WO2003038092A1

WO2003038092A1 - Method for identifying transport proteins

Info

Publication number: WO2003038092A1
Application number: PCT/AU2002/001480
Authority: WO
Inventors: Eugene J. Kim; Laurence Barker; Michael Burnet; Jan-Hinrich Guse; Kattie Luyten; Georgia Tsotsou
Original assignee: SYMPORE GmbH
Current assignee: SYMPORE GmbH
Priority date: 2001-11-01
Filing date: 2002-11-01
Publication date: 2003-05-08
Anticipated expiration: 2004-05-01

Abstract

The present invention relates to methods for identifying proteins with transport activities and methods of screening for compounds that interact with the transport proteins. In one aspect, the present invention relates to a method for identifying a transport protein, the method comprising: (i) transforming yeast cells with a library of expression constructs, wherein one or more of the expression constructs comprises a polynucleotide encoding a putative transport protein; (ii) contacting the transformed yeast cells with a fluorescent substrate for the transport protein under conditions such that cells comprising an expression construct encoding a transport protein express the transport protein and thereby bind or internalise the fluorescent substrate; and (iii) isolating transformed yeast cells that exhibit uptake of the fluorescent substrate.

Description

Method for identifying transport proteins

Field of the Invention

The present invention relates to methods for identifying proteins with transport activities and to method of screening for compounds that interact with the transport proteins.

Background of the Invention

Transport proteins are membrane proteins that catalyse the passage of molecules from one face of a membrane to another. These proteins occur in many forms and in all types of biological membranes. Each different protein is designed to transport a different class of chemical compounds (such as ions, sugars, or amino acids) and often only a specific molecular species of the class. The specificity of transport proteins was first indicated by studies in which single gene mutations were found to abolish the ability of bacteria to transport specific sugars across their plasma membranes. Similar mutations have now been discovered in humans suffering from a variety of inherited diseases affecting the transport of a specific solute in the kidney or intestine. Transport systems are of particular interest to the pharmaceutical industry in view of their potential involvement in cellular drug delivery or as targets for drug action. There are two main transport systems involved in the delivery of pharmaceutical agents into and through cells: Carrier mediated systems and receptor mediated systems. Carrier mediated systems are effected by transport proteins that are anchored to the cell membrane and function by transporting their substrates via an energy dependent flip-flop mechanism. Carrier proteins are involved in the transport of many important nutrients such as vitamins, sugars and amino acids and are typically involved in the transport of such molecules from the lumen to the intestine into the systemic circulation or across the blood brain barrier. Receptor-mediated transport systems differ from the carrier-mediated systems in that substrate binding triggers an encapsulation process that results in the formation of various transport vesicles to carry the substrate into and through the cell. Receptor mediated systems are capable of transporting a variety of compounds, including immunoglobulins, lectins, vitamins and metal ions. While novel transport proteins are clearly of interest, compounds that interact with these transporters are of particular importance to the pharmaceutical industry. For example, compounds that act as substrates for a transporter can be linked to a therapeutic agent in order to enhance uptake of the therapeutic agent by cells. Compounds that inhibit the activity of transport proteins are also useful pharmaceutical agents. For example, inhibitors of nutrient transporters can be used in the treatment of obesity or diabetes. Accordingly, a method that enables both the cloning of novel transport proteins and the screening of compounds that interact with these transport proteins would be particularly useful.

Schaffer and Lodish (1994) Cell 79: 427-436 describe a process for identifying transporters of long chain fatty acids. This process involves screening a cDNA library in mammalian (COS7) cells for uptake of a fluorescently labelled dodecanoic acid. The cells are sorted by a fluorescence- activated cell sorter and the plasmid cDNA recovered from the sorted cells and amplified in bacteria. However, this method suffers from the inherent disadvantages of using mammalian cells in cloning procedures. In addition, a highly targeted substrate was employed in this method which may have precluded the identification of transporters with broad substrate specificities, which are useful for transporter-mediated drug delivery. Furthermore, this publication does not teach or suggest a method of screening for compounds that modulate the activity of the cloned transporter.

Summary of the Invention The present inventors have now developed a high throughput method that allows for rapid identification of transport proteins. In a preferred embodiment, the method also enables screening for compounds that modulate the activity of the cloned transport proteins.

Accordingly, in a first aspect the present invention provides a method for identifying a transport protein, the method comprising:

(i) transforming yeast cells with a library of expression constructs, wherein one or more of the expression constructs comprises a polynucleotide encoding a putative transport protein; (ii) contacting the transformed yeast cells with a fluorescent substrate for the transport protein under conditions such that cells comprising an expression construct encoding a transport protein express the transport protein and thereby bind or internalise the fluorescent substrate; and

(iii) isolating transformed yeast cells that exhibit uptake of the fluorescent substrate. It will be appreciated that uptake of the fluorescent substrate by the transformed cells may be monitored by any suitable means. For example, if the transformed cells are pooled together and the fluorescent substrate is not quenched, cells which exhibit an increased internal fluorescence are indicative of uptake of the fluorescent substrate. Alternatively, if individual transformants or small groups of transformed cells are isolated in solution, the internalised fluorescence may be quenched and the fluorescence remaining in the solution may be counted, in which case a decrease in the total fluorescence in the original solution is indicative of the amount taken up by the isolated cells.

In a preferred embodiment of the first aspect, cells that exhibit uptake of the fluorescent substrate are detected by altered fluorescence properties.

More preferably, cells that exhibit uptake of the fluorescent substrate are detected by enhanced fluorescence within the cell when compared to untransformed yeast cells.

In a further preferred embodiment of the first aspect, the cells are sorted in step (iii) by fluorescence activated cell sorting (FACS).

In a further preferred embodiment of the first aspect, steps (ii) and (iii) are repeated in order to achieve further enrichment of the cells expressing the transport protein.

It will be appreciated by those skilled in the art that the polynucleotide encoding the transport protein may be further isolated from the enriched population of cells and characterised by, for example, sequencing.

In a further preferred embodiment of the first aspect, the method further comprises:

(iv) using the transformed cells from step (iii) in a screening process to identify one or more compounds that interact with the transport protein expressed by the cells.

In a further preferred embodiment, the screening process in step (iv) comprises the following steps:

(a) contacting the transformed yeast cells or a population of cells derived therefrom with a fluorescent substrate and a compound; and (b) monitoring the uptake of the fluorescent substrate by the yeast cells in the presence of the compound.

In a further preferred embodiment of the screening process, the population of yeast cells is washed after step (a) to remove residual fluorescent substrate from the cell composition.

In a further preferred embodiment of the first aspect, the method further comprises introducing into the transformed cells an expression construct encoding a transport protein enhancer.

In a further preferred embodiment of the method of the first aspect, each expression construct comprises a polynucleotide selected from the group consisting of a cDNA, genomic DNA or synthetic DNA molecule. Preferably, the polynucleotide is operably linked to one or more regulatory regions. The regulatory region may be a promoter or enhancer element that drives expression of the polynucleotide. In a further preferred embodiment of the first aspect, the yeast cells are genetically modified, for example, in order to reduce background transport activity and/or to reduce background fluorescence; enhance the fluorescent signal of the transformed cells; enhance plasma membrane targeting ability; enhance translation ability; alter intracellular post-translational modification; or enhance the ability of the cell to accept foreign DNA.

In a second aspect the present invention provides a kit for isolating a polynucleotide encoding a transport protein and screening for a compound which interacts with the transport protein, the kit comprising a fluorescent substrate for a transport protein. In a preferred embodiment of the second aspect, the kit further comprises a standard for determining background fluorescence. Preferably, the standard represents the level of fluorescence accumulation in a yeast strain exposed to the fluorescent substrate, where the yeast strain does not express the transport protein. In a third aspect the present invention provides a method for identifying a transport protein, the method comprising:

(i) transforming yeast cells with a library of expression constructs, wherein one or more of the expression constructs comprises a polynucleotide encoding a putative transport protein; (ii) incorporating into the transformed yeast cells a fluorescent indicator under conditions such that cells comprising an expression construct encoding a transport protein express the transport protein and thereby import or export a substrate that alters a signal generated by the fluorescent indicator; and

(iii) isolating transformed yeast cells that exhibit an altered signal generated by the fluorescent indicator when compared to untransformed yeast cells comprising the fluorescent indicator.

In a preferred embodiment of the third aspect, the method further comprises the step of

In yet a further preferred embodiment, the screening process in step (iv) comprises the following steps:

(a) contacting the transformed yeast cells comprising the fluorescent indicator with a compound; and (b) monitoring the change in signal generated by the fluorescent indicator in the presence of the compound.

In a further preferred embodiment of the third aspect, the fluorescent indicator is a ratiometric indicator.

More preferably, the fluorescent indicator is selected from the group consisting of indo-1 , BCECF, SBFI, cameleon, FLIP, Carboxy SNARF-1 , SPQ,

MQAE, MEQ, Lucigenin, di-4-ANEPPS, di-8-ANEPPS, DiOC2, JC-1 , JC-9, mag-indo-1 , FuraZin-1 , lndoZin-1 and FluoZin-1.

