IL167321A - Polynucleotide comprising a dna sequence encoding a glucose transporter 4, a yeast cell comprising such a polynucleotide and a process for preparing such a yeast cell - Google Patents

Abstract

A purified, isolated polynucleotide (I) that contains a DNA sequence encoding the human glucose transporter protein, GLUT4Val85Met (II), is new. Independent claims are also included for the following: (1) Saccharomyces cerevisiae cell in which all glucose transporters are no longer functional and no functional Erg4 protein is present; (2) method for preparing yeast cells that contain (I); (3) yeast cells in which all glucose transporters are no longer functional and which contain (I); (4) method for identifying compounds (A) that stimulate activity of GLUT4 protein; and (5) methods for identifying compounds (B) or (C) that inhibit the proteins encoded by the Fgy1 and ERG4 genes, respectively.

Description

^nm ητ T^uiN a^D *?¾»π D*H&W ΝΙΊ ,4 -itniBoait: A POLYNUCLEOTIDE COMPRISING A DNA SEQUENCE ENCODING A GLUCOSE TRANSPORTER 4, A YEAST CELL COMPRISING SUCH A POLYNUCLEOTIDE AND A PROCESS FOR PREPARING SUCH A YEAST CELL Pear! Cohen Zedek Latzer P-7764-1L The invention relates to yeast strains in which the human Glut 4 and Glut 1 transporters can be functionally expressed.

Most heterotropic cells transport glucose via special transporter proteins into the cell interior. The various organisms have developed different mechanisms mediating the transporting of glucose, such as, in particular, proton symport systems, Na<+> glucose transporters, binding protein-dependent systems, phosphotransferase systems, and systems for facilitated diffusion. In the eukaryotes, a family of glucose transporters which are encoded in mammals by the GLUT genes (GLUT=glucose transporter) and Saccharomyces cerevisiae by the HXT genes (HXT=hexose transporter) mediates glucose uptake via facilitated diffusion. Said transporters belong to a larger family of sugar transporters. They are characterized by the presence of 12 transmembrane helices and by a plurality of conserved amino acid radicals.

Glucose transport plays an important part in disorders associated with a defective glucose homeostasis, such as, for example, diabetes mellitus or Fanconi-Bicke! syndrome. The glucose transport in mammals has therefore been the subject of numerous studies. To date, thirteen glucose transporter-like proteins have been identified (GLUT1 to GLUT12, HM!T-H-myo-inositol transporter). Said transporters play key parts which include the uptake of glucose into various tissues, its storage in the liver, its insulin-dependent uptake into muscle cells and adipocytes and glucose measurement by the β cells of the pancreas. US 6,246,374 is directed to the identification of a further glucose transporter protein which is called GLUTX and methods for identifying compounds that modulate the activity of GLUTX.

GLUT1 mediates the transport of glucose into erythrocytes and through the blood-brain barrier, but is also expressed in many other tissues, while GLUT4 is limited to insulin-dependent tissues, primarily to muscle and fatty tissue. In said insulin-dependent tissues, controlling the targeting of GLUT4 transporters through intracellular compartments or plasma membrane compartments represents an important mechanism for regulating glucose uptake. In the presence of insulin, intracellular GLUT 4 is redistributed through the plasma membrane in order to facilitate glucose uptake. GLUT1 is likewise expressed in said insulin-dependent tissues, and its distribution in the cell is likewise influenced by insulin, albeit not as strongly. In addition, the relative efficacy with which GLUT1 or GLUT4 catalyze sugar transport is determined not only by the extent of the targeting of each transporter to the cell surface but also by their kinetic properties.

The fact that different glucose transporter isoforms are coexpressed and the rapid glucose metabolism have rendered studies on the role and the exact properties of each glucose transporter isoform in these insulin-dependent tissues complicated. In order to solve these problems, heterologous expression systems such as Xenopus oocytes, tissue culture cells, insect cells and yeast cells have been used. However, it turned out that a number of difficulties appeared in connection with these systems: too weak an activity of the heterologously expressed transporters, intrinsic glucose transporters in said systems, intracellular retention of a considerable proportion of the transporters or even production of inactive transporters.

Naturally occurring GLUT4 protein of mammals, in particular that of humans, can be expressed in a functional manner in strains of Saccharomyces cerevisiae under particular conditions.

