fragment or derivative thereof to an antigen. A further example is the interaction of an enzyme with a non-processible substrate analogue or the interaction of an enzyme which still binds its substrate but is enzymatically inactive. Additional examples of such specifically interacting structures are leucine zippers, calmodulin/M13 peptide, protein A/antibody constant regions, biotin ("strep-tag")/streptavidin, RNase/RNase S peptide and proteins derived from lipocalins.

A "signaling entity" denotes in connection with the present invention a three-dimensional structure usually comprising at least a domain that is capable of triggering the emission of a detectable signal. The signaling entity may directly emit said detectable signal. This is always the case for the second signaling entity (if it emits the signal) upon proper interaction with the first signaling entity or for the first signaling entity (if it emits the signal) upon proper interaction with the second signaling entity. Whereas in one embodiment of the invention, the first signaling entity may also emit a per se detectable signal (which is, however, not assessed for by said embodiment of the method of the invention), for example, to excite the detectable signal within the second signaling entity, it may also restore, alter or in other ways influence the emission of the signal from the second signaling entity. In another embodiment of the invention, these functions of the first and second signaling entities are inerchanged.

The method of the invention permits the convenient detection of compounds of various origins, functions and compositions within cells or cellular structures. Advantageously, the method allows various embodiments of the easy introduction of the detection constructs into the living cell, thus overcoming the time-consuming and laborious steps of the prior art methods referred to above. Also, the method of the invention allows, in many embodiments, that living cells or their progeny are assessed for said signal and can then be used for further analysis or manipulation. A possibility of how the method of the present invention may be performed is schematically shown in Figure 2.

If desired, nucleic acid encoding said fusion proteins may be introduced into said cells. Expression of said nucleic acid can easily be controlled employing appropriate means. In this way, expression may be induced at different developmental or differentiation statuses of said cell and the location of said compound be correlated with said different statuses.

Generally, it is advisable to introduce the fusion proteins or derivatives into the cell or express the corresponding nucleic acid once the compound or a precursor thereof is present/has been expressed within the cell. In this way, it is avoided that said compound prior to reaching its destination is picked up by said fusion proteins or derivatives thereof and carried to the targeted subcellular structures specified by the targeting structures of the fusion proteins. The cell employed for the analysis in accordance with the method of the invention is preferably a eukaryotic cell, more preferably a mammalian cell and most preferably a human cell such as a human tissue cell. The method of detecting the signal will essentially depend on the nature of the signal formed. Appropriate detection methods are well known in the art, for example, if the signal emitted is a fluorescent signal.

The method of the present invention finds a wide variety of applications: Thus, this method can be used in the examination of signal transduction pathways, where a change in protein localization is often involved.

In pharmacological studies it can be examined whether certain substances lead to or inhibit a change in the localization of candidate proteins. With a variation of this method, binding of substances to surface receptors and subsequent intemalization can be detected.

The method can be applied in medical research, for example in the examination of inflammatory processes, where nuclear transcription factors (e.g. NFKB, glucocorticoid receptor) play a crucial role.

In diagnostics, for example in blood cell diseases or tumors, cells could be cultured and transfected with plasmids encoding the fusion proteins to examine defects in protein transport/localization.

The method can be used for the examination of products of the chemical industry for their bioreactivity (e.g. hormonal effects).

Stably transfected cell lines or cell lines into which the fusion proteins or derivatives thereof or the nucleic acid encoding said fusion proteins have been otherwise introduced

(said cell lines thus containing the detection constructs) would abolish the need to transiently transfect them for each experiment and therefore represent useful tools for certain applications. Such cell lines also form embodiments of the present invention.

A further application is the construction of cDNA libraries from which the corresponding proteins may be expressed fused, e.g., to an epitope tag. They could be used to characterize new, tissue specific proteins e.g. in signal transduction pathways or gene regulation. The fundamental insights of the present invention also extend into other aspects of technology. Thus, a variation of this method can be used to monitor (ligand induced) endocytosis/receptor intemalization. In a preferred embodiment, fusion of the receptor with two different epitope tags and targeting two detection fusion proteins or derivatives e.g. to the endosomes is effected. Since the termini of the receptor polypeptide might be buried in the membrane or might be cytosolic (and thus not exposed to the endosome lumen after intemalization), in a further embodiment interacting amino acids at an internal position of the receptor are employed which may be either naturally occurring amino acid sequences or "tags" introduced into the receptor. This approach is intended to be employed in the functional screening for ligands of surface receptors (which lead to intemalization).

In a further aspect of the method of the invention, the assessment in step (c) is indicative of a signal transduction event. This aspect of the invention is particularly useful for screening for ligands of surface receptors. Thus, a surface receptor which is screened for interacting ligands would be coupled to a protein which directly translocates into' the nucleus and is detected there using the described fusion proteins or derivatives thereof. Alternatively, binding of a ligand would induce a signaling cascade, the known end-product of which would be detected in the nucleus (see Figure 9). In a variation of this method activated members of said signaling cascade may be detected in cellular substructures other than the nucleus. Such activated members may be, e.g., receptor- tyrosine-kinases (RTK) or G-protein coupled receptors activated upon ligand binding. In the first case, it is envisaged that the cytoplasmic domain of the RTK is fused to, e.g., two epitope-tags which in the inactive conformation of the RTK are not accessible for the detection proteins. Upon ligand binding, which activates the RTK and induces a conformational change of the cytoplasmic domain of the RTK, the epitope-tags become accessible for the detection proteins and subsequently are bound by the same. The thereby generated signal is indicative for RTK activation.

In the latter case, it is envisaged that the epitope-tags are inaccessible for the detection proteins due to the binding of the G protein trimer (α β γ). Upon receptor activation and dissociation of the G protein trimer, the epitope-tags become accessible for the detection proteins.

Alternatively, one (or both) of the interacting portions of said receptors has (have) a structure or conformation which is changing in response to ligand binding (e.g. phosphorylation/dephosphorylation) and is specifically bound by one (or both) detection proteins in one of these statuses.

The detection proteins may be expressed either in the cytoplasm or may be targeted to the cell membrane.

In accordance with the various embodiments presented throughout this invention, the signal intensity may be enhanced. This may be effected by e.g. cloning of various fusion protein encoding nucleic acids that will give rise to a number of fusion proteins arranged in a series.

Alternatively, signal intensity may be enhanced by generating a cascade of interacting fusion proteins wherein a third fusion protein or derivative thereof interacts with a portion of the first fusion protein or derivative thereof and a further fusion protein interacts with a portion of the second fusion protein or derivative thereof. This cascade may be further enhanced. The signals emitted by the fusion proteins or derivatives thereof may be of the same kind or may be different.

Furthermore, the present invention relates to a method for the detection of the fusion of two cells or two subcellular structures comprising the steps of: (a) introducing into a cell material comprising at least two fusion proteins or derivatives thereof or nucleic acid encoding said fusion proteins wherein one of said fusion proteins or derivatives thereof comprises (aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a first portion of a compound contained in or associated with one of said cells or subcellular structures; (ab) an amino acid sequence or a non proteinaceous structure representing or comprising a targeting signal for a cell or a subcellular structure; and (ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein further a second of said fusion proteins or derivatives thereof comprises (ad) an amino acid sequence or a non- proteinaceous structure capable of specifically interacting with a second portion of said compound wherein said first portion and said second portion are spatially distinct to allow the simultaneous interaction of said amino acid sequence or non-proteinaceous structure (aa) and said amino acid sequence or non-proteinaceous structure (ad) with said compound; (ae) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal specific for a different or the same cell or subcellular structure as said amino acid sequence or non-proteinaceous structure (ab); and (af) an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; wherein the specificities of said targeting signals (ab) and (ae) may be interchanged; (b) allowing expression of said nucleic acid, if applicable; and (c) assessing for a signal from said first or second signaling entity that is provided, restored, altered or influenced.

The term "wherein the specificities of said targeting signals (ab) and (ae) may be interchanged" means that in accordance with the method of the invention it is not decisive which fusion protein or derivative targets which cell or subcellular structure.

With essentially the same experimental setup, the method of the invention may thus also be extended to the assessment of fusion events of either cells or subcellular structures. For example, this embodiment of the invention can be used to monitor fusion processes of vesicles or other organelles, such as lysosomes or peroxisomes. In one embodiment, a free "linker" peptide which is expressed in one of the organelles and which is composed of two (or more than one for each) epitopes can be used in conjunction with the referenced detection method. Upon fusion of the cells or the subcellular structures/compartments, the two signaling entities are aligned in a close spatial arrangement such that detection of the signal emitted from the second signaling entity becomes possible. Preferably, said targeting signal (ab) or said targeting signal (ae) is specific for the same cell or subcellular structure which contains or is associated with said compound. This embodiment of the present invention is illustrated in Figure 3. The compound may per se be present in the cell or may be introduced into the cell, e.g. by way of a nucleic acid encoding the same, prior to, simultaneously with or after introduction of the material comprising the fusion proteins etc.

In addition, the present invention relates to a method for the detection of the fusion of two cells or two subcellular structures comprising the steps of: (a) introducing into a cell material comprising at least two fusion proteins or derivatives thereof or nucleic acid encoding said fusion proteins wherein one of said fusion proteins or derivatives thereof comprises (aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a portion of a second fusion protein or derivative thereof; (ab) an amino acid sequence representing or comprising a targeting signal for a cell or a subcellular structure; (ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein further said second fusion protein or derivative thereof comprises (ad) a portion capable of specifically interacting with said amino acid sequence or non-proteinaceous structure (aa); (ae) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal specific for a different cell or subcellular structure as in (ab); (af) an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; (b) allowing expression of said nucleic acid, if applicable; and (c) assessing for a signal from said first or second signaling entity that is provided, restored, altered or influenced.

This embodiment of the invention which is shown in Figure 4 is a variation to the hereinbefore described embodiment for assessing cell fusions or fusions of subcellular structures such as vesicles. The embodiment varies insofar from the previously disclosed embodiments that not both of the specifically interacting amino acid sequences of the fusion proteins or derivatives thereof interact with a third compound. Rather, one of the interacting sequences interacts with a target on the second fusion protein or derivative thereof. Fusion of the cells or subcellular structures will again bring the fusion proteins or derivatives thereof together and allow detection of the expected signal. Thus, in one embodiment one of the cells expresses an epitope tagged signaling entity in the cytoplasm, the other one a fusion protein comprising an amino acid sequence specifically recognizing a portion on the other construct, also in the cytoplasm. This approach could be applied in screening for fused cells e.g. in hybridoma technology. Another possible setup could be the expression of one detection protein comprising an amino acid sequence specifically interacting with a portion on the second construct in the plasma membrane of one cell and another detection protein comprising the "tag" for the specifically interacting amino acid sequence of the first detection construct in the plasma membrane of another cell. Thus, it might be possible to study cell-cell interactions.

The invention also relates to a method for the detection of ligand-induced receptor intemalization comprising the steps of: (a) introducing into a cell material comprising at least two fusion proteins or derivatives thereof or nucleic acid encoding said fusion proteins wherein one of said fusion proteins or derivatives thereof comprises an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a portion of a second fusion protein or derivative thereof; (ab) an amino acid sequence representing or comprising a targeting signal for a cell or a subcellular structure; (ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein further said second fusion protein or derivative thereof comprises (ad) a portion capable of specifically interacting with said amino acid sequence or non-proteinaceous structure (aa); (ae) an amino acid sequence or a non- proteinaceous structure representing or corpprising a receptor or a portion of a receptor that is capable of interacting with a ligand; (af) an amino acid sequence or a non- proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; (b) allowing expression of said nucleic acid, if applicable; and (c) assessing for a signal from said first or second signaling entity that is provided, restored, altered or influenced.

This embodiment of the method of the invention may be employed to investigate ligand induced receptor intemalization: a surface receptor is displayed as a first fusion protein with a signaling entity and an amino acid sequence to be specifically detected. Another construct contains the "binding" entity and a second signaling entity and is directed to, preferably, endosomes. Upon ligand binding, the first "receptor" fusion will be internalized and the second construct binds to it, allowing release of the desired signal (see Figure 5 for a schematic representation). ^■

Furthermore, the invention relates to a method for assessing the suitability of a signal sequence or a non-proteinaceous compound to direct a further compound into a subcellular structure comprising the steps of: (a) introducing into a cell a fusion polypeptide or derivative thereof comprising said signal sequence or said non- proteinaceous compound and said further compound, or a polynucleotide encoding said fusion polypeptide or derivative thereof comprising said signal sequence and said further compound, said cell comprising material comprising at least two fusion proteins or derivatives thereof or nucleic acid encoding said fusion proteins wherein one of said fusion proteins or derivatives thereof comprises (aa) an amino acid sequence or a non- proteinaceous structure capable of specifically interacting with a first portion of said compound, said non-proteinaceous compound or said signal sequence; (ab) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal for a subcellular structure; and (ac) an amino acid sequence or a non- proteinaceous structure representing or comprising a first signaling entity; wherein further a second of said fusion proteins or derivatives thereof comprises (ad) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a second portion of said compound, said non-proteinaceous compound or said signal sequence wherein said first portion and said second portion are spatially distinct to allow the simultaneous interaction of said amino acid sequence or non-proteinaceous structure (aa) and said amino acid sequence or non-proteinaceous structure (ad) with said compound; (ae) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal specific for the same subcellular structure as said amino acid sequence or non-proteinaceous structure (ab); and (af) an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; (b) allowing expression of said nucleic acid and/or said polynucleotide, if applicable; and (c) assessing for a signal from said first or second signaling entity that is provided, restored, altered or influenced. This embodiment of the present invention is illustrated in Figure 6 and 7.