In a fourth aspect the present invention provides a method for identifying a transport protein enhancer, the method comprising: (i) transforming yeast cells which express a transport protein with a library of expression constructs, wherein one or more of the expression constructs comprises a polynucleotide encoding a putative transport protein enhancer;

(ii) contacting the transformed yeast cells with a fluorescent substrate for the transport protein under conditions such that the cells express the transport protein and putative enhancer and the transport protein binds or internalises the fluorescent substrate; and

(iii) isolating transformed yeast cells that exhibit enhanced uptake of the fluorescent substrate when compared to yeast cells that express the transport protein but not the putative enhancer. In a fifth aspect the present invention provides a method for identifying a transport protein enhancer, the method comprising:

(i) transforming yeast cells which express a transport protein with a library of expression constructs, wherein one or more of the expression constructs comprises a polynucleotide encoding a putative transport protein enhancer;

(ii) incorporating into the transformed yeast cells a fluorescent indicator under conditions such that the cells express the transport protein and putative enhancer and the transport protein imports or exports a substrate that alters a signal generated by the fluorescent indicator; and

(iii) isolating transformed yeast cells that exhibit an altered signal generated by the fluorescent indicator when compared to yeast cells that express the transport protein but not the putative enhancer.

The method of the present invention provides an improved system for the rapid cloning and identification in yeast cells of novel transport proteins, and the immediate use of those yeast cells in high throughput screens for compounds that interact with the transport protein. In a preferred embodiment of the invention, FACS is used to identify and isolate yeast cells expressing the transport proteins of interest. While FACS has been used in the past to identify transport proteins expressed by mammalian cells, the present invention represents an advance which improves the ease, speed and economy of transporter identification.

Because yeast cells are smaller and have rigid cell walls, they can be sorted much more efficiently by FACS. Importantly, a much higher percentage of yeast cells is expected to survive passage through the flow cytometer compared to mammalian cells. This makes it possible to grow collected cells directly after sorting and then perform the enrichment process without the need to isolate the cloned DNA. It also makes it possible to use the sorted cells, once sufficiently pure, directly in a screening process to identify compounds that interact with the cloned transport protein.

The screening process is also more conveniently carried out using yeast cells. Unlike animal cells, the yeast cells do not have to be grown directly on the plate where the assay is to be performed. This means that the cells can be grown together, processed and then placed into the wells of a microtiter plate, ensuring a high level of consistency in cell properties and numbers. In addition, the diversity of biomolecules is such that it would be excessively laborious to mimic them all as opposed to mimicking important substructures and cloning the activity with a general substrate. Furthermore, the method is amenable to screening mixtures or combinations of substrates with compatible fluorescent reporters, which overcomes the necessity to provide substrates with nutritional, electrogenic, radiolabeled or regulatory properties normally necessary to clone or assay transport activities.

A key benefit of the present method is that there is no a priori need for knowledge of the selectivity of the required transport activity. The ability to use diverse compounds and detect low level transport events allows the isolation of transporters with sub-optimal substrates.

In addition, for the purposes of drug delivery through transport systems, it is often preferable to have knowledge of transporters of broad substrate specificity. In the context of the present invention, the fluorescent substrate or fluorescent indicator may be much more speculative with respect to the transporter, resulting in the identification of transport processes of unknown physiological function. The preferred methods of the present invention are also highly sensitive and can detect weak transport events, for example that of a fluorescent drug derivative. As will be appreciated by those skilled in the art, structural information relating to the new transport proteins identified by the method of the present invention will be useful in designing pharmaceutical agents that bind or are transported by these proteins. In addition, the present invention allows for the identification of substrates and/or ligands for the new transport protein. Substrates identified by these methods can potentially be linked to pharmaceutical agents in order to facilitate uptake of the pharmaceutical agents by a patient. Substrates for receptor-type transporters, for example, can be linked to a particle containing a pharmaceutical agent to form a pharmaceutical composition. Other ligands that are not themselves substrates are also useful. For example, such ligands may promote uptake of a substrate linked to the ligand. This may be effected where the ligand binds to a cellular outer surface molecule in proximity to a carrier or receptor for the substrate thereby bringing the substrate into contact with the carrier or receptor. Ligands which act as inhibitors of the new transport protein may also be useful as pharmaceutical agents. Brief description of the Figures

Figure 1 : Outline of a preferred transporter cloning strategy

Figure 2: Outline of a preferred screening method

Figure 3: Chemical Structures of preferred fluorescent substrates.

Figure 4: Results of FACS based enrichment of transformed yeast cells accumulating increased amount of fluorescent substrate.

Figure 5: Results of FACS based enrichment of yeast cells transformed with a genomic library of C. albicans, where after each round of FACS, the number of cells able to utilise dipeptides (as reflected by growth on selective medium) increases. Confirmation of the presence of peptide transporter was performed by PCR using primers against the CaPTR2 peptide transporter gene from C. albicans.

Detailed description of the Invention

Definitions

A "transport protein" is a protein that has a direct or indirect role in transporting a molecule into and/or through a cell. The term includes, for example, membrane-bound proteins that recognise a substrate and effect its entry into a cell by a carrier mediated transporter or by a receptor mediated transporter.

A compound that "interacts with" the transport protein includes but is not limited to compounds that (i) are directly transported by the transport protein; (ii) inhibit transport by the transport protein without being transported themselves; and (iii) enhance transport by the transport protein. A compound that interacts with the transport protein may be a ligand or a substrate for the transport protein.

A "substrate" of a transport protein is a compound whose uptake or passage through a cell is facilitated by the transport protein. A "ligand" of a transport protein includes substrates and other compounds that bind to the transport protein.

An "inhibitor" of a transport protein is a compound that binds to a transporter such that transport of the inhibitor and any ligand or substrate is reduced.

A "polynucleotide" may comprise DNA or RNA and may be single- stranded or double-stranded. The term encompasses polynucleotides which include within them synthetic or modified nucleotides.

The term "operably linked" means that the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under a condition compatible with the regulatory sequence.

"Fluorescent substrates" used in this invention is comprised of two components: a fluorescent or luminescent moiety (called 'reporter') and a 'transport tag', which may be any putative compound that has the potential to interact with the transport protein.

"Fluorescent indicators" used in this invention refer to fluorescent compounds whose optical properties change upon specific binding of substrates, thereby serving as reporters for concentration of their target substrates.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

General Techniques

Unless otherwise indicated, the recombinant DNA techniques utilised in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991 ), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (Editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present) and are incorporated herein by reference.

Polynucleotides

The polynucleotides used in the expression constructs are preferably derived from cDNA libraries or genomic libraries. The libraries may be derived from plants (Arabidopsis, Zea, Oryza), microorganisms (bacteria, fungi) or animals (humans, mice, rats, and cultured cells derived from animals), and may be derived from specific tissues and cell types (eg. intestine, liver, kidney, spleen, lung, heart, testis, skeletal muscle, bone, bone marrow, mucosa, pancreas, colon, breast, derma, endothelium, epithelium, myeloid tissue, connective tissue, erythrocytes, leukocytes, macrophage, cartilage, brain organs or brain tissue). The polynucleotides may also be derived from parasites and pathogens (eg. fungi, nematodes, insects, or blood-borne pathogens); abnormal human cells and tissues (eg. tumours, cysts, pulmonary tubercules); or cells that have been treated with a chemical/environmental inducer (eg. cytokines, hormones, drugs, lipopolysaccharides). Alternatively, the polynucleotides may constitute a family of mutant sequences generated, for example, by site directed mutagenesis, random mutagenesis, directed DNA shuffling, error prone PCR, of known transport protein sequences, vectors, promoters, regulators or hosts.

Expression constructs

An expression construct preferably comprises a polynucleotide comprising a coding sequence operably linked to a regulatory sequence that is capable of providing for the expression of the coding sequence.

The regulatory sequence may be, for example, a promoter, enhancer, transcription termination sequence or any other nucleic acid sequence that influences gene expression.

The promoter is preferably selected on the basis of the host cell to be used in the method of the present invention. For expression in yeast, exemplary promoters include GAL1, PH05, PMA1 and CUP1. In addition, it is possible to take advantage of the wealth of information concerning mRNA and protein expression levels for various yeast genes (see, for example, Gygi et al. (1999) Mol. Cell. Biology 19:1720-1730; and Velculescu et al. (1997) Cell 88:243-251 ) in order to achieve higher and more controlled expression of cloned transport proteins. Post-translational mechanisms controlling gene expression, such as translational control and control of protein half life, may be manipulated in order to achieve improved expression of cloned transporters. Information relating to genes that are known to be highly expressed, such as TDH2, TEF2; EN02 and PDC1, may be useful in this regard. In particular, promoters or enhancer signals derived from these genes may be used in the expression constructs used to clone transport proteins. Preferably, the expression construct is in the form of a vector. The vector may be for example, a plasmid capable of replicating in yeast cells. The vector may contain one or more selectable marker genes, for example, URA3, HIS3 or LEU2. Antibiotic markers can also be employed. In cases where it is desirable to identify transporters endogenous to yeast, the vector is preferably capable of integrating into the genome of the cells. The integrated vector may also contain selectable marker genes, as well as regulatory sequences which may influence expression of genes near the site of integration.