Yeast cells are unicell eukaryotic organisms. They are therefore, for some proteins, more suitable for expression than bacterial systems, in particular with regard to carrying out screen assays for identifying pharmaceutically active substances.

The present invention relates to a purified and isolated polynucleotide comprising a DNA sequence encoding a Glucose Transporter 4 containing at position 85 an amino acid exchange from valine to methionine (GLUT4V85M) protein having a glucose transporting activity.

Said protein contains at position 85 of the amino acid chain of the human GLUT4 protein an amino acid exchange from valine to methionine. This altered GLUT4V85M protein provides further alternatives for expressing a functional GLUT4 protein. A GLUT4 protein should be regarded as functional in connection with Saccharomyces cerevisiae if glucose uptake can be observed in a Saccharomyces cerevisiae strain whose glucose transporters in their entirety are inactive (=hxt{-)) after expression of said GLUT4 protein. Glucose uptake may be determined either by transport measurements by means of radioactively labeled glucose or by growth on medium with glucose as sole carbon source.

In a preferred embodiment, the purified and isolated polynucleotide comprising a DNA sequence which calls for a protein GLUT4V85M may include or comprise a sequence of the following groups: a) a nucleotide sequence according to Seq ID No. 1 , b) a nucleotide sequence which hybridizes to a sequence of Seq ID No. 1 under stringent conditions and which codes for a protein GLUT4V85M.

The purified and isolated polynucleotide preferably encodes a GLUT4V85M protein which has an amino acid sequence of Seq ID No. 2.

The purified and isolated polynucleotide comprising a DNA sequence which codes as discussed previously for a protein GLUT4V85M, may be operationally linked to a promotor. Suitable promotors are in particular prokaryotic or eukaryotic promoters such as, for example, the Lac-, trp-, ADH- or HXT7 promotor. The part of the polynucleotides, which codes for the protein GLUT4V85M is operationally linked to a promotor precisely if a bacterial or eukaryotic organism produces, by means of said promotor with the aid of a vector, an mRNA which can be translated into the protein GLUT4V85M. An example of such a vector is the vector p4H7GLUT4V85M (Seq ID No. 3). The protein GLUT4V85M may be expressed in yeast cells by means of said vector.

The above-described polynucleotide comprising a DNA sequence which codes for a protein GLUT4V85M is, in a preferred embodiment, suitable for replicating said polynucleotide in a yeast cell or for expressing the part of the polynucleotide, which encodes the protein GLUT4V85M, in a yeast cell to give the protein GLUT 4 V85M. A yeast cell from Saccharomyces cerevisiae is particularly suitable. For replication and expression in a yeast cell, the polynucleotide comprising a DNA sequence which calls for a protein GLUT4V85M is present in the form of a yeast vector. The polynucleotide region coding for the GLUT4V85M protein may be operationally linked to a yeast cell-specific promotor such as, for example, the ADH promotor (alcohol dehydrogenase promotor) or the HXT7 promotor (hexose-transporter promotor). The yeast sectors are a group of vectors which was developed for cloning of DNA in yeasts.

The invention furthermore relates to a yeast cell from Saccharomyces cerevisiae in which all glucose transporters are no longer functional (=hxt (-)) and which contains no functional Erg4 protein. Such a yeast cell is preferably a yeast cell deposited as Saccharomyces cerevisiae DSM 15187 with the DSMZ {Deutsche Samm!ung von Mikroorganismen und Zeiikulturen GmbH, Mascheroder Weg 16, 38124 Brunswick, Germany).

The invention also relates to a yeast cell in which all glucose transporters are no longer functional and which contains no functional Fgy1 and no functional Erg4 protein. The lack of an Erg4 protein or of an Fgy1 protein may be attributed in particular to an interruption of the corresponding coding genome sections or to a partial or complete removal of said coding genome sections.

Preference is given to using as yeast cell which contains no functional glucose transporters, no functional Fgy1 protein and no functional Erg4 protein, a yeast cell as deposited with the DSMZ as Saccharomyces cerevisiae DSM 15184.

A yeast cell as described above is preferably used for expressing a mammalian GLUT1 protein or a mammalian GLUT4 protein, in particular a protein from rats, mice, rabbits, pigs, cattle or primates. A preferred embodiment uses the yeast cell for expressing a human GLUT4 or GLUT1 protein.