Furthermore, the invention relates to a method for assessing the suitability of a polypeptide or a non-proteinaceous compound to direct a further compound into a subcellular structure comprising the steps of: (a) introducing into a cell said polypeptide or said non-proteinaceous compound, or a polynucleotide encoding said polypeptide, said cell comprising said further compound or a nucleotide sequence encoding said further compound, and material comprising at least two fusion proteins or derivatives thereof or nucleic acid encoding said fusion proteins wherein one of said fusion proteins or derivatives thereof comprises (aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a first portion of said compound or said non-proteinaceous compound; (ab) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal for a subcellular structure; and (ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein further a second of said fusion proteins or derivatives thereof comprises (ad) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a second portion of said compound, said non-proteinaceous compound or said signal sequence wherein said first portion and said second portion are spatially distinct to allow the simultaneous interaction of said amino acid sequence or non-proteinaceous structure (aa) and said amino acid sequence or non-proteinaceous structure (ad) with said compound; (ae) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal specific for the same subcellular structure as said amino acid sequence or non-proteinaceous structure (ab); and (af) an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; (b) allowing expression of said nucleic acid and/or said polynucleotide and/or said nucleotide sequence, if applicable; and (c) assessing for a signal from said first or second signaling entity that is provided, restored, altered or influenced.

The definitions provided for the embodiments above apply mutatis mutandis for the following embodiments.

Furthermore, the present invention relates to a method for the detection of a compound within a cell comprising the steps of: (a) introducing into a cell material comprising at least one fusion protein or derivative thereof or nucleic acid encoding said fusion protein wherein said fusion protein or derivative thereof comprises (aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a portion of said compound; (ab) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal for a subcellular structure; and (ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein said compound comprises or has attached thereto an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of either the first or the second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; wherein said compound or a precursor thereof is present or expressed in said cell before said material is introduced into or expressed in said cell or wherein said compound or precursor is introduced into said cell simultaneously with or after the introduction of said material or wherein said compound is present in said cell per se; (b) allowing expression of said nucleic acid, if applicable; and (c) assessing for a signal from said first or said second signaling entity that is provided, restored, altered or influenced. The amino acid sequence or non-proteinaceous structure representing or comprising said second signaling entity may have been transferred into said cell prior to the introduction of said material. As an amino acid sequence, it may be a portion of another fusion protein.

Moreover, the present invention relates to a method for the detection of the fusion of two cells or two subcellular structures comprising the steps of: (a) introducing into a cell material comprising at least one fusion protein or derivative thereof or nucleic acid encoding said fusion protein wherein said fusion protein or derivative thereof comprises (aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a portion of a compound contained in or associated with one of said cells or subcellular structures; (ab) an amino acid sequence or a non proteinaceous structure representing or comprising a targeting signal for a cell or a subcellular structure; and (ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein said compound comprises or has attached thereto an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of either the first or the second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; wherein the specificities of said targeting signals (ab) and (ae) may be interchanged; (b) allowing expression of said nucleic acid, if applicable; and (c) assessing for a signal from said first or second signaling entity that is provided, restored, altered or influenced. The term "wherein the specificities of said targeting signals (ab) and (ae) may be interchanged" means that in accordance with the method of the invention it is not decisive which fusion protein or derivative targets which cell or subcellular structure.

The present invention additionally relates to a method for the detection of the fusion of two cells or two subcellular structures comprising the steps of: (a) introducing into a cell material comprising at least one fusion protein or derivative thereof or nucleic acid encoding said fusion protein wherein said fusion protein or derivative thereof comprises (aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a portion of a second fusion protein or derivative thereof; (ab) an amino acid sequence representing or comprising a targeting signal for a cell or a subcellular structure; (ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein said cell comprises said second fusion protein or derivative thereof which comprises (ad) a portion capable of specifically interacting with said amino acid sequence or non-proteinaceous structure (aa); (ae) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal specific for a different cell or subcellular structure as in (ab); (af) an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; (b) allowing expression of said nucleic acid, if applicable; and (c) assessing for a signal from said first or second signaling entity that is provided, restored, altered or influenced.

The present invention also relates to a method for the detection of ligand-induced receptor intemalization comprising the steps of: (a) introducing into a cell material (i) comprising at least two fusion proteins or derivatives thereof or nucleic acid encoding said fusion proteins or (ii) one of said fusion proteins wherein the respective other fusion protein is contained in said cell, wherein one of said fusion proteins or derivatives thereof comprises (aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a portion of a second fusion protein or derivative thereof; (ab) an amino acid sequence representing or comprising a targeting signal for a cell or a subcellular structure; (ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein further said second fusion protein or derivative thereof comprises (ad) a portion capable of specifically interacting with said amino acid sequence or non-proteinaceous structure (aa); (ae) an amino acid sequence or a non-proteinaceous structure representing or comprising a receptor or a portion of a receptor that is capable of interacting with a ligand; (af) an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; (b) allowing expression of said nucleic acid, if applicable; and (c) assessing for a signal from said first or second signaling entity that is provided, restored, altered or influenced.

The present invention further relates to a method for assessing the suitability of a signal sequence or a non-proteinaceous compound to direct a further compound into a subcellular structure comprising the steps of: (a) introducing into a cell a fusion polypeptide or derivative thereof comprising said signal sequence or said non- proteinaceous compound, said further compound and a first signaling entity, or a polynucleotide encoding said fusion polypeptide or derivative thereof comprising said signal sequence, said further compound and said first signaling entity, said cell comprising material comprising at least one fusion protein or derivative thereof or nucleic acid encoding said fusion protein wherein said fusion protein or derivative thereof comprises (aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a portion of said compound, said non-proteinaceous compound or said signal sequence; (ab) an amino acid sequence or a non- proteinaceous structure representing or comprising a targeting signal for a subcellular structure; and (ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity; wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; (b) allowing expression of said nucleic acid and/or said polynucleotide, if applicable; and (c) assessing for a signal from said first or second signaling entity that is provided, restored, altered or influenced.

Further, the present invention relates to a method for assessing the suitability of a polypeptide or a non-proteinaceous compound to direct a further compound into a subcellular structure comprising the steps of: (a) introducing into a cell said polypeptide or said non-proteinaceous compound, or a polynucleotide encoding said polypeptide, said cell comprising said further compound and a first signaling entity or a nucleotide sequence encoding said further compound and said first signaling entity, and material comprising at least one fusion protein or derivative thereof or nucleic acid encoding said fusion protein wherein said fusion protein or derivative thereof comprises (aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a portion of said compound or said non-proteinaceous compound; (ab) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal for a subcellular structure; and (ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity; wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; (b) allowing expression of said nucleic acid and/or said polynucleotide and/or said nucleotide sequence, if applicable; and (c) assessing for a signal from said first or second signaling entity that is provided, restored, altered or influenced.

The present invention additionally relates to a method for the detection of one or more amino acid sequences or non-proteinaceous structures that interact with spatially distinct but closely arranged portions of a compound comprising (a) contacting said compound with at least two of said amino acid sequences or non-proteinaceous structures under conditions that allow an interaction to take place wherein (aa) one of said amino acid sequences or non-proteinaceous structures is connected with an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; and (ab) a second of said amino acid sequences or non-proteinaceous structures is connected with an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; and (b) assessing for a signal from said first or second signaling entity that is provided, restored, altered or influenced.

This embodiment of the invention, which is schematically shown in Figure 8, is particularly useful for providing suitable amino acid sequences that may be used in the earlier discussed embodiments of the invention. For example, parts of antibody derivatives such as scFv fragments from a phage display library, coupled to first or second signaling entities, may be contacted with compounds of interest. Binding of scFvs to distinct but spatially closely related epitopes on said compound will give rise to a detectable signal. Said scFvs may then be further selected, cloned and used for further analyses.

In a preferred embodiment of the method of the present invention said method further comprises producing said one or more detected amino acid sequences or non- proteinaceous structures.

Once suitable interacting sequences have been identified, production, preferably large- scale production of the detected sequences and corresponding molecules, respectively, may be instigated. Optionally, the sequences may be combined for production with further sequences referred to above for preparing fusion proteins or derivatives thereof directly applicable in the method of the invention.

In another preferred embodiment of the method of the present invention said amino acid sequences (aa) and (ab) are further connected to amino acid sequences or non- proteinaceous structures representing or comprising targeting signals for the same or different cells or the same or different subcellular structures.

As with the previous preferred embodiment, this embodiment is particularly useful for use in the main embodiment of the invention.

The invention also relates to a method for mapping epitopes comprising (a) contacting a compound under investigation for epitope mapping with two different amino acid sequences or non-proteinaceous structures wherein (aa) the first of said amino acid sequences or non-proteinaceous structures is connected with an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; and (ab) the second of said amino acid sequences or non-proteinaceous structures structures is connected with an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities. The above recited method may conveniently be used in the mapping of epitopes of, for example, proteins. Employing at least two and preferably a series of appropriate amino acid sequences such as sequences representing or comprising variable sequences of antibodies, epitopes of portions or complete proteins may be mapped. The close spatial relationship of two epitopes on a protein is given, if the expected signal from said second signaling entity is detectable.

The invention in another preferred embodiment relates to a method wherein either the first or second portion of said compound has a stage-specific conformation whereas the other portion has a non-stage-specific conformation.

This embodiment of the invention is particularly useful for assessing the differentiation status or the developmental status of a cell or tissue. For example, a first portion of the compound may be an epitope that is constitutively expressed in a protein, whereas a second portion represents a phosphorylation site within the same compound. Said compound may be phosphorylated or dephosphorylated during certain differentiation or developmental statuses of said cell or tissue. Also, the stage specific conformation may be indicative of a disease or the potential onset of a disease.

In an additional preferred embodiment of the method of the present invention both the first and second portion of said compound either have a stage-specific conformation or a non-stage-specific conformation.

This preferred embodiment of the invention may also be used for the detection of a differentiation status or a developmental status of a cell or tissue. In this regard, it is also referred to the particular advantages discussed in the connection with the preceding embodiment.

The first and second signaling entities may be selected from a variety of compounds. For example, it is envisaged in accordance with the present invention that said first and second signaling entities are structures or domains that provide, restore, alter or influence a signal upon dimerization, trimerization or forming a multi-domain structure.

Additionally, a signal may be generated by increased or decreased interaction of the two signaling entities due to the binding of said detection proteins to the compound under analysis.

The signal generated by said first and second signaling entities directly or indirectly leads to a phenotypical change of the cell under analysis.

Such change may allow the selection of cells which display said phenotypical change, e.g. by expression of a resistance gene.

In a preferred embodiment of the method of the present invention, said first and second signaling entities represent complementing portions of proteins (such as e.g. M15 beta- galactosidase/beta-galactosidase alpha peptide) which display altered signaling properties upon increased interaction, effected by binding to the compound under analysis.

In a more preferred embodiment of the method of the present invention said first and second signaling entity are an enzyme and its substrate.

Particularly preferred is that, upon interaction of the enzyme and its substrate, the substrate gives rise to a detectable color.

In an additional preferred embodiment of the method of the present invention said first and second signaling entity represent an luminescent entity.

In a particularly preferred embodiment of the method of the present invention said luminescent entity exhibits a fluorescent, phosphorescent, chemiluminescent or bioluminescent activity.

In a most preferred embodiment of the method of the present invention said first signaling entity has a luminescent activity with an excitation wavelength λ1 and an emission wavelength λ2 and wherein said second signaling entity has a luminescent entity with an excitation wavelength λ3 and emission wavelength λ4 wherein said emission wavelength λ2 and said excitation wavelength λ3 overlap, wherein further λ4 is distinguishable from λ1 and λ2, wherein further emission wavelength λ4 is detectable upon triggering excitation of λ1 and upon the close spatial arrangement of said first and said second luminescent entity.

This embodiment is most preferred since it allows the convenient manipulation of cells with fusion proteins wherein the fusion protein comprises a signaling entity that is excited with a given wavelength, then emits a second wavelength that excites the second signaling entity which, then, gives rise to a signal that has a specific wavelength that can be specifically detected using appropriate means. In other words, this system may be excited with one wavelength and gives a rise to a different wavelength that is not related to the incoming wavelength or the wavelength that is directly generated by the incoming wavelength. Systems that make use of this principle have been described and make, for example, use of an effect, designated "fluorescence resonance energy transfer" (FRET).

In an additional particularly preferred embodiment of the method of the present invention said first and second signaling entities are functional mutants, fragments or derivatives of Green Fluorescent Protein.

In accordance with the present invention, functional mutants, fragments or derivatives of Green Fluorescent Protein are particularly useful since they are already available on the market. They are particularly preferred if of smaller size than the full length GFP. One of the embodiments that is preferred and relies on the above has been described in Griffin, B. A. et al. (1998).

In another preferred embodiment of the method of the present invention said fusion proteins are members of an expressed nucleic acid library.

A search for appropriate fusion proteins is conveniently carried out from a source comprising a large number of such fusion proteins. An expressed nucleic acid library is a particularly suitable source. Such a library may be a cDNA library that is, for example, presented by a phage display. Alternatively, synthetic libraries comprising said fusion proteins may be employed. A number of different approaches may be employed when introducing the fusion proteins and optionally the compound into said cell. For example, if the fusion proteins are directed as proteins into the cell, microinjection may be used, although this embodiment is less preferred. Particle bombardment is a further possibility of introducing the desired proteins or encoding nucleic acids into the cell.

In a more preferred embodiment of the method of the present invention said introduction into said cells is effected by transfection.

Transfection of cells is well established in the art. Using this approach, large numbers of cells can be manipulated and later analyzed at the same time. Accordingly, this embodiment relying on the introduction of nucleic acid into the cells is particularly suitable for overcoming the above referenced prior art problems.

The compound employed in the method of the invention may be of heterogenous chemical nature. The invention in another preferred embodiment relates to a method wherein said compound is a protein or a linker.

In a particularly preferred embodiment of the method of the present invention said protein is a member of a signaling cascade or a gene regulatory protein.

In another preferred embodiment of the method of the present invention said compound or said non-proteinaceous structure is a carbohydrate, a lipid, a steroid, a vitamin, a phospholipid, a nucleic acid, a DNA or a pharmaceutically active agent.