Modification of yeast cells The use of yeast cells in the method of the present invention provides improved flexibility in terms of manipulating the host cell for the purposes of achieving improved assay/screening results.

It may also be possible to achieve more efficient targeting of transport proteins to the plasma membrane. Improved targeting may be important because in some cases, heterologous transporters are mistargeted to internal membranes when expressed in yeast. In addition, as it is feasible to alter the location of membrane proteins in yeast, it is possible to develop simple assays to study transporters normally localised to internal membrane structures. Many components involved in the cycle of yeast plasma membrane proteins have already been identified (see, for example, Luo and Chang (1997) The Journal of Cell Biology 138:731-746; and Wang and Chang (1999) The EMBO Journal

18:5972-5982) and may be useful in this regard.

An additional advantage of the yeast system is that the relative ease of genetic manipulation permits the use of random mutagenic strategies to isolate cells with overall higher functional expression of the transport protein at the plasma membrane. Whether due to mistargeting or insufficient activation, mutations within the yeast genome resulting in higher overall transport activity at the plasma membrane can be identified using genetic complementation or by screening for cells with increased accumulation of fluorescence substrate.

Another advantage of the yeast system is that it is possible to manipulate yeast to reduce background transport levels in host cells. For example, if the test compounds are sugar based compounds, then the assay may be improved by reducing the activity of native sugar transporters in the host cell. A deletion strain devoid of hexose transporters, for example, has already been described (Wieczorke et al. (1999) FEBS Lett. 464:123-128). The ability to readily modify yeast cell size may also be an advantage in the method of the present invention. In particular, increasing the cell size may improve the fluorescent signal from cells expressing transport proteins. Yeast cell size may be readily increased by manipulating the ploidy of the cells. For example, the conversion of haploid cells to diploid or polyploid cells, which can be readily achieved through established experimental procedures, results in a significant increase in cell size (see, for example, Bertl et al. (1998) Eur. J. Phvsiol. 436:999-1013).

It may also be possible to increase the fluorescent signal in yeast cells expressing transporters by genetically removing efflux transporters or their regulators. Bauer et al. (1999; Biochim. Biophys. Acta 1461 :217-236 describe a family of transporters known as "ABC" transporters. Many of these transporters have been implicated in drug efflux in animals and in yeast. By reducing the activity of these transporters through deletion of the genes encoding transporters or their regulators, it should be possible to achieve an enhanced signal within host yeast cells.

Additional improvements, particularly in expressing heterologous membrane proteins, may involve introducing homologous or heterologous molecular 'chaperones', or accessory proteins whose function is to promote the folding and/or maturation of proteins. These chaperones can be general (ie. they may assist in the folding of many proteins) or may be very specific for a small class of proteins. Examples of chaperones include CD147 (Kirk ef al (2000) The EMBO Journal 19:3896-3904) and the binding protein BiP (Harmsen et al (1996) Appl. Microbiol. Biotechnol. 46:365-370; and Robinson et a/ (1996) J. Biol. Chem. 271 :10017-10022). In some cases, transport proteins require specific lipid compositions within the phospholipid bilayer for optimal functionality. In this respect, it is a number of yeast mutants which have altered membrane compositions would be known to those skilled in the art. Genes involved in membrane biosynthesis and turnover, therefore, are additional targets for modification for optimal functional expression of transport proteins. Additional changes in overall functional expression of transport proteins may be mediated by altering growth conditions. It has been previously noted that several heterologous proteins are expressed with higher overall activity when grown at different temperatures, or by alteration of the pH of growth medium. Finally, chemical additives present in the growth and assay media may improve expression of cloned transport proteins. For example, a synthetic small-molecule dithiol (+)-trans-1 ,2-bis (2-mercaptoacetamido) cyclohexane (BMC) has been shown to catalyse protein folding in vivo (Woycechowsky et al. (1999) Chemistry & Biology 6:871-879). This is one example of an additive that may enhance expression of cloned transport proteins in yeast. Alternatively, enzyme stabilisers, such as glycerol, may be included in the growth medium (see, for example, Figler et al. (2000) Arch. Biochem. Biophys. 376:34-46).

Fluorescent substrates A series of compounds may be produced in which a fluorescent or luminescent moiety (called 'reporter') and a transport tag are combined. The 'transport tag' may be any putative compound that has the potential to interact with the transport protein.

The reporter and the transport tag can be the same entity (see, for example, Figure 3A), directly connected or overlapping (see, for example, Figure 3B, 3K and 3L) or linked by a spacing element (see, for example, Figures 3C-J). A spacing element can be one atom or a chain of atoms. In one embodiment, the spacing element allows an easy coupling reaction between the reporter and transport tag, where either can be an activated or not activated molecule. In addition, the spacing element can be designed for a tight junction of reporter and transport tag for directed cleavage. The cleavage can be achieved by any appropriate chemical or biological reaction. Preferably the cleavage is carried out by hydrolysis, oxidation, or reduction. Preferably the cleavage occurs under enzymatic catalysis, for example by the action of esterases, amidases, cytochrome P450. The coupling reaction may be achieved by, for example, the following:

1. The formation of an ester or amide from a carboxylic acid or an activated derivative thereof and an alcoholic function or a primary or secondary amine function, or the formation of an ether or a thioether or selenoether from an alcohol or a mercaptan or a selenol and an alkylating agent such as an alkyl halide or an ester of a sulfonic acid and an aliphatic alcohol, etc.

2. The formation of an amide or an ester or a hydrazide by reacting an activated carboxylic acid, as can be a carboxylic acid chloride or a carboxylic acid hydroxy-succinimide ester or an N-acyl isourea, with an alcohol or a primary or secondary amine or a hydrazide.

3. The formation of an amide or an ester by reacting a primary or secondary amine or the salt of a carboxylic acid with an activated alkyl halide.

4. The formation of a thioether by reacting a mercaptan with an activated alkyl halide, for example an allylic bromide or an iodoacetic acid amide.

5. The formation of a hydrazone by reacting a carbonyl compound as can be a ketone or an aldehyde with a hydrazine derivative or a sulfonic acid hydrazide.

6. The formation of a thiourea by reacting a primary or secondary amine with an isothiocyanate.

7. The formation of a disulfide by reacting two mercaptans or mercaptan derivatives with each other.

The fluorescent reporter molecule can be any molecule showing fluorescence or luminescence, preferably a small molecule with an excitation wavelength above 300 nm. These may be substituted coumarins, 5-amino- naphthalene-1 -sulfonic acid derivatives, fluorescein derivatives, luminarin derivatives, or 7-nitro benzofurazan derivatives or 1 ,2,3,4-tetrahydro-2,3- phthalazine-1 ,4-dione derivatives Rhodamine, BODIPY, tryptophan or 7-nitro benzofurazan derivatives.

The transport tag may be taken from the pool of fluorescent molecules as mentioned above or may be any other available molecule, which offers the possibility to react within a coupling reaction. Preferably, the putative transport tag has a molecular mass below 500 Da, and a logP value below 4 or is a fatty acid or is a steroid, or offers one or more functional groups suitable for specific recognition. Suitable functional groups include hydroxy functions, amine functions, carboxylic acid functions, ether functions, nonpolar groups, aromatic functions, ketone functions, (aldehyde functions), carbonic acid derived functions, double bonds, triple bonds, cyanide functions, amide functions, ester functions, oxime functions, hydroxamic acid functions, hydrazone functions, nitro functions, phenolic functions, anilinic functions, silicic acid derived functions, boric acid derived functions, and other functional groups known in the art.

Preferred transport tags are selected from the group consisting of an amino acid, peptide, vitamin, sugar, nucleotide, nucleobase, nucleoside, lipid, cholesterol, monoamine, polyamine, mono- or di-carboxyl moiety, sulphate, phosphate, silicate, carbamate, carbamate sugar phosphate, phospholipid, fatty acid, metal chelate, alcohol, ketone, diol, tri-azole, imidazole, and any therapeutic or diagnostic compound or combinations of any of these groups.

The formation of fluorescent putative transport substrates is designed to create a maximal variety of putative transport tags coupled to a limited pool of fluorescent labels. The limited number of fluorophores facilitates subsequent analyses using these molecules because it reduces settings required for fluorescence measurements. This leads to an easy technical handling with only a few settings required for the fluorescence measurement. It also allows the synthesis of combinatorial matrices of combinations of putative transport tags and fluorescent reporters. Examples of suitable fluorescent substrates are depicted in Figure 3.