A Saccharomyces cerevisiae yeast cell whose glucose transporters in their entirety and also the Erg4 protein are no longer functional may contain a polynucleotide of the present invention, which comprises a DNA sequence coding for a protein GLUT4V85M. Said yeast cell can also express the GLUT4V85M protein and thus contain said protein.

A yeast strain of this kind, containing a polynucleotide which comprises a DNA^ sequence coding for the GLUT4V85M protein, is preferably the Saccharomyces cerevisiae DSM 15185 yeast strain which has been deposited with the DMSZ.

A yeast cell whose glucose transporters in their entirety and also the Erg4 protein are no longer functional and which contains a polynucleotide comprising a DNA sequence which calls for a protein GLUT4V85M may be prepared, for example, by a) providing a yeast cell whose glucose transporters in their entirety and also the Erg4 protein are no longer functional, b) providing an isolated and purified polynucleotide which comprises a DNA sequence coding for the GLUT4V85M protein and which can be replicated in the yeast cell, c) transforming the yeast cell from a) with the polynucleotide from b), d) selecting a transformed yeast cell, e) where appropriate expressing the GLUT4V85 protein.

An isolated and purified polynucleotide which comprises a DNA sequence coding for the GLUT4V85M protein is preferably a vector which can be replicated in a yeast cell and in which said DNA sequence was cloned. An example of such a vector is p4H7GLUT4V85M (Seq ID No. 3).

The invention also relates to a yeast cell whose glucose transporters in their entirety and whose proteins for Fgy1 and Erg4 are no longer functional and which contains a polynucleotide which comprises a DNA sequence coding for the GLUT4V85M protein. Said yeast cell can also express the GLUT4V85M protein and thus contain said protein. A yeast strain of this kind is preferably the Saccharomyces cerevisiae DSM 15186 deposited with the DSMZ.

A yeast cell whose glucose transporters in their entirety and also the proteins Fgy1 and Erg4 are no longer functional and which contains a polynucleotide comprising a DNA sequence which codes for the GLUT4V85M protein may be prepared, for example, by a) providing a yeast cell whose glucose transporters in their entirety and also the proteins Fgy1 and Erg4 are no longer functional, b) providing an isolated and purified polynucleotide which comprises a DNA sequence coding for the GLUT4V85M protein and which can be replicated in the yeast cell, c) transforming the yeast cell from a) with the polynucleotide from b), d) selecting a transformed yeast cell, e) where appropriate expressing the GLUT4V85M protein.

The abovementioned isolated and purified polynucleotide which comprises a DNA sequence coding for the GLUT4V85M protein is preferably a vector which can be replicated in a yeast cell and in which said DNA sequence was cloned. An example of such a vector is p4H7GLUT4V85M (Seq ID No. 3).

The invention also relates to a yeast cell whose glucose transporters in their entirety are no longer functional and which contains a polynucleotide comprising a DNA sequence which calls for the GLUT4V85M protein.

Said yeast cell can also express the GLUT4V85M protein and thus contain said protein. A preferred yeast strain of this kind is the Saccharomyces cerevisiae 15188 yeast strain deposited with the DS Z.

A yeast cell whose glucose transporters in their entirety are no longer functional and which contains a polynucleotide comprising a DNA sequence which codes for the GLUT4 V85 protein may be prepared, for example, by a) providing a yeast cell whose glucose transporters in their entirety are no longer functional, b) providing an isolated and purified polynucleotide which comprises a DNA sequence coding for the GLUT4V85M protein and which can be replicated in the yeast cell, c) transforming the yeast cell from a) with the polynucleotide from b), d) selecting a transformed yeast cell, e) where appropriate expressing the GLUT4V85M protein.

An isolated and purified polynucleotide which comprises a DNA sequence coding for the GLUT4V85M protein is preferably a vector which can be replicated in a yeast cell and in which said DNA sequence was cloned. An example of such a vector is p4H7GLUT4V85M (Seq ID No. 3).

The invention also relates to a protein having the amino acid sequence according to Seq ID No. 2. Said protein is a human GLUT4 protein in which a valine has been replaced by a methionine in position 85 of the amino acid chain.