As regards pharmaceutically active agents, the method of the invention is advantageous for following up the processing within a cell or their interaction with various organelles/compartments or other substructures of the cell. Thus, the method of the invention contributes to the elucidation of the mode of action of new agents designed as pharmaceuticals prior to in vivo testing. As regards the first and second portions of said compound that are bound by the interacting amino acid sequences, the present invention generally envisages two different options: either these portions are externally attached to the compound of interest as heterologous parts thereof, such as tags. Thus, in a further preferred embodiment of the method of the present invention said first and/or second portion of said compound is a tag.

Alternatively, they may be naturally comprised portions known to have a close spatial relationship. In an additional preferred embodiment of the method of the present said first and/or second portion of said compound are therefore portions naturally occurring in said compound.

In addition, in a different preferred embodiment of the method of the present invention said amino acid sequence capable of specifically interacting with said portions of said compound or said second fusion protein are derived from or represent antibody variable regions, protein-protein interaction domains, (portions of) receptor-ligand systems or enzyme substrate systems.

Particularly preferred are antibody variable regions as binding partners to portions of said compounds. Fragments or derivatives of antibodies comprising antibody variable regions comprise Fab-, F(ab')₂- or scFv-fragments.

In a particularly preferred embodiment of the method of the present invention said amino acid sequences comprise single-chain Fv fragments. ScFv fragments are particularly preferred because of their relatively small size.

The construction of scFv against epitope tags is published (- myc-tag in Fuchs, P. et al.

(1997); -> his-tag in Lindner, P. et al. (1997)) and their genes are used for the detection constructs. scFv genes against any other suitable epitope tag or against suitable epitopes of the protein of interest itself can be generated by phage display technology. scFv have been successfully targeted to different organelles and proven to be functional in these environments. Cystein residues of scFv targeted to mitochondria and secretory compartments (ER) were found to be in an oxidized state, while those targeted to the cytosol were found to be reduced. The latter may explain the poorer (but still acceptable) efficiency of cytosolic scFv (Biocca, S. et al. (1995)). In another case, only ER-targeted scFv was expressed at high level in COS cells, while the levels of scFv targeted to the nucleus and cytosol was low. Addition of sequences of a mouse Ig constant domain improved cytosolic expression moderately. (Jannot, C.B. and Hynes, N.E. (1997)). scFv constructs containing an endoplasmatic reticulum (ER) or frans-Golgi network (TGN) retention signal were targeted correctly to the respective organelle, while an scFv without such signal was secreted. The half-life of the scFv was >24h (scFv-ER) and <9h (scFv-TGN) and both of them were able to bind their target protein (Zhou, P. et al. (1998)). Targeting vectors for the expression of scFv in different organelles have been constructed. These include vectors for the expression in the ER, the nucleus, mitochondria, the cytoplasm and as secreted proteins. The function of these vectors has been assessed by immunofluorescence of transiently transfected COS cells (Persic, L. et al. (1997)).

In plant cells, scFv have been targeted to (and were functional in) the cytosol, apoplastic space, and ER (reviewed in Conrad, U. and Fiedler, U. (1998); Fiedler, U. et al. (1997)). C-terminal addition of a (KDEL)-ER-retention signal was shown to improve cytosolic scFv expression (Schouten, A. et al. (1997); Schouten, A. et al. (1996)).

The invention in a different preferred embodiment relates to a method wherein said first and said second fusion protein are encoded by the same nucleic acid molecule.

In a particularly preferred embodiment of the method of the present invention said nucleic acid molecule is a bicistronic vector.

In still another preferred embodiment of the method of the present invention said first and said second fusion protein are encoded by the different nucleic acid molecules. In yet another preferred embodiment of the method of the present invention said expression is inducible.

Inducible expression is particularly useful due to the fact that the timely arrangement of expression of nucleic acid encoding the fusion proteins is possible. Thus, it is usually preferred to have the compound first locate to its destined location within the cell and only then induce expression of the detection constructs (i.e. the fusion proteins or derivatives thereof or the corresponding coding nucleic acid). As has been outlined above, this arrangement of events precludes the pickup of the compound and its carrying off to compartments of the cell specified by the targeting portions of the fusion proteins. This embodiment of the invention is also particularly useful to follow the route of a protein inside a cell by inducing expression at different time points.

In a particularly preferred embodiment of the method of the present invention said subcellular structure is the nucleus, nucleolus, cytoplasm, cytoskeleton, chromatin, a mitochondrion, a microtubulus, a centriole, a nuclear pore, a ribosome, a microfilament, a perixosome, a proteasome, a lysosome, vacuole, chloroplast, thylakoid, membrane, the' Golgi apparatus or the endoplasmatic reticulum. The following is on overview on these subcellular structures with a special view on targeting modules that may be used in the fusion proteins or derivatives thereof for targeting these subcellular structures.

Mitochondria

In most cases, mitochondrial matrix proteins encoded in the nucleus carry an N-terminal signal peptide which is proteolytically removed after import into mitochondria. Those signal peptides are usually 20 to 35 residues in length, more hydrophilic than export signals and rich in hydroxylated and basic amino acids, lacking acidic ones and can fold into amphiphilic α-helix or α-sheet. These signal peptides can direct heterologous proteins to mitochondria. This is true for proteins supposed to reside in the mitochondrial matrix. The signals for the inner/outer membrane or the intermembrane space are somewhat more complex, including composite signal sequences where an import signal is followed by an export signal or a stop-transfer signal (e.g. import from cytoplasm to mitochondrial outer or inner membrane or mitochondrial intermembrane space). There seem to be more determinants for correct and efficient transport of proteins to mitochondria. It has been shown that yeast mutants deficient in a certain component of the mitochondrial transport pathway could correctly deliver mitochondria-supposed wt proteins, but not heterologous proteins carrying a MTS (mitochondrial targeting signal) at their N-terminus. Comparison of the 3' UTR of mitochondrial proteins revealed a consensus sequence which enhances the transport to mitochondria (Juretic, N. and Theus, M. (1991 )).

Examples for Mitochondria Targeting Signals (MTS):

Mitochondrial matrix:

- 16 amino acid MTS of human 3-oxoacyl-CoA thiolase (hOACTL) (Abe, H. et al.

(1993)) aa-sequence: MRLLRGVFVVAAKRTP

DNA-sequence: ATG CGT CTG CTC CGC GGT GTG TTT GTA GTT GCT GCT

AAG CGA ACG CCC This signal has been used to direct GFP to the mitochondrial matrix (Zhang, C. et al. (1998)).

- the presequence of omitine transcarbamylase has been used to direct GFP to mitochondria (Kanazawa, M. et al. (1997)) -» F1 β (β-subunit of the mitochondrial ATPase (Garrett, J.M. et al. (1991)))

Mitochondrial inner membrane:

-> cytochrome c oxidase subunit Va is targeted to the inner membrane (Gartner, F. et al.

(1995)), a reporter construct containing the leader peptide was shown to be directed to the yeast mitochondrial matrix and to some extent to plant chloroplasts (Huang, J. et al.

(1990)) -> the cytochrome c oxidase subunit IV presequence

(LSLRQSIRFFKPATRTLCSSRYLL) was used as a fusion to GFP (Kohler, R.H. et al.

(1997))

-> cytochrome d is bound to the inner membrane, facing the intermembrane space; leader peptide: FSNLSKRWAQRTLSKSFYSTATGAASKSGKLTEKLVTAGVAAAGITA

STLLYADSLTAEA

Mitochondrial intermembrane space:

-> cytochrome b2, a protein of the yeast intermembrane space, is synthesized with an

80 residue bipartite presequence (Beasley, E.M. (1993))

- cytochrome c peroxidase

- Rizzuto R. et al. (1995) describe the targeting of GFP to mitochondria which included an N-terminal epitope tag

"Mitochondrial" Diseases:

-> Reye's syndrome, Keams-Sayre syndrome

- a fusion of frataxin (the first intron of this protein is mutated in Friedreich's ataxia) with

GFP localizes to mitochondria (Priller, J. et al. (1997))

Peroxisomes

The C-terminal peroxisomal targeting signal (PTS1 ) tripeptide (S/A/C/K/N)- (K/R/H/Q/N/S)-L is sufficient for targeting of proteins (and even gold particles conjugated to a peroxisomally targeted protein (Walton, P.A. (1995)) into peroxisomes (Subramani, S. (1996); Amery, L. et al. (1998)). A second sequence, PST2, is composed of a conserved nonapeptide with the sequence (R/K)-(L/V/I)-(X5)-(H/Q)-(L/A) and is used by a smaller subset of matrix proteins (Subramani, S. (1993)). Although naturally located at the N-terminus of proteins, the PTS2 signal also functions at internal location in passenger proteins (Faber, K.N.; Subramani, S. unpublished data). Other sequences, located internally within peroxisomal matrix proteins such as Candida tropicalis acyl CoA oxidase (Small, G.M. et al. (1988)) and Saccharomyces cerevisiae catalase (Kragler, F. et al.(1993)) and camitine acetyl transferase (Elgersma, Y. et al. (1995)), have been described but their generality is undocumented. Another PTS has been described that is near the N-terminus of ScPebl p (S. cerevisiae peroxisome biogenesis protein 1), which is the receptor for the PTS2 sequence (Zhang, J.W. and Lazarow, P.B. (1996)).

Peroxisomal membrane targeting signals (mPTSs) have been defined for a few proteins. The best studied of these is the mPTS in the Candida boidinii peroxisomal membrane protein (PMP)47, in which the mPTS lies in a 20 amino acid loop that is located between putative transmembrane domains four and five, in a protein predicted to have six transmembrane domains (McNew, J.A. and Goodman, J.M. (1996)). Another mPTS was localized to the first 40 amino acids of PpPas2p (Pichia pastoris peroxisome assembly protein 2) (Wiemer, E.A.C. et al. (1996)), which is a homologue of the peroxisomal membrane protein ScPas3p (Hόhfeld, J. (1991)). Comparison of the mPTSs in PMP47 and PpPas2p revealed only a stretch of basic amino acids common to both.

Examples for PTSs: PTS1 (C-terminal)

-> (S/A/C/K/N)-(K/R/H/Q/N/S)-L (Subramani, S. (1996); Amery, L. et al. (1998)) PTS2 (N-terminal)

- (R/K)-(LΛ /I)-(X5)-(H/Q)-(UA) (Subramani, S. (1993)) internal PTS

- Candida tropicalis acyl CoA oxidase (Small, G.M. et al. (1988)) -> Saccharomyces cerevisiae catalase (Kragler, F. et al.(1993)) -> Saccharomyces cerevisiae camitine acetyl transferase (Elgersma, Y. et al. (1995)) mPTS

-> Candida boidinii PMP47 (McNew, J.A. and Goodman, J.M. (1996)) -> Pichia pastoris PpPas2p (Wiemer, E.A.C. et al. (1996)) Human disease caused by peroxisomal defects:

- human PTS1 receptor (PTS1 R or PXR1 (peroxisome targeting signal receptoM ) responsible for Zellweger syndrome and neonatal adrenoleukodistrophy in some, but not all, patients (Dodt, G. et al. (1995); Wiemer, E.A. et al. (1995))

- Restoration by a 35 K membrane protein of peroxisome assembly in a peroxisome- deficient mammalian cell mutant (Tsukamoto, T. et al. (1991))

-> Peroxisome assembly factor-2, a putative ATPase cloned by functional complementation on a peroxisome-deficient mammalian cell mutant (Tsukamoto, T. et al. (1995)) -» RCDP disease:

Differential protein import deficiencies in human peroxisome assembly disorders (Motley, A. et al. (1994))

- Identification of three distinct peroxisomal protein import defects in patients with peroxisome biogenesis disorders (Slawecki, M.L. et al. (1995))

-^Transport of microinjected proteins into peroxisomes of mammalian cells: inability of Zellweger cell lines to import proteins with the SKL tripeptide peroxisomal targeting signal (Walton, P.A. (1992))

- Presence of cytoplasmic factors functional in peroxisomal protein import implicates organelle-associated defects in several human peroxisomal disorders (Wendland, M. and Subramani, S. (1993))

Golqi apparatus

At present, there is no evidence for proteins resident in the Golgi apparatus being localized to the Golgi lumen, but instead all the proteins are either integral membrane proteins or peripheral membrane proteins on the cytoplasmatic face of the Golgi. The single transmembrane domain (TMD) of enzymes such as the glycosyltransferases are sufficient to confer Golgi localization when transplanted into another protein (reviewed in Colley, K.J. (1997) Glycobiology 7: 1-13). For the proteases of the trans Golgi network (TGN) (e.g. furin in mammalian cells, and DPAP-A, Kexl p and Kex2p in yeast), short, tyrosine-containing sequences (e.g. YQRL (Zhan, J. et al (1998)) in the cytoplasmic tail, similar to those required for endocytosis, have been shown to be crucial for specifying TGN localization (Wilcox, CA. et al. (1992); Nothwehr, S.F. et al. (1993); Bos, K. et al. (1993) and Schafer, W. et al. (1995)). Some viruses use the Golgi as their site of assembly, either by budding through the Golgi membrane (e.g. coronaviruses), or by directly wrapping their cores in Golgi membranes (e.g. poxviruses). Their membrane proteins have been examined for Golgi-targeting signals and in some cases it seems that mechanisms similar to those involved for targeting of endogenous proteins are used (TMD-mediated retention or cytoplasmic retrieval signals specifying TGN localization). However, in both the multispanning M-protein of coronaviruses, and a single-spanning protein from Uukuniemi virus, a cytoplasmic signal has been found that can specify Golgi localization without recycling through the cell surface (Anderson, A.M. et al. (1997) and Locker, J.K. et al. (1994)).