Fluorescence indicators

A number of fluorescence indicators exist whose optical properties report the concentration of a chemical, for example H⁺, Ca²⁺, Na⁺, K⁺, Mg²⁺, and Zn2+. Examples of such indicators include fura-2, indo-1 , fluo-3, SBFI, PBFI, FuraZin-1 , lndoZin-1 , FluoZin-1 ,FluoZin-2 and RhodZin-1. While different in structure, the basic principle behind such fluorescence indicators is direct binding of the substrate leading to a change in the optical properties of the indicator which can be measured by monitoring their fluorescence. Two general classes of indicators are known: those whose fluorescence intensity change, and others whose fluorescence spectrum change in response to interaction with their substrates. The latter are also known as ratiometric dyes, because shift in fluorescence spectrum results in an overall change in the ratio of fluorescence intensities at two given wavelengths. Changes in spectrum can occur at the excitation wavelength, the emission wavelength or both. Such ratiometric dyes are particularly useful because the measured ratio changes are much less affected by variations in indicator concentration, cell thickness compared with measurements of fluorescence intensity at a single wavelength. For use with flow cytometers, dyes which shift in emission spectrum are particularly useful as fluorescence can be monitored simultaneously using a single light source.

Fluorescence indicators which report changes in Na+, pH, and membrane potential are preferred. Many transporters use a gradient of Na+ or pH as driving forces for transport of other substrates. Furthermore, many transport proteins mediate a net movement of charged molecules, resulting in an overall change in membrane electric potential. Detection of transport activity can therefore be measured either by monitoring the uptake of the substrate, or changes in internal pH, Na+ concentration and membrane potential are therefore useful means to measure activities of transport proteins which use these as driving forces. Other preferred properties of fluorescence indicators for this invention are: the dissociation constant (Kd) for the ligand should approximately match the expected physiological concentration range of the ligand; the indicator should exhibit strong discrimination against other ligand-like compounds which might be present in the cell; the indicator should show relatively strong fluorescence; the indicator should exhibit a large wavelength shift upon ligand binding; the chemical properties of the indicator should be such that it does not easily leak out of cells once loaded; derivatives of indicators should exist which allow them to be easily loaded into cells, and which are cleaved in the cytoplasm to efficiently trap the indicator in the cell. More recently, fluorescent indicators have been developed which are composed entirely of protein, eliminating the need to load the chemicals into cells prior to measurement. Instead, these indicators can be expressed in cells by transforming the cells with a nucleic acid expression construct encoding the indicator protein. The use of such nucleic acid encoded indicators provides much more uniform distribution of the indicator within cells, and has the additional advantage that the indicator can be targeted to specific compartments within the cell. The use of these dyes in living cells is well known to those skilled in the art, and has been extensively used to measure the changes in concentration of the target chemicals. Examples of such fluorescent indicators include cameleon (Miyawaki et al. (1997) Nature, 388, 882-887) cAMP(Zaccolo et al. (2000) Nat. Cell Biol. 2. 25-29). and FLIP (Fehr et al. (2002) Proc. Natl. Acad. Sci. USA, 99, 9846-9851 ), which report changes in the concentration of calcium, cAMP and hexose sugars, respectively. The cameleon construct, which uses two variants of the green fluorescent protein connected by a protein domain which undergoes conformational changes upon calcium binding. The resulting cameleon's optical properties change in response to changes in calcium concentration.

Isolation of yeast cells expressing transporters by fluorescence activated cell sorting (FACS) Flow cytometry is an analytical procedure for measuring the characteristics of individual cells (Shapiro H.M. (1994) Practical Flow Cytometry, 3rd Ed., Wiley-Liss New York). In the flow cytometry procedure, cells are injected into a fluid stream such that they are lined up in single file by hydrodynamic forces and carried through the focus of an intense light source such as a laser beam. Light scattering from the cells is measured by light detectors in two directions (see, for example, Figure 4A). Light scattered in the direction of the light beam is termed forward light scatter (FSC). This scatter generally increases in intensity with the size of the cells, although other factors such as refractive index can also influence the scatter. Light scattered at right angles to the light beam, or side scatter (SSC), is scattered from structures within the cells and is a measure of cell internal granularity. It is particularly influenced by the size and shape of the cell nucleus. In addition, the light can excite fluorescence from cell components or from dyes added to the cells either to stain cell components or attached to antibodies that recognise specific cell components. As with side scatter, the fluorescence emission is measured by light detectors placed at 90° to the illuminating light beam. Optical filters are used in the light path to discriminate between fluorescence emission from different dyes that emit distinct wavelengths of light. Therefore several different dyes can be measured simultaneously, provided that their spectral characteristics are sufficiently separate. The electrical pulses from the light detectors are processed electronically and analysed by computer software that allows the collected data to be displayed and cell populations with unique characteristics to be identified and analysed in isolation from other cells in the sample.

Because flow cytometry measures the characteristics of individual cells, it gives quantitative data on a sample of the cell population that can give a statistical measurement of the characteristics of the population. It can also identify sub-populations based on their light scatter or fluorescence properties. Flow cytometry is very rapid, as data rates of up to100,000 cells per second can be processed by modern machines. Therefore, it is feasible to perform flow cytometry of a very complex mixture of cells containing, for example, a cDNA expression library of up to 10⁸ independent transformants.

After cell sub-populations have been identified by flow cytometry, it is possible to physically sort them into tubes or microtiter plates using a flow cytometer with sorting capability. Cell sorting may be accomplished by electrostatic separation of fluid droplets containing the cells or by fluidic/mechanical sorting, depending on the machine design and manufacturer.

Generation of a detectable fluorescent signal for the cell populations expressing a transport protein can be made using a number of different means. One method is to employ a fluorescent substrate analog of the transport protein so that the fluorescent substrate is taken up preferentially into cells expressing the transport protein of interest. Alternatively, cells can be loaded with a fluorescence indicator which reports changes in the intracellular substrate concentration of the transport substrate.

Collected sub-populations of cells exhibiting enhanced or reduced fluorescence relative to background controls can be re-grown and re-sorted following another uptake reaction with the fluorescent substrate. Alternatively, plasmid DNA can be isolated from the collected sub-populations and re- transformed into yeast cells of the same or different genetic background. The newly transformed cells can then be subjected to another round of uptake reaction / flow cytometry. Enrichment of cells can be monitored by FACS, and upon sufficient enrichment, collected cells can be isolated and measured individually for uptake of fluorescent substrate using a microtiter plate based assay. Once confirmed, the polynucleotide sequence encoding the putative transporter can be isolated from the yeast cells and further analysed by, for example, sequencing. Once cells functionally expressing a transport protein of interest have been isolated, further optimisations can be performed using FACS. For example, cells containing a transport protein of interest can be transformed with a cDNA expression library and cells displaying higher fluorescence can be sorted. Alternatively, cells containing a transport protein of interest may be treated with one or more mutagens to cause random mutations within the genome. The resulting mixture of cells may be subsequently analysed by flow cytometry and cells displaying higher fluorescence can be sorted. Alternatively, cells expressing the transport protein of interest can be randomly mutagenized and cells showing higher overall fluorescence intensity can be collected by cell sorting.

Screening process for the identification of chemicals interacting with transport proteins

The method of the present invention allows the identification of compounds (such as substrates or ligands) that interact with the transport protein. In general, the method involves contacting a cell or multiple cells expressing the transport protein with the fluorescent substrate in the presence of a putative ligand or substrate and determining the effect of the compound on the uptake of the fluorescent substrate by the cell(s). Preferably, a parallel control reaction is also performed, in which the uptake of the fluorescent substrate by the cell(s) in the absence of the putative ligand or substrate or in the absence of the transport protein is determined. An enhanced signal when compared to that of the control reaction indicates that the putative compound is an agonist of the transport protein. Conversely, a reduced signal in the assay conducted in the presence of the putative compound when compared to the signal in the control reaction indicates that the putative compound is a substrate or an inhibitor of the transport protein. Discrimination between these two possibilities (substrate or inhibitor) may be achieved in a confirmatory assay involving direct uptake of the putative compound in the absence of the fluorescent substrate and direct measurement by, for example, analytical TLC (see, for example, Example 8), LC/MS, GC/MS, Fluorescence spectroscopy, HPLC with absorbance, fluorescence and conductivity detection, or any other sensitive analytical tool.

The screening process may utilise various techniques to selectively detect signal from inside the cell rather than signal arising from complexes located at the cell surface or in solution. One means for achieving this is to simply wash the cells to remove unincorporated complexes.

Another approach is to spin the cells to be assayed through a layer of oil, and count the fluorescence in the cell pellet.