For the targeting to the cytoplasmic face of the Golgi membrane, it has been possible to identify protein regions that are necessary and sufficient for correct localization. For three such proteins, endothelial nitric oxide synthase, glutamate decarboxylase and SCG10, this region comprises the first 30-35 N-terminal residues (Solimena, M. et al. (1994); DiPaolo, G. et al. (1997) and Liu, J.W. et al. (1997)). None of these regions are related by sequence, but all three are fatty acetylated (which is also true for many other proteins located to other membranes). The first 35 amino acids of endothelial nitric oxide synthase (eNOS) have been used to target GFP to the Golgi apparatus. It was found that the unique (Gly-Leu)5 repeat located between the palmitoylation sites (Cys-15 and - 26) of eNOS is necessary for palmitoylation and thus localization, but not for N- myristoylation, membrane association and NOS activity (Liu, J. W. et al. (1997)). Fusions of GFP to Golgi proteins allowed to follow the membrane diffusion of these Golgi-retained chimeras after photobleaching of living cells and revealed that both medial and trans enzymes are highly mobile (Cole, N.B. et al. (1996)). Although there is no convincing model for how the retention in / correct localization to the Golgi apparatus is achieved, it is obvious that certain proteins are indeed localized in (or at least their localization is focussed to) defined substructures. Consequently, fusion of a heterologous protein to the appropriate Golgi-targeted protein (or part of it) should enable to target this chimera to discrete suborganellar Golgi structures. The review by Lippincott-Schwartz, J. et al. (1998) describes recent studies of Golgi-targeted GFP chimeras. The targeting of GFP to neuroendocrine secretory granules as fusion with chromogranin B (a soluble marker protein of neuroendocrine secretory granules) is described in Kaether, C. et al. (1997). Secretion is programmed by a cleavable leader of 15-30 amino acids. An example is given by the presequence of bovine growth hormone: MAAGPRTSLLLAFALLCLPWTQVVGA LPVC.

Endoplasmatic Reticulum (ER)

Miyawaki, A. et al. (1997) used the N-terminal calreticulin signal sequence (Crsig) and a C-terminal ER retention signal to direct GFP chimeras to the ER.

Crsig: MLLSVPLLLGLLGLAAAD (Kendall, J.M. et al. (1992))

ER retention signal: KDEL (Kendall, J.M. et al. (1992))

In Yeast: HDEL or DDEL

The signal for localization in the ER membrane is KKXX (e.g. KKMP (Zhan, J. et al (1998)) at the C-terminus (which is the cytoplasmic tail) of a protein.

Nucleus

There is no strict consensus sequence for NLSs (reviewed in Hicks, G.R. and Raikhel, N.V. (1995)). In general, they are rich in the basic amino acids arginine and lysine and may contain residues as proline, that disrupt helical domains (Chelsky, D. et al. (1989)). The different NLSs can be (roughly) organized in three groups:

The first group are the SV40(largeTantigen)-like NLSs, defined as single peptide regions containing basic residues.

SV40 NLS: Pro-Lys-Lys-Lys-Arg-Lys-Val (Kalderon, D. et al. (1984);

Lanford, R.E. and Butel, J.S.

(1984)) Polyoma T antigenl : Pro-Lys-Lys-Ala-Arg-Glu-Asp

Polyoma T antigen2: Pro-Val-Ser-Arg-Lys-Arg-Pro-Arg-Pro

SV40VP1 : Ala-Pro-Thr-Lys-Arg-Lys-Gly-Ser

The SV40 NLS has been shown to function in animals and yeast (Forbes, D.J. (1992)) and plants (Lassner, M.W. et al (1991), van der Krol, A.R. and Chua, N.-H. (1991 ), Varagona, M.J. and Raikhel, N.V. (1994)).

The second group is typified by the NLS within Xenopus protein nucleoplasmin (Lys-Arg- X10-Lys-Lys-Lys-Lys) (Dingwall, C. et al. (1988), Chatterjee, S. and Stochaj, U. (1998)). The nucleoplasmin signal is composed of two peptide regions containing basic residues that are separated by a spacer of ten residues. Mutation of basic residues in one of the regions does not inhibit nuclear translocation, whereas mutations in both peptide regions block nuclear translocation (Robbins, J. et al. (1991 )). Since the length of the spacer can vary, many NLSs can be classified as bipartite. In fact it has been suggested that this class of NLSs is the most common (Dingwall, C. and Laskey, R. (1991)). The third and most unusual class of NLSs is defined by the N-terminal signal of the yeast protein Matα2, containing (in addition to the basic residues) one or more hydrophobic residues with unclear significance. The actual yeast Matα2 NLS does not function in mammals.

Matα2-NLS Lys-lle-Pro-lle-Lys (Hall, M.N. et al. (1984))

In endogenous plant proteins, all three classes are found, for example:

Class: Protein: Name of NLS: peptide sequence (basic amino acids underlined):

Bipartite Opaque-2 (NLS B) RKRKESNRESARRSRYRK

SV40-like R (NLS M) MSERKRREKL

Matα2-like R (NLS C) MISEALRKAIGKR

Some proteins of plant pathogens do have functional NLSs (e.g. VirD2 and VirE2 of A tumefaciens, squash leaf curl-virus movement proteins BR1 and BL1 and potyvirus proteins Nla and Nib).

Miyawaki, A. et al. (1997) used a C-terminal NLS to direct GFP chimera to the nucleus. This signal leads to efficient transport to the nucleus, excluding the nucleoli.

NLS: PKKKRKVEDA (Miyawaki, A. et al. (1997))

Nucleolus

Proteins directed to the nucleolus (e.g. yeast NSR1 , Nopp140, NAP57) contain acidic and serine-rich domains with which they interact with NLSs and which also bind RNA (reviewed in Xue, Z. and Melese, T. (1994)). Proteasomes

Proteasomes are proteolytic complexes involved in non-lysosomal degradation (e.g. of ubiquitinated proteins or proteins for presentation by MHC class I molecules) which are localized in both the cytoplasm and the nucleus. Reits, E.A.J. et al. (1997) describe the fusion of GFP to the C-terminus of proteasome subunit LMP2 and the localization thereof in proteasomes. A FRET approach might be used to determine proteins directed to proteasomes upon stimulation (fusion of GFP1 to LMP2 and fusion of a protein of interest to GFP2).

Lvsosomes

Lysosomal proteins are assembled on membrane-bound ribosomes of the ER and carried to the cis Golgi cistemae where they are recognized by enzymes that add a phosphate group to mannose sugars of the N-linked carbohydrate chains. (The signal for the addition of N-linked oligosaccharides to an asparagine residue in the ER is Asn- X-Ser or Asn-X-Thr (where X is any amino acid except proline)). These phosphorylated mannose residues serve as a signal for lysosomal sorting. Lysosomal proteins pass from trans Golgi via late endosomes to lysosomes.

The 35 amino acid cytoplasmic tail of the adhesion receptor P-selectin is subdivided into stop transfer, C1 and C2 domains. A KCPL sequence within C1 mediates sorting from endosomes to lysosomes (Blagoveshchenskaya, A.D. et al. (1998A)). Two sequences within the C2 domain (YGVF and DPSP) seem to be lysosome avoidance signals, since their substitution results in enhanced lysosomal sorting (Blagoveshchenskaya, A.D. et al. (1998B)).

The invariant chain targets newly synthesized MHC II molecules to a lysosome-like compartment. The transmembrane domain and the sequences DQRDLI and EQLPML in the cytoplasmic tail are sufficient for this targeting (Odorizzi, C.G. et al. (1994)). The C-terminal sequence KFERQ has been reported to be a lysosomal targeting signal (Zhan, J. et al. (1998)).

Usually lysosomes are detected via the presence of the enzyme acid phosphatase. It might be difficult to functionally import heterologous proteins to lysosomes, due the low pH and the presence of proteases. However, there are examples of proteins which are stable in this environment (e.g. MHC II and the resident enzymes). Receptors which are supposed to be recycled need to be stable at acidic pH. This is largely achieved by multiple disulfide bridges. Labeling of lysosomes using fusions of different lysosomal proteins with GFP has been reported (Burd, C.G. and Emr, S.D. (1998); Brown, F.D. et al. (1998); Gerhardt, B. at al. (1998); Via, L.E. et al. (1998); Wacker, I. et al. (1997); Wubbolts, R. et al. (1996)).

There are many human disorders resulting from defects in lysosomal function. Some of them are due to incorrect targeting of enzymes to the lysosomes. An example is the I- cell disease which is caused by a deficiency in N-acetylglucosamine phosphotransferase, resulting in non-phosphorylation of mannose residues. Thus lysosomal enzymes are secreted instead of targeted to lysosomes. While in most cases of lysosomal storage disorders (with Tay-Sachs disease being the most prominent), enzyme mutations (that are transported to the lysosome but are not functional) are described to be responsible, it is rather probable that defects in correct targeting are among the causes for some of these diseases as well.

Vacuole

The vacuole-resident protease aminopeptidase I (AP I) is targeted from the cytosol to the vacuole, rather than via the secretory route utilized by most characterized vacuolar enzymes (Klionsky, D.J. et al. (1992)). AP I is synthesized as a precursor containing an N-terminal extension necessary for correct vacuolar sorting. This propeptide region is predicted to form two α helices (Martinez, E. et al. (1997)). The first helix is amphipathic in nature and is critical for correct sorting of the enzyme (Oda, M.N. et al. (1996)). Upon delivery of the protein to the vacuole, the proregion is cleaved resulting in the mature- sized enzyme. The active form of AP I is a homodecamer of 600 kDa which is formed rather fast during delivery. This large size suggests that AP I targeting occurs by a vesicle-mediated process, rather than by transport via a translocation channel (Scott,

S.V. and Klionsky, D.J. (1998)). Examination of AP I precursors was done in mutant yeast strains (Scott, S.V. et al. 1997)).

Examples for vacuole-targeting of GFP by fusion with:

-> EmrE (a small E.coli multidrug transporter) (Yelin, R. et al. (1999))

-> yeast Ste2; although GFP was fused to the cytoplasmic tail of the receptor, it also accumulated in the lumen of the vacuole (Stefan, C.J. and Blumer, K.J. (1999))

-> the vacuole targeting peptide of tobacco chitinase A (DiSansebastiono, G.P. et al.

(1998))

-> Yeb3p (a yeast armadillo protein); this fusion lead to incorporation into the vacuole membrane; fusion with the first 69 amino acids of Yeb3p was sufficient for vacuolar membrane targeting but lead also to targeting to the plasma membrane (Pan, X. and

Goldfarb, D.S. (1998))

Chloropiasts and Thylakoids

Signal sequences for the targeting to chloropiasts are usually N-terminal peptides of >25 residues composed of charged amino acids. The s-subunit of the protein ribulase-1 ,5- bisphosphat-carboxylase is targeted to the chloroplast stroma by a 44 amino acid presequence. Ferredoxin is another example of a chloroplast-lumen targeted protein.

The chlorophyll a/b binding proteins are targeted to the light harvesting complexes of the thylakoid membrane. Plastocyanin is targeted to the thylakoid lumen by two distinct targeting signals (one for the targeting to the chloroplast, the other one for the targeting to the thylakoid lumen).

Transfer peptides (substrates of ΔpH-dependent translocase) spinach 23K AQKQDDNEANVLNSGVSRRLALTVLIGAAAVGSKVSPADA wheat 23K AQKNDEAASDAAVVTSRRAALSLLAGAAAIAVKVSPAAA spinach 16K -AQQVSAEAETSRRAMLGFVAAGLASGSFVKAVLA maize 16K --ASAEGDAVAQAGRRAVIGLVATGIVGGALSQAARA barley PSI-N --AAAKRVQVAPAKDRRSALLGLAAVFAATAASAGSARA cotton PSII-T --VQMSGERKTEGNNGRREMMFAAAAAAICSVAGVATA

Arabidopsis PSII-T -TPSLEVKEQSSTTMRRDLMFTAAAAAVCSLAKVAMA

Signal peptides (Sec substrates) wheat 33K AFGVDAGARITCSLQSDIREVASKCADAAKMAGFALATSALLV SGATA spinach 33K AFGVESASSSGGRLSLSLQSDLKELANKCVDATKLAGLALATS ALIASGANA

spinach PSI-F -QENDQQQPKKLELAKVGANAAAALALSSVLLSSWSVAPDAA MA barley PSI-F -SGDNNNSTATPSLSASIKTFSAALALSSVLLSSAATSPPPAAA barley PC AS LG KKAASAAVAM AAG AM LLGGSAM A spinach PC ASLKNVGAAVVATAAAGLLAGNAMA

(Robinson, C. et al. (1998))

It might be possible as well to target proteins to other plastids in plant cells (e.g. etioplasts, chromoplasts, amyloplasts or elaioplasts).

Membrane

The targeting of proteins to the cell membrane can be achieved by a famesylation signal.

A signal peptide for the linkage of a myristic acid to the N-terminus is Gly-Ser-Ser-Lys-

Ser-Lys-Pro-Lys. Other

It might be worthwhile to target detection proteins to specific subcellular structures, like actin or tubulin filaments. It is envisaged that the method of the present invention can be extended to other cellular structures than organelles/vesicles.

In an additional preferred embodiment of the method of the present invention at least said step of assessing is effected by using a high throughput system.

Possibly, one of the greatest advantages of the present invention is its applicability in high throughput systems (HTS).

Using HTS, measurement can be done using FACS scan or automated reading devices in microplates. All light sources and optical equipment needed are well established and can be easily used for this method.

In basic research, for example for the examination of gene regulatory proteins for which a nuclear localization is a prerequisite of their action, this method could represent a reasonable alternative to existing methods, because of the higher throughput due to the automated analysis and the possibility to study the effects of a large number of substances on protein localization.

In general, due to the automated analysis and thus the ability to examine millions of cells/samples instead of dozens, an application of the method in large scale screening strategies could be extraordinarily useful.

Moreover, the present invention relates to a kit comprising at least two fusion proteins or derivatives thereof or nucleic acid, encoding upon expression said fusion proteins wherein one of said fusion proteins or derivatives thereof comprises (a) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a first portion of said compound; (b) an amino acid sequence or a non proteinaceous structure representing or comprising a targeting signal for a subcellular structure; and (c) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein further a second of said fusion proteins or derivatives thereof comprises (d) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a second portion of said compound wherein said first portion and said second portion are spatially distinct to allow the simultaneous interaction of said amino acid sequence or non-proteinaceous structure (a) and said amino acid sequence or non-proteinaceous structure (d) with said compound; (e) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal specific for the same subcellular structure as said amino acid sequence or non- proteinaceous structure (b); and (f) an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities.

The kit of the present invention in this as well as in the following embodiments is particularly useful for carrying out the method of the invention. The various components of the kit may be bottled in one or in more containers.

The present invention also relates to a kit comprising at least two fusion proteins or derivatives thereof or nucleic acid, encoding upon expression said fusion proteins wherein one of said fusion proteins or derivatives thereof comprises (a) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a first portion of a compound contained in or associated with one of said cells or subcellular structures; (b) an amino acid sequence or a non proteinaceous structure representing or comprising a targeting signal for a different cell or a subcellular structure; and (c) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein further a second of said fusion proteins or derivatives thereof comprises (d) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a second portion of said compound wherein said first portion and said second portion are spatially distinct to allow the simultaneous interaction of said amino acid sequence or non-proteinaceous structure (a) and said amino acid sequence or non-proteinaceous structure (d) with said compound; (e) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal specific for a different or the same cell or subcellular structure as said amino acid sequence or non-proteinaceous structure (b); and (f) an amino acid sequence or a non- proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; wherein the specificities of said targeting signals (b) and (e) may be interchanged.

Further, the present invention relates to a kit comprising at least two fusion proteins or derivatives thereof or nucleic acid, encoding upon expression said fusion proteins wherein one of said fusion proteins or derivatives thereof comprises (a) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a portion of a second fusion protein or derivative thereof; (b) an amino acid sequence representing or comprising a targeting signal for a cell or a subcellular structure; (c) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein further said second fusion protein or derivative thereof comprises (d) a portion capable of specifically interacting with said second amino acid sequence or non-proteinaceous structure (a); (e) an amino acid sequence or a non- proteinaceous structure representing or comprising a targeting signal specific for a different cell or subcellular structure as in (b); (f) an amino acid sequence or a non- proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities.

Moreover, the present invention relates to a cell into which the fusion proteins or derivatives thereof or the nucleic acid encoding said fusion proteins referred to in the above have been stably introduced. Furthermore, the invention relates to a cell comprising (a) a compound as described above and, optionally (b) at least one of the fusion protein(s) or derivative(s) thereof or the nucleic acid encoding said fusion protein(s) referred to herein above.

A preferred embodiment of the invention relates to a cell into which said compound and/or said fusion proteins or derivatives thereof or the nucleic acid encoding said fusion proteins have been stably introduced.

Moreover, the invention relates to a cell stably transfected with nucleic acid encoding at least two pairs of fusions proteins or derivatives thereof as described above wherein each pair of fusion proteins or derivatives thereof has a targeting signal that is specific for a different cell or subcellular structure as compared to the targeting signal of the other pairs of fusion proteins and wherein each pair of fusion proteins or derivatives thereof generates a signal that is different from any signal generated by the other pairs of fusion proteins.

Furthermore, the invention relates to a method of assessing the localization of a compound comprising introducing said compound into said cell of the invention and assessing the generation of a signal.

With the above cell and method, respectively, the localization of a compound such as a protein within a cell can conveniently be determined.

In addition, the invention relates to a vector encoding a nucleic acid molecule as specified above.

The contents of the various documents cited in this specification are herewith incorporated by reference.

The Figures show: Figure 1 : A. Schematic representation of a fusion protein that can be used in the presented method in conjunction with those of fig 1 B. and C. N: amino terminus; C: carboxy terminus; ET-1 : epitope tag #1 ; ET-2: epitope tag #2; Px: protein to be detected. In this case, epitope tag #1 is recognized by the single chain antibody variable region (scFv) #1 shown in fig. 1 B. Epitope tag #2 is structurally different from epitope tag #1 and is recognized by scFv #2 shown in fig. 1 C. As mentioned before, binding of scFv to epitope tags is a convenient way to effect highly specific and tight protein protein interactions. The two epitope tags shown here are fused to the amino terminus of the protein under investigation but carboxy terminal fusions are possible as well. The number and arrangement of the epitope tags may be varied. Instead of one or both epitope tags shown here, epitopes of Px may be used for the interaction with the fusion proteins shown in fig. 1 B. and C. It is important, that the epitope tags (or the epitopes of Px itself) are spatially close to each other for the signaling as described in the following to take place.

B. Schematic representation of a fusion protein that can be used in the presented method in conjunction with those of fig! 1A. and C. N: amino terminus; C: carboxy terminus; scFv-1 : single chain antibody variable region #1 ; NLS: nuclear localization signal; GFP-1 : green fluorescent protein #1. ScFv-1 is raised against epitope tag #1 of the fusion protein shown in fig. 1A.. The NLS directs the fusion protein to the nucleus and GFP-1 is a variant of the wild type green fluorescent protein which is excited by light of the wavelength λ1 and which emits light of the wavelength λ2, different from λ1.

C. Schematic representation of a fusion protein that can be used in the presented method in conjunction with those of fig. 1A. and B.. N: amino terminus; C: carboxy terminus; scFv-2: single chain antibody variable region #2; NLS: nuclear localization signal; GFP-2: green fluorescent protein #2. ScFv-2 is raised against epitope tag #2 of the fusion protein shown in fig. 1A.. The NLS directs the fusion protein to the nucleus and GFP-2 is a variant of the wild type green fluorescent protein which is excited by light of the wavelength λ3 and which emits light of the wavelength λ4, different from λ1 , λ2 and λ3, where λ2 and λ3 overlap, such that fluorescent resonance energy transfer (FRET) is possible upon close spatial arrangement of the two fusion proteins shown in fig. 1 B. and C. which is achieved by the simultaneous binding of both to their respective epitope tags in the fusion protein shown in fig. 1A.. This binding is possible if the fusion protein shown in fig. 1 A. is localized in the nucleus.

Figure 2: Flowchart of an experiment performed using the presented method. ET1- ET2-Px: fusion protein as shown in fig. 1A.; DetProtl : detection protein #1 , fusion protein as shown in fig. 1 B.; DetProt2: detection protein #2, fusion protein as shown in fig. 1C; C: cytoplasm; N: nucleus. Cells are transfected with nucleic acids encoding all three fusion proteins. The fusion protein ET1-ET2-Px is expressed constitutively and does or does not translocate to the nucleus upon stimulation (right panel and left panel, respectively). The expression of the fusion proteins DetProtl and DetProt2 is induced. As shown in figure 1 B. and C, they contain a nuclear localization signal and therefore will be localized in the nucleus. Only if the fusion protein ET1-ET2-Px has translocated to the nucleus upon stimulation, both the fusion proteins DetProtl and 2 will bind to their respective epitope tag, bringing the signaling entities (GFP-1 and GFP-2 as described in fig. 1 B. and C.) in close proximity such that fluorescent resonance energy transfer (FRET) can occur and, upon excitation of GFP- 1 , the emission wavelength of GFP-2 can be measured (right panel). Figure 3: Example of the use of the presented method for the detection of cell fusions. ET1 -ET1 : fusion of two interaction domains (or epitopes), each recognized by an interaction domain (e.g. scFv) included in DetProt-1 and DetProt-2; DetProt-1 : detection protein #1 , as shown in fig. 1 B. with the difference that no localization signal is present; DetProt-2: detection protein #2, as shown in fig. 1C. with the difference that no localization signal is present; C: cytoplasm; N: nucleus.

The fusion protein ET1-ET2 is expressed in one cell and will be present in the cytoplasm, since it does not contain a localization signal directing it to any organelle. The fusion proteins DetProt-1 and DetProt-2 are expressed in a second cell. Like the fusion protein ET1 -ET2, they will be localized in the cytoplasm. If the two cells fuse, DetProt-1 will bind to ET1 and DetProt- 2 will bind to ET2, bringing the signaling entities (GFP1 and GFP2) in close proximity to allow FRET between them. Thus, the emission wavelength of GFP2 will be measured upon excitation of GFP1. FRET will not be measured in the cell in which only DetProt-1 and -2 are expressed, since the distance between is too large for FRET to occur. For the detection of the fusion of two different subcellular structures (e.g. organelles), the fusion protein ET1-ET2 contains a targeting signal which directs it to the desired organelle. The fusion proteins DetProt-1 and -2 contain both the same targeting signal, directing them to another organelle. If both organelles fuse, the interaction of the fusion protein ET1-ET2 with the fusion proteins DetProt-1 and -2 is effected and a signal can be measured analogous to the signal obtained by the fusion of two cells described above.

Figure 4: Example of the use of the presented method for the detection of cell fusions. ET2: epitope tag #2; DetProt-1 : detection protein #1 , fusion protein consisting of a signaling entity as described before which in this application is fused to ET2; DetProt-2: detection protein #2, fusion protein as described in the legend of fig. 3; C: cytoplasm; N: nucleus. The fusion protein ET2-DetProt-1 is expressed in one cell and the fusion protein DetProt-2 is expressed in another cell. Upon the fusion of both cells, DetProt-2 binds to ET2, bringing the signaling entities of DetProt-1 and -2 in close proximity and a FRET signal can be measured. Analogously to the situation described in fig. 3, the fusion of two subcellular structures (e.g. organelles) can be detected by including a targeting signal specific for an organelle in the fusion protein ET2-DetProt-1 and including another targeting signal for a different organelle in the fusion protein DetProt-2.

Figure 5: Example of the use of the presented method for the detection of ligand- induced receptor intemalization. ET1-ET2-receptor: fusion protein consisting of a surface receptor and the two epitope tags described in fig. 1A.; DetProt-1 and -2: fusion proteins as shown in fig. 1 B. and C. with the difference that the NLS is replaced by a signal sequence directing them to endosomes; L: ligand specific for the receptor portion of ET1-ET2-receptor; M: cell membrane; E: endosome; C: cytoplasm.

A surface receptor is expressed as fusion with the epitope tags ET1 and ET2. Upon ligand binding, the receptor is internalized into an endosome. The fusion proteins DetProt-1 and -2 are found in the endosome, according to their localization signal. Binding of DetProt-1 and -2 to their respective epitope tags allows FRET between their signaling portions to be measured.

Figure 6: Example of the use of the presented method for the detection of signal sequences capable of targeting a heterologous protein to a predefined subcellular structure. ET1 : epitope tag #1 , interaction domain for the interaction domain of the second fusion protein; sign.seq.1 , 2, 3, 4, 5, signal sequences whose capability to direct a heterologous protein to a defined subcellular organelle is to be determined; GFP1 : signaling entity of fusion protein #1 ; scFv: single chain antibody as an example for an interacting protein domain; NLS: nuclear localization signal as an example for the targeting to the nucleus; GFP2: signaling entity of fusion protein #2; C: cytoplasm; N: nucleus.

The nucleic acids encoding fusion protein #2 and fusion protein #1 which carries one of several signal sequences, whose function is to be determined are transfected into a cell. The fusion protein #1 is synthesized and localizes in the compartment defined by (the functionality of) its signal sequence. In the example shown here, the signal sequences #3 and #5 are functional and direct the fusion protein to the nucleus. The expression of fusion protein #2 is induced and, after binding to fusion protein #1 , a discrete signal (e.g. by FRET) is emitted by the cells harboring functional signal sequences in their fusion protein #1 (#3 and #5). Again, the signal sequences to be characterized can be fused to two interaction domains (ET1 and ET2) and two fusion proteins as shown in fig. 1 B. and C. can be employed to generate the detectable signal if co- localized with the signal sequence-ET1/2 fusion protein.

Figure 7: Example of the use of the presented method for assessing the suitability of a compound to direct a further compound into a subcellular structure. DetProt-1 / -2: detection protein #1 and #2, fusion proteins corresponding to fig. 1 B. and C, which are capable to interact with two spatially close but distinct epitopes of compound 1 ; C: cytoplasm; N: nucleus. A compound (here: compound 2 and 3, respectively), whose capability to direct another compound (here: compound 1) into a subcellular structure (here: the nucleus) is introduced into a cell together with compound 1. Additionally, the fusion proteins 1 and 2 are transfected into the same cell. According to the capability of compound 1 and 2 to direct compound 1 to the nucleus, the fusion proteins 1 and 2 whose expression is induced and which are directed to the same subcellular structure by their NLS, will bind to compound 1 and generate a detectable signal. Compound 1 may be added to the cell or may be endogenous. It may be a pharmaceutical agent which e.g. leads to the correct (or desired) localization of a protein whose localization is to be altered.

Figure 8: An example for the use of the presented method for the detection of one or more amino acid sequences that interact with spatially distinct but closely arranged portions of a compound.

P1-4: portions one to four of the compound under investigation; ID1-6: interaction domains one to six, the interaction of which with the compound is to be determined; SE1/2: signaling entity one and two, respectively, where the signaling properties of SE2 are influenced upon close spatial arrangement with SE1.

The compound may be a protein, the ID may be a scFv and the SE a functional mutant of GFP, as described in the legend of fig. 1. Given that ID1 is known to interact with a portion of the compound (P1), the ID binding to a distinct but close portion of the compound is determined by assessing the signal of SE2. In the case of fluorescent signaling entities and FRET as a means of influencing the signaling properties of SE2 by SE1 , only ID2/SE2 will be detected, since the remaining IDs either bind at distant portions of the compound (ID3 and 4) or do not bind at all (ID5 and 6). By mixing the compound together with pairs of ID1/SE1 and one of ID2- 6/SE2, the detected ID can be isolated easily and characterized.

Figure 9: Example of the use of the presented method for the detection of ligands of cellular surface receptors.

L: ligand; R: receptor; SCM-1/-2/-x: members of the signaling cascade activated by the ligand-bound receptor; M: cell membrane; C: cytoplasm; N: nucleus.