Yet another approach is to expose the cells being assayed to a quenching solution that quenches signal from the uninternalised fluorescent substrate. Alternatively, the internalised fluorescence may be quenched and the fluorescence remaining in solution counted, in which case the decrease in the total fluorescence in the original solution is indicative of the amount taken up by the cells. In another variation of the screening process, the internalised fluorescence may be detected using fluorescence resonance energy transfer (FRET), where the internalised fluorescent compound gives its energy to another fluorescent compound (already present in the cell), which gives a secondary fluorescence of a different wavelength. In a further variation of the screening process, a conditional fluorescence reporter compound is used. A signal from a reporter of this type is not generated until the reporter is internalised within the cell. Methods using such conditional reporters may utilise a fluorophore and a quencher moiety pair. Outside the cell, the quencher moiety is disposed relative to the fluorophore such that it quenches fluorescence from the fluorophore. Once the reporter is internalised within the cell, the internal conditions are such that the quencher no longer effectively quenches the fluorophore. This may be achieved, for example, by enzymatic cleavage of the quencher from the fluorophore.

Alternatively, the conditional reporter may be a compound that is not fluorescent but is activated upon entry into the cell such that it becomes fluorescent. This activation may be effected by enzymes present within yeast cells which are capable of modifying the conditional reporter. Such enzymes include, for example, amidases and esterases (see, for example, Degrassi et al. (1999) App. Environ. Microbiol. 65:3470-3472). The following examples are provided to illustrate certain aspects of the invention and are not to be construed to limit the invention. Example 1

Manipulation of Yeast Cells

Deletion of the PTR2 gene of yeast strain YSYA003 to abolish endogenous dipeptide transport

A. Amplification of the deletion construct PTR2-KanMX-PTR2 by PCR

Template: pUG6 (Gϋldener et al. (1996) Nucleic Acids Res 24:2519-24)

Primers:

OSYA117 5' ttc ttc ttt tga att aga tea eta ata aac tet tat aat get caa ccT TCG

TAG GCT GCA GGT CGA C 3' (SEQ ID NO:1 )

OSYA118 5' tga aac aaa taa cag cag cac aga aaa etc ccg tea acg taa tat ggG

CAT AGG CCA CTA GTG GAT CTG 3' (SEQ ID NO:2)

The sequences in capital letters allow PCR amplification of the KanMX4 marker. The sequences in italics correspond to the borders of the genomic region to be deleted.

PCR reactions:

2 μL Template plasmid DNA: 10 ng (5 ng / μL)

1 μL 5' primer: OSYA117 (20 pmol / μL)

1 μL 3' primer: OSYA118 (20 pmol / μL)

1.75 μL dNTPs (10 mM each)

5 μL Buffer 2 10 X

0.7 μL Expand HF Enzyme

Total volume 50 μL

Place tubes in thermocycler and perform the following program:

94°C 3 Min. 15 Sec.

30 cycles

cycle 68°C 7 Min. Ethanol precipitation of PCR products

1. Transfer PCR reaction to 1.5 mL microcentrifuge tube.

2. To 50 μL of PCR reaction add 5 μL 3 M NaOAc pH 5.2 and mix.

3. Add 120 μL cold absolute ethanol, mix by inversion and incubate for about 15 Min. at -20°C, centrifuge at 4°C for 15 Min. at top speed.

4. Discard supernatant and wash pellet with 500 μL 70% ethanol.

5. Centrifuge 5 Min. at top speed. Remove supernatant and dry pellet. 7. Resuspend in 11 μL TE or H₂0. Check recovery using 0.5 μL on an agarose gel.

B. Yeast transformation

Yeast strain YSYA003 (MATa his3A1 leu2A0 met15A0 ura3A0) is transformed with 2-3 μg of the amplified PCR product (PTR2-KanMX-PTR2) according to the transformation method described in Example 2, though only one-tenth of the transformation mix is used. To allow expression of the resistance marker transformants are incubated for 3 hours in 1 ml YPD at 29°C after heat shock. 2x 200 μl of cell suspension is subsequently plated onto YPD agar plates containing 200 mg/L G418. Transformants having the deletion construct integrated into the genome will give rise to large colonies. Correct deletion of the PTR2 gene is verified by PCR.

According to this method the following strain was constructed: YSYA005 MA 7a his3A 1 leu2A0 met15A0 ura3A0 ptr2v.kanMX4

Example 2

Library Transformation of Yeast Cells

Materials Single-stranded Carrier DNA (2 mg/mL)

1. Weigh out 200 mg of high molecular weight DNA (Deoxyribonucleic acid Sodium Salt Type III from Salmon Testes, Sigma D1626) into 100 mL of TE buffer (10 mM Tris-CI pH 8.0, 1.0 mM EDTA). Disperse the DNA into solution by drawing it up and down repeatedly in a 10 mL pipette. Mix vigorously on a magnetic stirrer for 2-3 hours or until fully dissolved. If convenient, leave the covered solution mixing at this stage overnight in a cold room.

2. Aliquot the DNA and store at -20°C.

3. Prior to use, an aliquot should be placed in a boiling water bath for at least 5 Min. and quickly cooled in an ice water slurry.

Lithium Acetate Stock Solution (1.0 M.) Sterilise by autoclaving.

Polyethylene glycol Solution 50% (w/v) PEG MW 3350 (Sigma P3640)

Sterilise by autoclaving

YPD Growth Medium (YPD)

Yeast Extract 10.0 g / L Bacto Peptone 20.0 g / L

Dextrose 20.0 g / L

Bacto Agar 20.0 g / L (use only when making plates)

Yeast Growth Medium (SDG - URA ) YNB 1.70 g / L

(NH₄)₂S0₄ 5.00 g / L

Glucose (or galactose) 2 % (w/v)

CSM -URA 0.77 g / L adjust pH to 5.8 with NaOH

Transformation (based on method of Agatep et al. (1998) Technical Tips Online)

1. Inoculate 5 mL of liquid YPD and incubate with shaking overnight at 29°C. 2. Inoculate 50 mL of warm YPD to an OD₆oo of ~ 0.2.

3. Incubate the culture at 29°C on a shaker at 200 rpm for 3 to 5 hours, to an ODeoo of ~ 0.8.

4. Harvest the culture in a sterile 50 mL centrifuge tube at 3000 x g (5000 rpm) for 5 Min. 5. Pour off the medium, resuspend the cells in 25 mL of sterile water and centrifuge again.

6. Pour off the water, resuspend the cells in 1.0 mL 100 mM LiAc.

7. Pellet the cells at 3000 rpm for 3 Min. and remove the LiAc with a micropipette.

8. Resuspend the cells to a final volume of 500 μL (2 x 10⁹ cells/mL) - about 400μL of 100 mM LiAc.

9. Boil a 1.0 mL sample of SS-DNA for 5 Min. and quickly chill in ice water. 10. Vortex the cell suspension, pellet the cells and remove the LiAc with a micropipette.

11. Add the following ingredients in the order listed to provide a basic "transformation mix":

2.4 mL PEG (50% w/v) 360 μL 1.0 M. LiAc

500 μL SS-DNA (2.0 mg / mL) X μL Library Plasmid DNA (1 μg) 340-X uL Sterile ddH?Q

3600 μL TOTAL

12. Vortex vigorously until the cell pellet has been completely mixed (max 1 Min.).

13. Incubate at 30°C for 30 Min.

14. Heat shock in a water bath at 42°C for 30 Min. 15. Centrifuge at 3000 rpm for 3 Min. Sec. and remove the transformation mix with a pipette.

16. Pipette 10 mL of sterile water into the tube and resuspend the pellet by pipetting it up and down gently.

17. Plate 200 μL of the transformation mix onto large SDG - URA plates for a total of 50 plates.

18. Incubate the SDG - URA plates for 2 - 4 days to recover transformants. Example 3

Uptake reaction in Test Tubes (Preparation for FACS)

Uptake & Wash buffer 10 mM Na-Citrate buffer pH 5.0

Uptake Reaction

1. Grow cells overnight in SDG - URA medium to an OD₆oo of ~ 0.4 to

0.8. 2. Wash cells with sterile MilliQ H₂0 and resuspend in Uptake Buffer at a final OD₆oo of 2.0.

3. Aliquot 2 mL of cells for each time point and place tubes in a water bath pre-warmed at 29°C at least 5 Min. prior to start of reaction. Add glucose to a final concentration of 50 mM. 4. Initiate uptake reaction by addition of 10 - 50 μM reporter compound, and incubate for 15 - 180 Min. at 29°C with orbital shaking.

5. Terminate reaction by pelleting cells by centrifugation at 3000 x g for 10 Min. at 4°C.

6. Wash cells twice in uptake medium and resuspend to the same OD₆oo of 2.0.

7. Perform parallel uptake reactions with positive control (YSY005 + pDR-StSUT1), negative (YSY005 + pDR195) control cells. Trapped fluorescence is measured using a Shimadzu RF-5301 PC spectrofluorometer. 8. Perform flow cytometry immediately, or cells can be stored at 4°C in the dark for up to 36 hours.