A surface receptor gets activated by its ligand and transduces this signal into the cell. Members of a signal transduction cascade (here: SCM-1 and -2, e.g. SOS/Ras) pass the signal to another member of the cascade (here: SCM-x, e.g. a MAP kinase) which then translocates to another cellular compartment (here: the nucleus). This translocation is detected by two fusion proteins according to fig.1 B. and C, whose interaction domains (scFvl and 2 in fig. 1 B. and C.) recognize and bind to sequences on the protein that has entered the nucleus (SCM-x). This protein may as well contain one or more sequences of heterologous nature that interact with the interaction domains of the fusion proteins shown in fig. 1B. and C. The member of the signal transduction cascade which translocates may be a natural component of the cascade influenced by a given receptor or may be (part of) a heterologous signaling cascade which has been engeneered into the cell under analysis. The method shown here can be used to screen for ligands of e.g. receptor tyrosine kinases.

The examples illustrate the invention.

Example 1 : Design of a Targeting Construct

A targeting construct is prepared that comprises a fusion of the cDNA of the protein of interest (PX) with two different (sequentially) arranged epitope tags (ET1 and ET2). His-, FLAG-, HA1- or Myc-tags are well characterized epitope tags, against which monoclonal antibodies and partially (His, Myc) also scFv are available. In the following, some epitope tags and their amino acid sequences useful for incorporation into the targeting construct are listed:

FLAG DYKD(DDK) (Hopp, T.P. et al. (1988); Sassenfeld, H.M.

(1990); Knappik, A. and Plϋckthun, A. (1994)) Myc EQKLISEEDL (Munro, S. and Pelham, H.R.B. (1986); Ward,

E.S. et al. (1989)) KT3 TPPPEPET (Martin, G.A. et al. (1990, 1992)) α-tubulin peptide EEF (Skinner, R.H. et al. (1991 )) T7gene10peptide MASMTGGQQMGR (Rosenberg, A.H. et al. (1987); Lutz-

Freyermuth, C. et al. (1990))

(Strep SAWRHPQFGG detection with streptavidin; only C-terminal

(Schmidt, G.M.T. and Skerra, A. (1993)) SNWSHPQFEK C- and N-terminal)

The binding affinity of the mAb M1 to the FLAG peptide is strongly dependent on Ca²⁺ ions (Hopp, T.P. et al. (1996)). Thus, and as a further aspect of the present invention, it might be possible to develop a Ca²⁺ sensor using the method referred to above.

It has to be determined experimentally, which epitope tags are best and which order of ET1 , ET2 and PX is most suitable. In general, the following combinations are possible:

N-terminal fusions:

N-term-ET1 -ET2-PX-C-term

N-term-ET2-ET1 -PX-C-term C-terminal fusions:

N-term-PX-ET1 -ET2-C-term

N-term-PX-ET2-ET1 -C-term

It cannot be excluded that constructs which contain a PX flanked by the epitope tags could be suitable as well, although the distance between the epitope tags might be too large for efficient detection, preferably by FRET between the detection proteins. On the other hand, the increased distance might decrease possible steric hindrance and/or allow multiple detection proteins to bind to their respective epitope tags, thus increasing FRET efficiency. However, like this, the list of possible arrangements of the PX fusion protein will also contain the following orders:

N-term-ET1 -PX-ET2-C-term N-term-ET2-PX-ET1 -C-term It is possible that a PX fusion protein containing more than one of each epitope tag could lead to higher FRET efficiency, due to the binding of multiple detection proteins. On the other hand, this might lead to a decrease in FRET efficiency due to quenching effects. This further lengthens the list of possible orders of the PX fusion protein:

N-terminal fusions:

N-term-(ET1 )n-(ET2)n-PX-C-term

N-term-(ET2)n-(ET1 )n-PX-C-term

N-term-(ET1 -ET2)n-PX-C-term

N-term-(ET2-ET1 )n-PX-C-term

N-term-(ET1 )n-(ET2)n-(ET1 )n-(ET2)n -PX-C-term C-terminal fusions:

N-term-PX-(ET1 )n-(ET2)n-C-term

N-term-PX-(ET2)n-(ET1 )n-C-term

N-term-PX-(ET1 -ET2)n-C-term

N-term-PX-(ET2-ET1 )n-C-term

N-term-PX-(ET1 )n-(ET2)n-(ET1 )n-(ET2)n-C-term

Of course, the same operation can be done for PX fusion proteins having N- and C- terminally fused epitope tags.

To find out the optimal order of the epitope tags and PX, in a ligation reaction, all four components ET1 , ET2, PX and the plasmid expression vector can be mixed together as blunt-ended fragments (or staggered ended fragments, produced by the same enzyme or displaying the same overhangs, respectively). Although there will be an influence of the amount (which can be varied) of the single fragments added, there will be a reasonable chance to get circular plasmids consisting of many different orders of inserts. These can be further analyzed in terms of optimal suitability for allowance of FRET between the detection proteins. To reduce background of religated vector DNA and to direct the ligation reaction vaguely in one direction (e.g. get only N- or C-terminal fusions (by introducing a staggered 5'- or 3'-end in PX); or start or stop the insert with ET1 or ET2 (by introducing staggered 5'- ends or 3'-ends in ET1 or ET2; etc...)), one (or more) of the three inserts can bear a staggered end and the vector e.g. a staggered end and a blunt end. The DNA encoding the epitope tags is synthesized rather than excised from an existing plasmid. Thus, any appropriate restriction site can be introduced. However, all the mentioned factors have to be determined empirically. This is possible for the person skilled in the art without undue burden.

Furthermore, in accordance with the present invention, scFv against epitopes of the protein of interest itself are generated by phage display technology. In this case, scFv genes are generated either by immunization of mice with the protein of interest or by synthetic scFv libraries. A phage display is performed and phages containing binding scFv are collected. The scFv genes are fused to the GFP mutants and expressed. The resulting proteins are combined (as pairs) and assayed for FRET. The proteins displaying FRET can be used as detection constructs for the wild-type PX thus excluding any possible influence of the epitope tags on the (spatial) behavior of the protein of interest as well as the need to transfect the tagged protein into the cells. It i.s very convenient to measure FRET directly between the phages which display the scFv and which are bound to the protein of interest, enabling to select them not only for binding but also for possibility of FRET in one experiment. For this approach, the scFv are displayed as fusions with the GFP mutants. The display of GFP/surface protein- fusions has been shown by the art to be possible in baculovirus (Autographa californica nuclear polyhedrosis virus (AcNPV) (Mottershead, D. et al. (1997) and references therein: Schnell, M.J. et al. (1996) (reporting non-specific incorporation of heterologous membrane proteins on the surface of Spodoptera frugiperda insect cells into the vesicular stomatitis virus (VSV) particle; Boublik, Y. et al. (1995) (reporting fusion of glutathione-S-transferase or the HIV gp120 protein to the N-terminus of gp64 (of AcNPV) and incorporation into virus particles). Further examples of eucaryotic (peptide) display systems using animal viruses are: poliovirus: Rose, C. et al. (1994); rhinovirus: Resnick, D.A. et al. (1995); sindbis virus: London, S.D. et al. (1992) and using plant viruses are: cowpea mosaic virus: Porta, C. et al. (1994) and tobacco mosaic virus: Turpen, T.H. (1995)).There are further reports of the surface display of GFP in the potato virus X by fusion to the N-terminus of the coat protein (Oparka, K.J. et al. (1995); Santa Cruz, S. et al. (1996)). All of these prior art teachings may be usefully employed in the method of the invention.

Example 2: Generation of Detection Construct 1

The detection construct 1 is a fusion of the cDNAs of a scFv raised against ET1 (or against an epitope of the protein of interest itself) with a mutant of the green fluorescent protein (GFP1), possessing the excitation wavelength λ1 and the emission wavelength λ2. Additionally it contains a strong targeting signal, directing it to defined organelles. Its expression is inducible.

A lot of work has been done to improve GFP for expression in mammalian cells, to generate mutants with different spectral properties, improved brightness and/or FRET suitability and to generate mutants optimized for FACS analysis (Cormack, B.P. et al. (19^'96); Heim, R. and Tsien, R.Y. (1995); Heim R. et al. (1994); Mitra R.D. et al. (1995); Zolotukhin, S. et al. (1996); Yang, T.T. et al. (1996); Kimata, Y. et al (1997); Delagrave, S. et al, (1995); Yang, T.T. et al. (1998); Stauber, R.H. et al. (1998)). Mutants of GFP are commercially available, e.g. from CLONTECH (..Living Colors™") under license from Columbia University and from Quantum Biotechnologies („AutoFluorescent Proteins, AFPs™"). CLONTECH offers GFP mutants useful for the present invention, for example the N-terminal protein fusion expression vectors pEBFP- N1 , pEGFP-N1 , -N2, -N3 (for all three reading frames), pEYFP-N1 , pECFP-N1 , the C- terminal protein fusion expression vectors pEBFP-C1 , pEGFP-C1 , -C2, -C3, pEYFP-C1 , pECFP-C1 , the bicistronic expression vectors plRES-EGFP, plRES-EYFP and the selection marker vectors pHygEGFP, pNeoEGFP as well as vectors for the targeting of GFP mutants to different organelles/structures, for example pEGFP-F (farnesylated/plasma membrane), pE(G/Y)FP-Actin, pE(G/Y)FP-Tub(ulin), pE(C/Y)FP- Mito(chondria) and pE(C/Y)FP-Nuc(leus). Pairs of GFP mutants suitable for FRET are EGFP (enhanced green fluorescent protein, excitation: 488 nm, emission: 507 nm) / EYFP (enhanced yellow fluorescent protein, excitation: 513 nm, emission: 527 nm) and ECFP (enhanced cyan fluorescent protein, excitation: 433 and 453 nm, emission: 475 and 501 nm) / EYFP. The mutants excited in the blue spectrum might be considered less preferred because of the higher energy of blue light that could damage the cells under analysis. However, this risk is greatly reduced in the present method because the cells are not analyzed by fluorescence microscopy, leading to long exposure to the excitation wavelength, but by automated means, using an „excitation-flash" rather than a long exposure. So further pairs for FRET are EBFP (enhanced blue fluorescent protein, excitation: 380 nm, emission: 440 nm) / ECFP or EBFP / EGFP.

Fluorescence resonance energy transfer has been described for all of these pairs (Periasamy, A. et al. (1999); Biondi, R.M. (1998); Day, R.N. (1998); Miyawaki, A. et al. (1997); Mitra, R.D. et al. (1996); Heim, R. and Tsien, R. (1995); Mahajan, N.P. et al. (1998); Gordon, G.W. et al. (1998); Xu, X. et al. (1998); Prescott, M. et al. (1997)). It was found that the absorbance and fluorescence of GFP are pH dependent. However, for the GFP mutant S65T, the fluorescence spectral shape, lifetime and circular dichroic spectra were almost completely pH independent in the range of pH 5-8. It seems that down to a pH of approximately 5, the observed slight changes in the spectral properties were due to simple protonation events, while at pH <5 protonation and conformational changes seem to occur (Kneen, M. et al. (1998)).

There seems to be no fundamental problem to visualize GFP in plants. Autofluorescence of endogenous proteins either has different, non-overlapping spectral properties or is too weak to interfere with detection of GFP.

Example 3: Generation of Detection Construct 2

Analogous to detection construct 1 , the detection construct contains a fusion of a scFv raised against ET2 (or against an epitope of the protein of interest, selected for the possibility of FRET with detection construct 1) with a second mutant of the green fluorescent protein (GFP2) the excitation wavelength λ3 of which overlaps the emission wavelength λ2 of GFP1 and which has an emission wavelength λ4 distinguishable from λ1 and λ2. The construct contains the same targeting signal as detection construct 1 and the same inducible promoter.

Alternatively, the two detection constructs can be organized in a bicistronic manner under the control of an inducible promoter. This ensures the same expression modus for both detection constructs, thus reducing the risk of differences in expression due to surrounding sequences in cell lines which contain the detection constructs stably integrated into their genome and providing the desired 1 :1 ratio of expression of both detection constructs.

It is envisaged in accordance with the present invention that all bioluminescent/fluorescent proteins can be used as donors for FRET, including for example luciferases (oxygenases using molecular oxygen to oxidize a substrate (a luciferin) with the ultimate formation of a product molecule in an electronically excited state) or any protein using any sort of substrate which can be processed or not to produce light or which fluoresces upon excitation. Plants or bacteria might be a good source of light producing/emitting proteins/compounds (Murphy, J.T. and Lagarias, J.C. (1997)). It is also envisaged to include the enzyme in one of the detection products and the substrate in the other one. It is noteworthy that a luminescent protein can be divided in two non-functional parts, which, upon interaction of the scFv parts of the detection proteins with their epitopes come in sufficient proximity to restore the luminescent protein function (which then processes a substrate). (Analogous to the possibility to restore the binding of metothrexate by dehydrofolatreductase divided into two parts upon close spatial arrangement.) Alternatively, a fluorescent protein can be divided and regain its fluorescent properties upon interaction of the scFv parts of the detection proteins with their epitopes. In these cases, an interaction domain should be included (e.g. transient disulfide bond, dimerization interphase), to assure tight contact. This in turn calls for a mechanism to avoid interaction in the absence of the epitopes. This can be achieved by addition of sequences which lead e.g. to the complexion with heat shock proteins. Example 4: Detection of Protein Localization

The three constructs are transfected into cells, resulting in the synthesis of the respective fusion proteins after promoter induction. The detection proteins will be localized in an organelle according to their targeting signal (e.g. in the nucleus). Upon excitation with wavelength λ1 , only wavelength λ2 but not λ4 will be measured, if the protein of interest is not localized in the same compartment as the detection proteins. This is due to the strong decrease of FRET efficiency as a function of the sixth power of the distance between two fluorophores.

If the protein of interest is located in the same compartment as the two detection proteins, the detection proteins will bind with their scFv portions to the epitope tags, bringing the two GFP mutants in close proximity. Thus FRET is possible and upon excitation with λ1 , the emission wavelength λ4 of GFP2 will be detected. The measurement can be done using FACS scan or automated reading devices in microplates. All light sources and optical equipment needed are well established and can be easily used for this method.