Example 4

Enrichment of Cells by Flow Cytometry

Equipment Used: MoFlo cytometer from Cytomation (Fort Collins, USA) with the following lasers:

UV laser (Excitation: 350-363 nm), emission monitored at 460 / 40 nm (FL1). Argon laser (Excitation: 488 nm), emission monitored at 530 / 30 nm (FL2).

For Sheath Fluid, use FACSFIow (BD Immunocytometry Systems, San Jose, USA). Nozzle diameter: 70 μm. Sort rate: between 10000 and 70000 per second, depending on application.

Keep cells in the dark at 4°C until sorting.

1. Set up instrument using control cells. Use the same substrate and uptake conditions against cells functionally expressing characterised transporters, as well as empty vector as positive and negative controls. Use biparametric plots of Forward Scatter (FSC) and Side Scatter (SSC) to eliminate dead cells or cells with unusual morphology (see, for example, Figure 4A).

2. Use positive control cells to gauge the efficiency of uptake reaction. Analysis should be done using a FL1 vs. FL2 biparametric logarithmic plot, with a minimum of 50000 events. Adjust the gain on FL1 and FL2 so that ~ 99% of negative control cells fall within the 10° to 10¹ range (see, for example, Figure 4B). A 'comet tail' appearance (i.e., one starts to detect increased fluorescence in the FITC wavelengths among cells with high FL1 uptake, resulting in a slight upward curling of cells, as in Figure 4C) indicates a good uptake reaction. In some cases, subtraction of cell autofluorescence was performed to distinguish cells with weak fluorescence.

3. Before applying library samples, run the sorter with just the Sheath Fluid for at least 5 Min. to make sure no control cells are trapped in the tubing of the machine. Also, thoroughly clean sample inlet valve to ensure it is free of previous cell samples.

4. Collect cells with highest fluorescence values (the threshold will depend on the round of enrichment, but generally choose the top 0.02%, 0.1%, and 2% in the first, second and third rounds, respectively) into sterile tubes filled with 0.5 mL SDG - URA medium. Once collection is complete, make sure to vortex tube briefly, as many cells are deposited on the walls of the tubes and may dry. 5. As a confirmatory step, the collected library cells (between 10000 and 100000 cells) should be immediately resorted using the same gate.

6. Once again, collect the cells after the second sort in liquid SD-URA medium, and grow again for another round of uptake reaction/flow cytometry.

Alternatively, plasmid DNA is extracted from yeast cells as a mixture(see Example 6) and retransformed (see Example 2).

7. After two or three enrichment rounds, a portion of the collected cells should be plated on SDG - URA plates. Once grown, 100 - 2000 cells should be grown in liquid culture and tested either individually or in pools of less than 10 colonies for uptake of reporter compound using assays in microtiter plates (see, for example, Example 5). Note that each round of uptake/flow cytometry typically achieves -30 - 200 fold enrichment of transport competent clones, so three rounds of enrichment are usually required to achieve sufficient level of clonal purity in order to confidently isolate a clone with a representation of 10^"6 in the input library.

Example 5 Uptake Assay of Yeast Cells in Microtiter Plates

1. Uptake assay protocol for sugar transport

Strains Strain YSYA045 (MatD ura 3-52, leu 2-3, 112, his3A200, tφ1A901, Iys2-801, suc2A9) YSYA045 + pDR195-StSUT1 YSYA045 + pDR195

Uptake & Wash buffer: 10 mM Na-Citrate buffer pH 4.0

Stop buffer:

50 mM Tris-CI pH 8.0 Uptake Assay:

1. Grow culture overnight (70 - 100 mL SDG - URA; 100 μL overnight culture for pDR195, 200 μL ON-Culture for pDR195-StSUT1) to an ODeoo of 0.4 to 0.8 in a 250 mL bevelled Erlenmeyer flask at 29°C with orbital shaking at 200 RPM.

2. Collect cells by centrifugation at 3000 rpm, 4°C for 5 Min.

3. Wash pellet 2x with equal volume of 10 mM Na-citrate buffer pH 4.0 (3000 RPM, 4°C, 5 Min.)

4. Resuspend pellet in 10 mM Na-citrate buffer pH 4.0 to a final ODeoo of 2.0.

5. Add 100 μL cell suspension OD₆oo 2.0 per well (Filterplatte, Corning 0.2 μm PVDF), and shake for 5 Min., 300 rpm on a Heidolph shaker at 12°C pre-incubation.

6. Add of 5 μL of 20% carbon source (glucose or galactose) per well (to a final concentration of 50 mM per well) and incubate for 5 Min. at 12°C and 300 rpm on a Heidolph shaker.

7. Add competitor compound to 0 to 1 mM, and incubate for 3 Min. at 12°C and 300 rpm on a Heidolph shaker.

8. Add 5 μL 200 μM Esculin (6,7-Dihydroxycoumarin 6-glucoside) or Fraxin (8-[β-D-Glucopyranosyloxy]-7-hydroxy-6-methoxycoumarin) per well to a final concentration of 10 - 50 μM and incubate for 15 - 30 Min. at 12°C and 300 rpm on a Heidolph shaker.

9. Add 200 μL of ice cold Stop Buffer and mix well.

10. Remove the supernatant by filtering, using an microcentrifuge vacuum manifold (~ 15 in. Hg).

11. Wash 3x with 300 μL of 10 mM Na-Citrate buffer pH 4.0 (again with vacuum manifold as in step 10).

12. Resuspend cells in 100 μL of uptake buffer by vigorous vortexing for 10 Min. 13. Measure fluorescence (make sure the cells are well resuspended), with a Gemini fluorescence plate reader from Molecular Devices. See Table for wavelength values. 2. Uptake of Fluorescent Peptide-Like Substrates into Yeast Cells

Strain YSYA05 (Mata, his3D1 leu2D0 met15D0 ura3D0 ptr2::kanMX4) YSYA05 + pDR195-AtNTR1 YSYA05 + pDR195 YSYA05 + pYES2-TaPEPT1

Uptake & Wash Buffer:

10 mM Na-Citrate, 0.1 mM MgCI₂, pH 5.0

Assay:

1. Grow a fresh overnight cultures as described earlier for sucrose transporters, but in 10 mL of SDG -URA medium.

2. The next morning dilute the culture 1 : 50-1 :20 and grow further until OD₆oo is between 0.3 and 0.8.

3. Wash cells 3X in ice-cold water and resuspend in uptake buffer to an ODeoo of 2.0. 4. Transfer 100 μL of cell suspension to the wells of a 96-well filter plate

(Corning Incorporated 3504, 96 Well White Polystyrene Plate, 0.2 μM

PVDF). 5. Add glucose to a final concentration of 1% [w/v] to the cell suspension and incubate the microtiter plate in a shaker (200 RPM) at 28 °C for 5 Min. 6. Add test compound from 0 to 10 mM, and incubate for 3 Min. at 12°C and

300 rpm on a Heidolph shaker.

Initiate uptake by addition of (final concentrations): 225 μM Ala-AMC (Figure 3K) for YSYA005-pDR-AtNTR1 , 745 μM Sym 0159 (Figure 3L) for YSYA005-pYES(PMA1 )-TaPepT1 ;

7. Incubate plates in the shaker at 28°C for 30 Min. to 3 Hr.

8. Stop uptake reaction by removing the incubation solution by filtration.

9. Wash harvested cells three times with 300 μL uptake buffer. 10. Resuspend cells in 100 μL uptake buffer. 11. Measure sample fluorescence on a Spectra Max Gemini XS (Molecular Devices).

Example β Extraction of Plasmid DNA from Yeast Cells

Reagents;

SCE buffer

1.2 M sorbitol, 10 mM EDTA 0.1 M Na-citrate, pH 7.0

Lysis buffer

2% SDS, 50 mM Tris-CI (pH 8.0), 10 mM Na-EDTA

Potassium acetate 5M (KOAc)

Just before starting, prepare fresh "Ready-to-use SCE" by mixing: lO mL SCE 10 mg lyticase 80 μL β-mercaptoethanol

Protocol:

1. Grow 1 mL yeast liquid culture to saturation at 29°C in selective medium

(SDG - URA).

2. Spin down, add 600 μL "Ready-to-use SCE". Incubate at 37°C for 45 Min.

3. Spin down, add 300 μL SCE. 4. Add 600 μL lysis buffer and resuspend.

5. Add 300 μL KOAc and let stand for 30 Min. at 4°C.

6. Spin 10 Min. at 10000g.

7. Extract sample with 1 volume phenol/chloroform/isoamylalcohol (25:24:1), vortex and centrifuge 10 Min. at 10000g. 8. Transfer supernatant to new tube and add glycogen to 1 mg / mL and 2.5 volumes ice cold EtOH.

9. Spin 10 Min. at 10000g.

10. Discard supernatant and allow pellet to dry.

11. Resuspend pellet in 20 μL MilliQ H₂0 or TE Example 7

Colony PCR of FACS Sorted Yeast Cells

Reagents

Lyticase Solution:

5U / μL solution in H₂0, stored at -20°C

1. Grow a 1 mL overnight yeast culture to a high OD (saturated culture) either in individual culture tubes or in microtiter plates. 2. Place 20 μL of the culture into a microcentrifuge tube.

3. Add 10 μL of lyticase solution and incubate at 30°C for 20 Min.

4. Freeze samples at -85°C for at least 10 Min.

5. Perform PCR on the cell lysate with the following reaction mixture:

10X Taq Buffer 5 μL 25 mM MgCI₂ 5 μL

25 mM dNTPs 1 μL

5' pYES2 primer (20 pmol / μL) 1 μL

3' pYES2 primer (20 pmol / μL) 1 μL

MilliQ H₂0 31 μL Cell lysate 5 μL

Taq polymerase (1 U / μL) 1 μL

Total Volume 50 μL

6. Place tubes in thermocycler and perform the following program: 95°C 5 Min.

30 cycles

72°C 10 Min.

4°C D

7. Run 10 μL samples on agarose gels directly or after restriction digestion. Example 8

Confirmation of Test Compound Uptake by Thin Layer Chromatographv

1. Uptake reaction was performed in test tubes as described in Example 3, except that the test compounds were used as substrates instead of the reporter compound.

2. Two mL of cell suspension at OD₆oo of 2.0 were washed twice with uptake buffer and then pelleted. The supernatant was discarded and the pellet was resuspended in 0.5 mL of MeOH, along with a few glass beads (glass beads, 212-230 microns, Sigma).

3. The mixture was vortexed vigorously for 2 Min., followed by sonication for 2 Min. The sample was kept overnight at -20 °C.

4. After thawing, the sample was vortexed again for 2 Min. The samples were then centrifuged and the supernatant was concentrated by evaporation.

5. The concentrated sample was spotted on a silica gel plate (silica gel 60 F254 on aluminium sheets, Merck Darmstadt). Samples were resolved using a suitable solvent mixture such as 1 :1 (v/v) methanol:chloroform. 6. UV light was employed for visualisation in the case of UV-active compounds.

Otherwise, a standard staining method such as the ninhydrin reagent (0.3g per 100mL butanol and 3 mL acetic acid) for amino compounds, or molybdo-phosphoric acid (5g per 100 mL ethanol) for oxidisable compounds was employed. The samples resolved on TLC plates were visualised by dipping the plates into the respective solutions and heating until spots were visible.

TLC can be replaced as the analytical method by any other qualitative or quantitative technique available including all forms of high performance liquid chromatography and gas chromatography equipped with any of a range of detection systems including all forms of mass selective detectors, absorbance, ionisation, fluorescence, conductivity, and NMR. All of these techniques allow for reduced sample size and high throughput based on automated plate based sample preparation and injection. Example 9

Cloning of a Peptide Transporter cDNA from Triticum aestivum

1. cDNA expression library from roots of 3 day old seedlings of Triticum aestivum (in pYES2 vector) were transformed into YSYA005 cells (Example 2) using the procedure described in Example 2 and selected on SD-URA plates, resulting in > 1.1 X 10⁶ transformants.

2. Transformants were scraped from petri dish plates and pooled. The collected, pooled cells were stored at -85°C in SD-URA medium containing 15% (v/v) glycerol or used to seed liquid culture in SD-URA medium. 10 mL of culture medium was seeded with ~10⁷ cells and grown overnight. Parallel cultures of YSYA005 transformed with pYES2 empty vector were seeded for use as negative control.

3. The next morning, 250 mL Erlenmeyer flasks containing 50 mL of pre- warmed SD-URA medium were seeded with the overnight culture to a starting ODeoo of 0.075 and 0.150 and grown with vigorous shaking at 28°C until reaching an OD600 of 0.4 - 0.8.

4. Uptake reaction was performed essentially as described in Example 3, using 500 μM Gly-DMCA-OH (Figure 3B). Uptake was performed with 3 mL of washed cells resuspended at an ODeoo of 2.0, and was allowed to take up substrate for 120 min. at 28°C with vigorous shaking in darkness, and cells were subsequently rinsed twice in uptake buffer without substrate. Cells were either used immediately for sorting or stored in darkness at 4°C overnight.

5. Flow cytometry was performed essentially as described in Example 4. The instrument was first set up using the YSYA005-pYES2 control cells so that the UV fluorescence (FL2) of > 99% cells was between 100 and 101 fluorescence units. The first sort of library transformed cells was performed at high speed (40,000-50,000 cells per second) using the 'enrich 1-cell' mode. Approximately 130,000 cells were collected in a test tube containing 2 mL of SD-URA medium, using a gate set so that the most fluorescent 0.3% of cells were collected. After collection, the tube was vortexed briefly and centrifuged to concentrate cells. 6. Cells were re-sorted in 'purify 1-cell' mode using the same gate. Approximately 40,000 cells were collected, again into culture tubes containing SD-URA medium (supplemented with 30 mg/L of G418 antibiotic). Cells were vortexed briefly and grown at 28°C with shaking.

7. Once grown, aliquots of cells were used for two additional rounds of uptake and sorting as performed in Steps 3- 6, except that cells were sorted in the 'purify 1-cell' mode' without an immediate re-sorting, and approximately 10,000 and 2,000 cells were collected in the second and third rounds, respectively, which had the top 0.3% of UV fluorescence (FL2). After the second sort, cells were collected in tubes containing 2 mL SD -URA medium containing 30 mg/L G418. After the third sort, collected cells were plated on SD-URA petri dishes. 22 colonies were picked and analysed further for (a) uptake as described in Example 5, (b) DNA using procedures described in Example 6. Results of the uptake and DNA analysis are shown in Figure 4G.

Example 10

Cloning of a Peptide Transporter from a Candida albicans genomic library

1. A genomic library from the fungus C. albicans was transformed into YSYA005 cells (Example 2) using the procedure described in Example 2 and selected on SD-URA plates, resulting in > 2.3 X 10⁵ transformants.

3. The next morning, 250 mL Erlenmeyer flasks containing 50 mL of pre- warmed SD-URA medium were seeded with the overnight culture to a starting ODeoo of 0.075 and 0.150 and grown with vigorous shaking at 28°C until reaching an OD₆₀₀ of 0.65. 4. Uptake reaction was performed essentially as described in Example 3, using 500 μM Gly-DMCA-OH (Figure 3B). Uptake was performed with 3 mL of washed cells resuspended at a final OD₆oo of 2.0, and was allowed to take up substrate for 120 min. at 28°C with vigorous shaking in darkness, and cells were subsequently rinsed twice in uptake buffer without substrate. Cells were either used immediately for sorting or stored in darkness at 4°C overnight.

5. Flow cytometry was performed essentially as described in Example 4. The instrument was first set up using the YSYA005-pYES2 control cells so that the UV fluorescence (FL2) of > 99% cells was between 10° and 10¹ fluorescence units. The first sort of library transformed cells was performed at high speed (40,000-50,000 cells per second) using the 'enrich 1-cell' mode. Approximately 130,000 cells were collected in a test tube containing 2 mL of SD-URA medium, using a gate set so that the most fluorescent 0.3% of cells were collected. After collection, the tube was vortexed briefly and centrifuged to concentrate cells.

6. Cells were re-sorted in 'purify 1-cell' mode using the same gate. Cells were sorted once again using the same gate parameters, and ~ 2000 cells were collected in a tube containing 2 mL of SD - URA medium supplemented with 30 mg/L G418 and grown further for additional rounds of FACS. At the same time, ~10,000 cells were also plated onto petri dish plates containing either non-selective medium (SD - URA medium containing 30 mg/L G418 and 2% Bacto-agar), and selective medium (0.17% (w/v) yeast nitrogen base, 0.5% (NH₄)₂ S0₄, 2% Dextrose, 100 mg/L Leucine, 100 μM His-Met, 30 mg/L G418) in order to monitor levels of enrichment after each sorting round. Under non- selective conditions, all collected cells should grow, whereas only cells containing functional dipeptide transporters are expected to grow in selective medium (See Figure 5a).

7. Once the liquid culture is grown, aliquots of cells were used for two additional rounds of uptake and sorting as performed in Steps 3 - 6, except that cells were sorted in the 'purify 1-cell' mode' without an immediate re-sorting, and approximately ~1 ,000-2,000 cells were collected which had the top 0.3% of UV fluorescence (FL2). As in step 6, cells were collected directly in tubes containing 2 mL SD -URA medium containing 30 mg/L G418, and also sorted directly onto petri dish plates containing selective and unselective medium. Figure 5a shows that, after the third sort, the percentage of cells containing functional peptide transporter exceeds 25%.

8. Confirmation of the presence of peptide transporter was performed by retransformation of the plasmid into YSYA005 cells and testing for growth on selective dipeptide medium, as well as by PCR analysis. Plasmid was isolated from 35 yeast colonies growing on control, non-selective and selective medium essentially as described in Example 6, and transformed into E. coli using standard procedures. Ampicillin resistant colonies were further grown and plasmid DNA isolated. The purified plasmid DNA was used to re-transform YSYA005 cells as described in Example 2, and retested for growth on selective dipeptide medium (Figure 5B, top). Transformants derived from two negative control cells were grown alongside 10 transformants derived from colonies which grew on selective media.