References

Abe, H. et al. (1993) Biochim. Biophys. Acta 1216: 304-306

Amery, L et al. (1998) Biochem. J. 336: 367-371

Andersson, A.M. et al. (1997) J. Virol. 71 : 4717-4727

Beasley, E.M. et al. (1993) EMBO J. 12: 2303-2311

Biocca, S. et al. (1995) Biotechnology 13: 1110-1115

Biondi, R.M. et al. (1998) Nucleic Acids Res. 26(21): 4946-4952

Blagoveshchenskaya, A.D. et al. (1998A) J. Biol. Chem. 273: 2729-2737

Blagoveshchenskaya, A.D. et al. (1998B) J. Biol. Chem. 273: 27896-27903

Bos, K. et al. (1993) EMBO J. 12: 2219-2228

Boublik, Y. et al. (1995) Bio/Technology 13: 1079-1084

Brown, F.D. et al. (1998) Curr. Biol. 8: 835-838

Burd, C.G. and Emr, S.D. (1998) Mol. Cell 2: 157-162

Chalfie, M. et al. (1994) Science 263: 802-805

Chatterjee, S. and Stochaj, U. (1998) Biotechniques 24(4): 668-674

Chelsky, D. et al. (1989) Mol. Cell. Biol. 9: 2487-2492

Cody, C.W. et al. (1993) Biochemistry 32: 1212-1218

Cole, N.B. et al. (1996) Science 273: 797-801

Colley, K.J. et al. (1997) Glycobiology 7: 1 -13

Conrad, U. and Fiedler, U. (1998) Plant Mol. Biol. 38: 101 -109

Cormack, B.P. et al. (1996) Gene 173: 33-38

Crameri, A. et al. (1996) Nature Biotechnol. 14: 315-319

Day, R.D. (1998) Mol. Endocrin. 12: 1410-1419

Delagrave, S. et al. (1995) Biotechnology 13(2): 151 -154

Dingwall, C. and Laskey, R. (1991) Trends Biochem. Sci. 16: 478-481

Dingwall, C. et al. (1988) J. Cell. Biol. 107: 841-849

DiPaolo, G. et al. (1994) J. Biol. Chem. 272: 5175-5182

DiSansebastiano, G.P. et al. (1998) Plant J. 15: 449-457

Dodt, G. et al. (1995) Nat. Genet. 9: 115-125 Elgersma, Y. et al. (1995) EMBO J. 14: 3472-3479

Fiedler, U. et al. (1997) Immunotechnology 3: 205-216

Forbes, D.J. (1992) Annu. Rev. Cell Biol. 8: 495-527

Fuchs, P. et al. (1997), Hybridoma, 16 (3): 227-233

Galbraith, D.W. et al. (1995) Methods Cell Biol. 50: 1-12

Garrett, J.M. et al. (1991 ) Mol. Gen. Genet. 225: 483-489

Gartner, F. et al. (1995) J. Biol. Chem. 270: 3788-3795

Gerhardt, B. at al. (1998) J. Biol. Chem. 273: 15818-15829

Gordon, G.W. et al. (1998) Biophys. J. 74(5): 2702-2713

Griffin, B.A. et al. (1998) Science 289: 269-272

Haas, J. et al. (1996) Curr. Biol. 6: 315-324

Hall M.N. et al. (1984) Cell 36: 1057-1065

Heim R. et al. (1994) Proc. Natl. Acad. Sci. USA 91 : 12501 -12504

Heim, R. and Tsien, R.Y. (1995) Curr. Biol. 6: 178-182

Hicks, G.R. and Raikhel, N.V. (1995) Annu. Rev. Cell Dev. Biol. 11 : 155-188

Hόhfeld, J. (1991 ) J. Cell Biol. 114: 1167-1178

Hopp, T.P. et al. (1988) Bio/Technology 6: 1204-1210

Hopp, T.P. et al. (1996) Mol. Immunol. 33: 601-608

Huang, J. et al. (1990) Plant Cell 2: 1249-1260

Inouye, S. and Tsuji, F.I. (1994A) FEBS Letters 341 : 277-280

Inouye, S. and Tsuji, F.I. (1994B) FEBS Letters 351 : 211 -214

Jannot, C.B. and Hynes, N.E. (1997) Biochem. Biophys. Res. Comm. 230: 242-246

Juretic, N. and Theus, M. (1991 ) FEBS Lett. 290: 4-8

Kaether, C. et al. (1997) Eur. J. Cell Biol. 74: 133-142

Kalderon, D. et al. (1984) Nature 311 : 33-38

Kanazawa, M. et al. (1997) Biochem. Biophys. Res. Commun. 239: 580-584

Kendall, J.M. et al. (1992) Biochem. Biophys. Res. Commun. 189: 1008-1026

Kimata, Y. et al. (1997) Biochem. Biophys. Res. Commun. 232(1 ): 69-73

Klionsky, D.J. et al. (1992) J. Cell Biol. 119: 287-299

Knappik, A. and Plϋckthun, A. (1994) Biotechniques 17: 754-761

Kneen, M. et al. (1998) Biophys. J. 74: 1591-1599 Kohler, R.H. (1997) Plant J. 11 : 613-621

Lassner, M.W. et al. (1991 ) Plant Mol. Biol. 17: 229-234

Lindner, P. et al. (1997), BioTechniques 22: 140-149

Lippincott-Schwartz, J. et al (1998) Trends Cell Biol. 8: 16-20

Liu, J.W. et al. (1997) J. Cell Biol. 137: 1525-1535

Llopis, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95(12): 6803-6808

Locker, J.K. et al. (1994) J. Biol. Chem. 269: 28263-28269

London, S.D. et al. (1992) Proc. Natl. Acad. Sci. USA 89:207-211

Lutz-Freyermuth, C. et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6393-6397

Mahajan, N.P. et al. (1998) Nat. Biotechnol. 6: 547-552

Martin, G.A. et al. (1990) Cell 63: 843-849

Martin, G.A. et al. (1992) Science 255: 192-194

Martinez, E. et al. (1997) J. Mol. Biol. 267: 1124-1138

McNew, J.A. and Goodman, J.M. (1996) Biochem. Sci. 21 : 54-58

Mitra R.D. et al. (1995) Gene 173: 13-17

Miyawaki, A. et al. (1997) Nature 388: 882-887

Motley, A. et al. (1994) J. Cell Bio. 125: 755-767

Mottershead, D. et al. (1997) Biochem. Biophys. Res. Comm. 238: 717-722

Munro, S. and Pelham H.R.B. (1986) Cell 46: 291-300

Murphy, J.T. and Lagarias, J.C. (1997) Curr. Biol. 7(11 ): 870-876

Nothwehr, S.F. et al. (1993) J. Cell Biol. 121 : 1197-1209

Oda, M.N. et al. (1996) J. Cell Biol. 132: 999-1010

Odorizzi, C.G. et al. (1994) J. Cell Biol. 126: 317-330

Oparka, K.J. et al. (1995) Protoplasma 189: 133- 141

Ormό, M. et al. (1996) Science 273: 1392-1395

Pan, X: and Goldfarb, D.S. (1998) J. Cell Sci. 111 : 2137-2147

Periasamy, A. et al. (1999) Methods Cell. Biol. 58: 293-314

Persic, L. et al. (1997) Gene 187: 1-8

Porta, C. et al. (1994) Virology 202: 949-955

Prasher, D.C. et al. (1992) Gene 111 : 229-233

Prescott, M. et al. (1997) FEBS Lett. 411 (1): 97-101 Priller, J. et al. (1997) Ann. Neurol. 42: 265-269

Reits, E.A.J. et al. (1997) EMBO J. 16: 6087-6094

Resnick, D.A. et al. (1995) J. Virol. 69: 2406-2411

Rizzuto, R. et al. (1995) Curr. Biol. 5: 635-642

Robbins, J. et al. (1991) Cell 64: 615-623

Robinson, C. et al. (1998) Plant Mol. Biol. 1-2: 209-221 )

Rose, C. et al. (1994) J. Gen. Virol. 75: 969-977

Rosenberg, A.H. et al. (1987) Gene 56: 125-135

Santa Cruz, S. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 6286-6290)

Sassenfeld, H.M. (1990) Trends Biotechnol. 8: 88-93

Schafer, W. et al (1995) EMBO J. 14: 2424-2435

Schmidt, G.M.T. and Skerra, A. (1993) Protein Eng. 6: 109-122

Schnell, M.J. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 11359-11365

Schouten, A. et al. (1996) Plant Mol. Biol. 30: 781-793

Schouten, A. et al. (1997) FEBS Lett. 415: 235-241

Scott, S.V. and Klionsky, D.J. (1998) Curr. Opin. Cell Biol. 10: 523-529

Scott, S.V. et al (1997) J. Cell Biol. 138:37-44

Skinner, R.H. et al. (1991) J. Biol. Chem. 266: 14163-14166

Slawecki, M.L et al. (1995) J. Cell Sci. 108: 1817-1829

Small, G.M. et al. (1988) EMBO J. 7: 1167-1173

Solimena, M. et al. (1994) J. Cell Biol. 126: 331-341

Spada, S. et al. (1998), J. Mol. Biol. 283 (2): 395-407

Stauber, R.H. et al. (1998) Biotechniques 24(3): 462-466, 468-471

Stefan, C.J. and Blumer, K.J. (1999) J. Biol. Chem. 274: 1835-1841

Subramani, S. (1993) Annu. Rev. Cell Biol. 9: 445-478

Subramani, S. (1996) Curr. Opin. Cell Biol. 8: 513-518

Toby, G. et al. (1998) Biotechniques 24(4): 637-640

Tsukamoto, T. et al. (1991) Nature 350: 77-81

Tsukamoto, T. et al. (1995) Nat. Genet. 11 : 395-401

Turpen, T.H. (1995) Biotechnology 13: 53-57 van der Krol, A.R. and Chua, N.-H. (1991) Plant Cell 3: 667-675 Varagona, M.J. and Raikhel, N.V. (1994) Plant J. 2: 207-214 Via, L.E. et al. (1998) J. Cell Sci. 111 : 897-905 Wacker, I. et al. (1997) J. Cell Sci. 110: 1453-1463 Walten, P.A. (1995) Mol. Bio. Cell 6: 675-683 Walton, P.A. (1992) Mol. Cell. Biol. 12: 531 -541 Wang, S. and Hazelrigg, T. (1994) Nature 369: 400-403 Ward, E.S. et al. (1989) Nature 341 : 544-546

Wendland, M. and Subramani, S. (1993) J. Clin. Invest. 92: 1462-1468 Wiemer, E.A. et al. (1995) J. Cell. Biol. 130: 51-65 Wiemer, E.A.C. et al. (1996) J. Biol. Chem., (in press) Wilcox, CA. et al. (1992) Mol. Biol. Cell 3: 1353-1371 Wubbolts, R. et al. (1996) J. Cell Biol. 135: 611-622 Xu, X. et al. (1998) Nucleic Acids Res. 26(8): 2034-2035 Xue, Z. and Melese, T. (1994) Trends Cell Biol. 4: 414-417 Yang, F. et al. (1996) Nat. Biotechnol. 14(10): 1246-1251 Yang, T.T. et al. (1996) Nucleic Acids Res. 24(22): 4592-4593 Yang, T.T. et al. (1998) J. Biol. Chem. 273(14): 8212-8216 Yelin, R. et al. (1999) J. Bacteriol. 181 : 949-956 Zhan, J. et al. (1998) Cancer Immunol. Immunother. 46: 55-60 Zhang, J.W. and Lazarow, P.B. (1996) J. Cell Biol. 132: 325-334 Zhou, P. et al. (1998) J. Immunol. 160: 1489-1496 Zolotukhin, S. et al. (1996) J. of Virol. 70: 4646-4654

Claims

A method for the detection of a compound within a cell comprising the steps of: (a) introducing into a cell material comprising at least two fusion proteins or derivatives thereof or nucleic acid encoding said fusion proteins wherein one of said fusion proteins or derivatives thereof comprises

(aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a first portion of said compound;

(ab) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal for a subcellular structure; and

(ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein further a second of said fusion proteins or derivatives thereof comprises

(ad) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a second portion of said compound wherein said first portion and said second portion are spatially distinct to allow the simultaneous interaction of said amino acid sequence or non-proteinaceous structure (aa) and said amino acid sequence or non-proteinaceous structure (ad) with said compound;

(ae) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal specific for the same subcellular structure as said amino acid sequence or non- proteinaceous structure (ab); and

(af) an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; wherein said compound or a precursor thereof is present or expressed in said cell before said material is introduced into or expressed in said cell or wherein said compound or precursor is introduced into said cell simultaneously with or after the introduction of said material or wherein said compound is present in said cell per se;

(b) allowing expression of said nucleic acid, if applicable; and

(c) assessing for a signal from said first or second signaling entity that is provided, restored, altered or influenced.