9. PCR analysis was performed on the same DNA preparation to test for the presence of, CaPTR2, the peptide transporter gene previously isolated from C. albicans. Compared with control cells, cells able to grow on selective medium all contain the peptide transporter gene CaPTR2 (Figure 5b). PCR analysis was performed essentially as described in Example 7 using the following primers and conditions:

CaPTR2 forward primer: OSYA566 TGA TGA CTA CAA TCC CAA AGG G (SEQ ID NO:3)

CaPTR2 reverse primer: OSYA579 CCT AAT CCA CTA TCG GCC AAA TTG

(SEQ ID NO:4)

Approximately 2-5 ng of plasmid DNA isolated from E. coli was used as template DNA, derived from 4 negative control yeast colonies (pYES2- transformed), 5 yeast colonies which were not selected for enhanced fluorescence, and 26 colonies which grew on selective medium.

PCR conditions:

PCR was performed with the following reaction mixture: 10X Taq Buffer 5 μL

25 mM MgCI₂ 5 μL

25 mM dNTPs 1 μL

CSY566 (20 pmol / μL) 1 μL

CSY579 primer (20 pmol / μL) 1 μL

MilliQ H₂0 35 μL

Template DNA 1 μL

Taq polymerase (1 U / μL) 1 μL

Total Volume 50 μL

6. Tubes were placed in the thermocycler and the following program was used: 95°C 5 Min.

35 cycles

72°C 10 Min.

4°C

Approximately 10 μL of PCR samples were separated on 1.5% agarose gels (See Example 5B). All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

Claims:

1. A method for identifying a transport protein, the method comprising: (i) transforming yeast cells with a library of expression constructs, wherein one or more of the expression constructs comprises a polynucleotide encoding a putative transport protein;

(ii) contacting the transformed yeast cells with a fluorescent substrate for the transport protein under conditions such that cells comprising an expression construct encoding a transport protein express the transport protein and thereby bind or internalise the fluorescent substrate; and (iii) isolating transformed yeast cells that exhibit uptake of the fluorescent substrate.

2. A method as claimed in claim 1 wherein the cells are isolated in step (iii) by fluorescence activated cell sorting (FACS).

3. A method as claimed in claim 1 or claim 2, wherein steps (ii) and (iii) are repeated in order to achieve further enrichment of the yeast cells expressing the transport protein.

4. A method as claimed in any one of claims 1 to 3 which further comprises: (iv) using the transformed cells from step (iii) in a screening process to identify one or more compounds that interact with the transport protein expressed by the cells.

5. A method as claimed in claim 4 wherein the screening process comprises the following steps:

(a) contacting the transformed yeast cells or a population of cells derived therefrom with a fluorescent substrate and a putative compound; and

(b) monitoring the uptake of the fluorescent substrate by the yeast cells in the presence of the putative compound.

6. A method as claimed in claim 5, wherein the yeast cells are washed after step (a) to remove residual fluorescent substrate.

7. A method as claimed in any one of claims 1 to 6 wherein the method further comprises introducing into the transformed cells an expression construct encoding a transport protein enhancer.

8. A method as claimed in any one of claims 1 to 7 wherein the polynucleotide encoding the transport protein is isolated from the transformed cells and characterised.

9. A method as claimed in claim 8 wherein the polynucleotide is characterised by sequencing.

10. A method as claimed in any one of claims 1 to 9, wherein each expression construct comprises a polynucleotide selected from the group consisting of a cDNA molecule, a genomic DNA molecule and a synthetic DNA molecule.

11. A method as claimed in claim 10, wherein the polynucleotide is operably linked to one or more regulatory regions.

12. A method as claimed in any one of claims 1 to 11 , wherein the host yeast cells are genetically modified in order to reduce background transport activity and/or to reduce background fluorescence.

13. A method as claimed in any one of claims 1 to 11 , wherein the host yeast cells are genetically modified in order to enhance the translation and/or stability of the transport protein.

14. A method as claimed in any one of claims 1 to 11 , wherein the host yeast cells are genetically modified in order to enhance the fluorescent signal of the transformed cells.

15. A method as claimed in any one of claims 1 to 14, wherein the fluorescent substrate comprises a fluorescent or luminescent moiety and a transport tag.

16. A method as claimed in claim 15 wherein the fluorescent or luminescent moiety is selected from the group consisting of substituted coumarins, 5-amino- naphthalene-1 -sulfonic acid derivatives, fluorescein derivatives, luminarin derivatives, Rhodamine, BODIPY, tryptophan and 7-nitro benzofurazan derivatives.

17. A method as claimed in claim 15 or claim 16, wherein the transport tag is selected from the group consisting of an amino acid, peptide, vitamin, sugar, nucleotide, nucleobase, nucleoside, lipid, cholesterol, monoamine, polyamine, mono- or di-carboxyl moiety, sulphate, phosphate, silicate, carbamate, carbamate sugar phosphate, phospholipid, fatty acid, metal chelate, alcohol, ketone, diol, triazole, imidazole, and any therapeutic or diagnostic compound or combinations of any of these groups.

18. A kit for identifying a transport protein and screening for a compound which interacts with the transport protein, the kit comprising a fluorescent substrate for a transport protein.

19. A method for identifying a transport protein, the method comprising:

(i) transforming yeast cells with a library of expression constructs, wherein one or more of the expression constructs comprises a polynucleotide encoding a putative transport protein;

(ii) incorporating into the transformed yeast cells a fluorescent indicator under conditions such that cells comprising an expression construct encoding a transport protein express the transport protein and thereby import or export a substrate that alters a signal generated by the fluorescent indicator; and

20. A method as claimed in claim 19 wherein the cells are isolated in step (iii) by fluorescence activated cell sorting (FACS).

21. A method as claimed in claim 19 or claim 20, wherein steps (ii) and (iii) are repeated in order to achieve further enrichment of the yeast cells expressing the transport protein.

22. A method as claimed in any one of claims 19 to 21 which further comprises:

23. A method as claimed in claim 22 wherein the screening process comprises the following steps:

(a) contacting the transformed yeast cells comprising the fluorescent indicator with a compound; and

(b) monitoring the change in signal generated by the fluorescent indicator in the presence of the compound.

24. A method as claimed in any one of claims 19 to 23 wherein the method further comprises introducing into the transformed cells an expression construct encoding a transport protein enhancer.

25. A method as claimed in any one of claims 19 to 24 wherein the polynucleotide encoding the transport protein is isolated from the transformed cells and characterised.

26. A method as claimed in claim 25 wherein the polynucleotide is characterised by sequencing.

27. A method as claimed in any one of claims 19 to 26, wherein each expression construct comprises a polynucleotide selected from the group consisting of a cDNA molecule, a genomic DNA molecule and a synthetic DNA molecule.

28. A method as claimed in claim 27, wherein the polynucleotide is operably linked to one or more regulatory regions.

29. A method as claimed in any one of claims 19 to 28, wherein the host yeast cells are genetically modified in order to reduce background transport activity and/or to reduce background fluorescence.

30. A method as claimed in any one of claims 19 to 28, wherein the host yeast cells are genetically modified in order to enhance the translation and/or stability of the transport protein.

31. A method as claimed in any one of claims 19 to 28, wherein the host yeast cells are genetically modified in order to enhance the fluorescent signal of the transformed cells.

32. A method as claimed in any one of claims 19 to 31 , wherein the fluorescent indicator is a ratiometric indicator.

33. A method as claimed in any one of claims 19 to 31 wherein the fluorescent indicator is selected from the group consisting of indo-1 , BCECF, SBFI, cameleon, FLIP, Carboxy SNARF-1 , SPQ, MQAE, MEQ, Lucigenin, di- 4-ANEPPS, di-8-ANEPPS, DiOC2, JC-1 , JC-9, mag-indo-1 , FuraZin-1 , lndoZin-1 , and FluoZin-1.

34. A method for identifying a transport protein enhancer, the method comprising:

(ii) contacting the transformed yeast cells with a fluorescent substrate for the transport protein under conditions such that the cells express the transport protein and putative enhancer and the transport protein binds or internalises the fluorescent substrate; and (iii) isolating transformed yeast cells that exhibit enhanced uptake of the fluorescent substrate when compared to yeast cells that express the transport protein but not the putative enhancer.

35. A method for identifying a transport protein enhancer, the method comprising:

(ii) incorporating into the transformed yeast cells a fluorescent indicator under conditions such that the cells express the transport protein and putative enhancer and the transport protein imports or exports a substrate that alters a signal generated by the fluorescent indicator; and (iii) isolating transformed yeast cells that exhibit an altered signal generated by the fluorescent indicator when compared to yeast cells that express the transport protein but not the putative enhancer.