A method for the detection of the fusion of two cells or two subcellular structures comprising the steps of:

(a) introducing into a cell material comprising at least two fusion proteins or derivatives thereof or nucleic acid encoding said fusion proteins wherein one of said fusion proteins or derivatives thereof comprises

(aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a first portion of a compound contained in or associated with one of said cells or subcellular structures;

(ab) an amino acid sequence or a non proteinaceous structure representing or comprising a targeting signal for a cell or a subcellular structure; and

(ad) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a second portion of said compound wherein said first portion and said second portion are spatially distinct to allow the simultaneous interaction of said amino acid sequence or non-proteinaceous structure (aa) and said amino acid sequence or non-proteinaceous structure (ad) with said compound; (ae) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal specific for a different or the same cell or subcellular structure as said amino acid sequence or non-proteinaceous structure (ab); and

(af) an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; wherein the specificities of said targeting signals (ab) and (ae) may be interchanged;

(b) allowing expression of said nucleic acid, if applicable; and

3. A method for the detection of the fusion of two cells or two subcellular structures comprising the steps of:

(aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a portion of a second fusion protein or derivative thereof;

(ab) an amino acid sequence representing or comprising a targeting signal for a cell or a subcellular structure;

(ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein further said second fusion protein or derivative thereof comprises

(ad) a portion capable of specifically interacting with said amino acid sequence or non-proteinaceous structure (aa); (ae) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal specific for a different cell or subcellular structure as in (ab);

(af) an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities;

(b) allowing expression of said nucleic acid, if applicable; and

A method for the detection of ligand-induced receptor intemalization comprising the steps of:

(ad) a portion capable of specifically interacting with said amino acid sequence or non-proteinaceous structure (aa);

(ae) an amino acid sequence or a non-proteinaceous structure representing or comprising a receptor or a portion of a receptor that is capable of interacting with a ligand; (af) an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities;

(b) allowing expression of said nucleic acid, if applicable; and

A method for assessing the suitability of a signal sequence or a non- proteinaceous compound to direct a further compound into a subcellular structure comprising the steps of:

(a) introducing into a cell a fusion polypeptide or derivative thereof comprising said signal sequence or said non-proteinaceous compound and said further compound, or a polynucleotide encoding said fusion polypeptide or derivative thereof comprising said signal sequence and said further compound, said cell comprising material comprising at least two fusion proteins or derivatives thereof or nucleic acid encoding said fusion proteins wherein one of said fusion proteins or derivatives thereof comprises

(aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a first portion of said compound, said non-proteinaceous compound or said signal sequence;

(ad) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a second portion of said compound, said non-proteinaceous compound or said signal sequence wherein said first portion and said second portion are spatially distinct to allow the simultaneous interaction of said amino acid sequence or non-proteinaceous structure (aa) and said amino acid sequence or non-proteinaceous structure (ad) with said compound;

(b) allowing expression of said nucleic acid and/or said polynucleotide, if applicable; and

A method for assessing the suitability of a polypeptide or a non-proteinaceous compound to direct a further compound into a subcellular structure comprising the steps of:

(a) introducing into a cell said polypeptide or said non-proteinaceous compound, or a polynucleotide encoding said polypeptide, said cell comprising said further compound or a nucleotide sequence encoding said further compound, and material comprising at least two fusion proteins or derivatives thereof or nucleic acid encoding said fusion proteins wherein one of said fusion proteins or derivatives thereof comprises (aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a first portion of said compound or said non-proteinaceous compound; (ab) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal for a subcellular structure; and

(b) allowing expression of said nucleic acid and/or said polynucleotide and/or said nucleotide sequence, if applicable; and

A method for the detection of a compound within a cell comprising the steps of: (a) introducing into a cell material comprising at least one fusion protein or derivative thereof or nucleic acid encoding said fusion protein wherein said fusion protein or derivative thereof comprises

(aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a portion of said compound;

(ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein said compound comprises or has attached thereto an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of either the first or the second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; wherein said compound or a precursor thereof is present or expressed in said cell before said material is introduced into or expressed in said cell or wherein said compound or precursor is introduced into said cell simultaneously with or after the introduction of said material or wherein said compound is present in said cell per se;

(b) allowing expression of said nucleic acid, if applicable; and

(c) assessing for a signal from said first or said second signaling entity that is provided, restored, altered or influenced.

8. A method for the detection of the fusion of two cells or two subcellular structures comprising the steps of: (a) introducing into a cell material comprising at least one fusion protein or derivative thereof or nucleic acid encoding said fusion protein wherein said fusion protein or derivative thereof comprises (aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a portion of a compound contained in or associated with one of said cells or subcellular structures;

(ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein said compound comprises or has attached thereto an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of either the first or the second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; wherein the specificities of said targeting signals (ab) and (ae) may be interchanged;

(b) allowing expression of said nucleic acid, if applicable; and

(a) introducing into a cell material comprising at least one fusion protein or derivative thereof or nucleic acid encoding said fusion protein wherein said fusion protein or derivative thereof comprises

(ab) an amino acid sequence representing or comprising a targeting signal for a cell or a subcellular structure; (ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein said cell comprises said second fusion protein or derivative thereof which comprises

(ae) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal specific for a different cell or subcellular structure as in (ab);

(b) allowing expression of said nucleic acid, if applicable; and

10. A method for the detection of ligand-induced receptor intemalization comprising the steps of:

(a) introducing into a cell material (i) comprising at least two fusion proteins or derivatives thereof or nucleic acid encoding said fusion proteins or (ii) one of said fusion proteins wherein the respective other fusion protein is contained in said cell, wherein one of said fusion proteins or derivatives thereof comprises

(ab) an amino acid sequence representing or comprising a targeting signal for a cell or a subcellular structure; (ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein further said second fusion protein or derivative thereof comprises

(ae) an amino acid sequence or a non-proteinaceous structure representing or comprising a receptor or a portion of a receptor that is capable of interacting with a ligand;

(b) allowing expression of said nucleic acid, if applicable; and

11. A method for assessing the suitability of a signal sequence or a non- proteinaceous compound to direct a further compound into a subcellular structure comprising the steps of:

(a) introducing into a cell a fusion polypeptide or derivative thereof comprising said signal sequence or said non-proteinaceous compound, said further compound and a first signaling entity, or a polynucleotide encoding said fusion polypeptide or derivative thereof comprising said signal sequence, said further compound and said first signaling entity, said cell comprising material comprising at least one fusion protein or derivative thereof or nucleic acid encoding said fusion protein wherein said fusion protein or derivative thereof comprises

(aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a portion of said compound, said non- proteinaceous compound or said signal sequence; (ab) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal for a subcellular structure; and

(ac) an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity; wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities;

12. A method for assessing the suitability of a polypeptide or a non-proteinaceous compound to direct a further compound into a subcellular structure comprising the steps of:

(a) introducing into a cell said polypeptide or said non-proteinaceous compound, or a polynucleotide encoding said polypeptide, said cell comprising said further compound and a first signaling entity or a nucleotide sequence encoding said further compound and said first signaling entity, and material comprising at least one fusion protein or derivative thereof or nucleic acid encoding said fusion protein wherein said fusion protein or derivative thereof comprises

(aa) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a portion of said compound or said non-proteinaceous compound;

13. A method for the detection of one or more amino acid sequences or non- proteinaceous structures that interact with spatially distinct but closely arranged portions of a compound comprising

(a) contacting said compound with at least two of said amino acid sequences or non-proteinaceous structures under conditions that allow an interaction to take place wherein

(aa) one of said amino acid sequences or non-proteinaceous structures is connected with an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; and

(ab) a second of said amino acid sequences or non-proteinaceous structures is connected with an amino acid sequence or a non- proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; and

(b) assessing for a signal from said first or second signaling entity that is provided, restored, altered or influenced.

14. The method of claim 13 further comprising producing said one or more detected amino acid sequences or non-proteinaceous structures.

15. The method of claim 13 or 14 wherein said amino acid sequences (aa) and (ab) are further connected to amino acid sequences or non-proteinaceous structures representing or comprising targeting signals for the same or different cells or the same or different subcellular structures.

16. A method for mapping epitopes comprising

(a) contacting a compound under investigation for epitope mapping with two different amino acid sequences or non-proteinaceous structures wherein

(aa) the first of said amino acid sequences or non-proteinaceous structures is connected with an amino acid sequence or a non- proteinaceous structure representing or comprising a first signaling entity; and

(ab) the second of said amino acid sequences or non-proteinaceous structures is connected with an amino acid sequence or a non- proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities.

17. The method of claims 1 , 2, 7 or 8 wherein either the first or second portion of said compound has a stage-specific conformation whereas the other portion has a non-stage-specific conformation.

18. The method of claim 1 , 2, 7 or 8 wherein both the first and second portion of said compound either have a stage-specific conformation or a non-stage-specific conformation.

19. The method of any one of claims 1 to 18 wherein said first and second signaling entity are an enzyme and its substrate.

20. The method of any one of claims 1 to 18 wherein said first and second signaling entity represent a luminescent entity.

21. The method of claim 20 wherein said luminescent entity exhibits a fluorescent, phosphorescent, chemiluminescent or bioluminescent activity.

22. The method of claim 20 or 21 wherein said first signaling entity has an excitable luminescent activity with an excitation wavelength λ1 and an emission wavelength λ2 and wherein said second signaling entity has an excitable luminescent entity with an excitation wavelength λ3 and emission wavelength λ4 wherein said emission wavelength λ2 and said excitation wavelength λ3 overlap, wherein further λ4 is distinguishable from λ1 and λ2, wherein further emission wavelength λ4 is detectable upon triggering excitation of λ1 and upon the close spatial arrangement of said first and said second luminescent entity.

23. The method of any one of claims 20 to 22 wherein said first and second signaling entities are functional mutants; fragments or derivatives of Green Fluorescent Protein.

24. The method of any one of claims 1 to 23 wherein said fusion proteins are members of an expressed nucleic acid library.

25. The method of any one of claims 1 to 18 wherein said introduction into said cells is effected by transfection.

26. The method of any one of claims 1 to 25 wherein said compound is a protein or a linker.

27. The method of claim 26 wherein said protein is a member of a signaling cascade or a gene regulatory protein.

28. The method of any one of claims 1 to 25 wherein said compound or said non- proteinaceous structure is a carbohydrate, a lipid, a steroid, a vitamin, a phospholipid, a nucleic acid, a DNA or a pharmaceutically active agent.

29. The method of any one of claims 1 to 28 wherein said first and/or second portion of said compound is a tag.

30. The method of any one of claims 1 to 28 wherein said first and/or second portion of said compound are portions naturally occurring in said compound.

31. The method of any one of claims 1 to 30 wherein said amino acid sequence capable of specifically interacting with said portions of said compound or said second fusion protein are derived from or represent antibody variable regions, protein-protein interaction domains, receptor-ligand systems or enzyme substrate systems.

32. The method of claim 31 wherein said amino acid sequences comprise single- chain Fv fragments.

33. The method of any one of claims 1 to 32 wherein said first and said second fusion protein are encoded by the same nucleic acid molecule.

34. The method of claim 33 wherein said nucleic acid molecule is a bicistronic vector.

35. The method of any one of claims 1 to 32 wherein said first and said second fusion protein are encoded by the different nucleic acid molecules.

36. The method of any one of claims 1 to 35 wherein said expression is inducible.

37. The method of any one of claims 1 to 15 and 17 to 36 wherein said subcellular structure is the nucleus, nucleolus, cytoplasm, cytoskeleton, chromatin, a mitochondrion, a microtubulus, a centriole, a nuclear pore, a ribosome, a microfilament, a perixosome, a proteasome, a lysosome, vacuole, chloroplast, thylakoid, membrane, the Golgi apparatus or the endoplasmatic reticulum.

38. The method of any one of claims 1 to 37 wherein at least said step of assessing is effected by using a high throughput system.

39. Kit comprising at least two fusion proteins or derivatives thereof or nucleic acid, encoding upon expression said fusion proteins wherein one of said fusion proteins or derivatives thereof comprises

(a) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a first portion of said compound;

(b) an amino acid sequence or a non proteinaceous structure representing or comprising a targeting signal for a subcellular structure; and

(c) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein further a second of said fusion proteins or derivatives thereof comprises

(d) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a second portion of said compound wherein said first portion and said second portion are spatially distinct to allow the simultaneous interaction of said amino acid sequence or non- proteinaceous structure (a) and said amino acid sequence or non- proteinaceous structure (d) with said compound;

(e) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal specific for the same subcellular structure as said amino acid sequence or non-proteinaceous structure (b); and

(f) an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities.

40. Kit comprising at least two fusion proteins or derivatives thereof or nucleic acid, encoding upon expression said fusion proteins wherein one of said fusion proteins or derivatives thereof comprises

(a) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a first portion of a compound contained in or associated with one of said cells or subcellular structures;

(b) an amino acid sequence or a non proteinaceous structure representing or comprising a targeting signal for a different cell or a subcellular structure; and

(e) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal specific for a different or the same cell or subcellular structure as said amino acid sequence or non-proteinaceous structure (b); and

(f) an amino acid sequence or a non-proteinaceous structure representing or comprising a second signaling entity, wherein the signaling properties of said first or second signaling entity are provided, restored, altered or influenced upon close spatial arrangement of said first and said second signaling entities; wherein the specificities of said targeting signals (b) and (e) may be interchanged.

41. Kit comprising at least two fusion proteins or derivatives thereof or nucleic acid, encoding upon expression said fusion proteins wherein one of said fusion proteins or derivatives thereof comprises

(a) an amino acid sequence or a non-proteinaceous structure capable of specifically interacting with a portion of a second fusion protein or derivative thereof;

(b) an amino acid sequence representing or comprising a targeting signal for a cell or a subcellular structure;

(c) an amino acid sequence or a non-proteinaceous structure representing or comprising a first signaling entity; wherein further said second fusion protein or derivative thereof comprises

(d) a portion capable of specifically interacting with said second amino acid sequence or non-proteinaceous structure (a);

(e) an amino acid sequence or a non-proteinaceous structure representing or comprising a targeting signal specific for a different cell or subcellular structure as in (b);

42. A cell into which the fusion proteins or derivatives thereof or the nucleic acid encoding said fusion proteins referred to in any of the preceding claims have been stably introduced.

43. A cell comprising (a) a compound as described in any of the preceding claims and, optionally (b) at least one of the fusion protein(s) or derivative(s) thereof or the nucleic acid encoding said fusion protein(s) referred to in any of the preceding claims.

44. The cell of claim 43 into which said compound and/or said fusion proteins or derivatives thereof or the nucleic acid encoding said fusion proteins have been stably introduced.

45. A cell stably transfected with nucleic acid encoding at least two pairs of fusions proteins or derivatives thereof as described in any of the foregoing claims wherein each pair of fusion proteins or derivatives thereof has a targeting signal that is specific for a different cell or subcellular structure as compared to the targeting signal of the other pairs of fusion proteins and wherein each pair of fusion proteins or derivatives thereof generates a signal that is different from any signal generated by the other pairs of fusion proteins.

46. A method of assessing the localization of a compound comprising introducing said compound into said cell of claim 43 and assessing the generation of a signal.

47.- A vector encoding a nucleic acid molecule as specified in any of the preceding claims.