The invention also relates to a method for identifying a compound which stimulates the activity of a GLUT4 protein, which method comprises the steps a) providing a yeast cell whose glucose transporters in their entirety and also Erg4 protein are no longer functional and which contains a polynucleotide comprising a DNA sequence which codes for a protein GLUT4V85M, b) providing a chemical compound, c) contacting the yeast of a) with the chemical compound of b), d) determining glucose uptake by the yeast of c), e) relating the detected value of the glucose uptake of d) to the detected value of glucose uptake in a yeast cell as claimed in a) which has been contacted with a chemical compound as claimed in b), with a compound which causes an increase in the amount of glucose taken up in the yeast as claimed in d) stimulating the activity of said GLUT4 protein. Compounds which stimulate the activity of the GLUT4V85M protein can be assumed to stimulate also the GLUT4 activity.

The invention also relates to a pharmaceutical which contains a compound which has been identified by the method described above and furthermore to additives and excipients for formulating a pharmaceutical. Furthermore, the invention relates to the use of a compound which has been identified by the method described above for producing a pharmaceutical for the treatment of type I and/or II diabetes.

The invention also relates to a pharmaceutical comprising a compound which has been identified by the method described above and to additives and excipients for formulating a pharmaceutical. Furthermore, the invention relates to the use of a compound identified by the method described above for producing a pharmaceutical for the treatment of diabetes.

The invention furthermore relates to the use of a compound identified by a method described above for producing a pharmaceutical for the treatment of diabetes. ^ The present invention also comprises a method for identifying a compound which inhibits the protein encoded by the Erg4 gene, which method comprises the steps: a) providing a yeast cell whose glucose transporters in their entirety and no longer functional and which contains a polynucleotide comprising a DNA sequence which codes for the GLUT4V85M protein and can be replicated in a yeast cell, b) providing a chemical compound c) contacting the yeast of a) with the chemical compound of b), d) determining glucose uptake by the yeast of c), e) relating the detected value of the glucose uptake of d) to the detected value of glucose uptake in a yeast cell as claimed in a) which is not contacted with a chemical compound as claimed in b), with a compound which causes an increase in the amount of glucose taken up in the yeast as claimed in d) stimulating the activity of a protein Erg4.

The invention furthermore relates to a method for identifying a compound inhibiting the corresponding protein of the Fgy1 gene, which comprises the steps: a) providing a yeast cell whose glucose transporters in their entirety and whose Erg4 protein are no longer functional and which contains a GLUT4 protein, b) providing a chemical compound c) contacting the yeast of a) with the chemical compound of b), d) determining glucose uptake by the yeast of c), e) relating the detected value of the glucose uptake of d) to the detected value of glucose uptake in a yeast cell as claimed in a) which is not contacted with a chemical compound as claimed in b), with a compound which causes an increase in the amount of glucose taken up in the yeast as claimed in d) stimulating the activity of a protein Fgy1.

The invention also relates to a pharmaceutical comprising a compound which has been identified by the method described above and to additives and excipients for formulating a pharmaceutical.

The invention may be illustrated in more detail below with respect to technical details. ^ Hybridization means the assembling of two nucleic acid single strands having complementary base sequences to double strands. Hybridization may take place between two DNA strands, one DNA and one RNA strand and between two RNA strands. In principle, it is possible to prepare hybrid molecules by heating the nucleic acids involved which may initially be in double-stranded form, by boiling, for example, in a waterbath for 10 minutes, until they disintegrate into single-stranded molecules without secondary structure. Subsequently, they can be cooled slowly. During the cooling phase, complementary chains pair to give double-stranded hybrid molecules. Under laboratory conditions, hybridizations are usually carried out with the aid of hybridization filters to which single-stranded or denaturable polynucleotide molecules are applied by blotting or electrophoresis. It is possible to visualize the hybridization using appropriate complementary polynucleotide molecules by providing said polynucleotide molecules to be hybridized with a radioactive i r fluorescent label. Stringency describes the degree of matching or alignment of 5 particular conditions. High stringency has higher demands on matching than low stringency. Depending on the application and objective, particular conditions with different stringency are set for the hybridization of nucleic acids. At high stringency, the reaction conditions for the hybridization are set in such a way that only complementary molecules which match very well can hybridize with one another. 10 Low stringency enables molecules also to partially hybridize with relatively large sections of unpaired or mispaired bases.

The hybridization conditions are to be understood as being stringent, in particular, if the hybridization is carried out in an aqueous solution containing 2 x SSC at 68°C for 15 at least 2 hours, followed by washing first in 2x SSC/0.1 % SDS at room temperature for 5 minutes, then in 1 x SSC/0.1 % SDS at 68 °C for 1 hour and then in 0.2% SSC/0.1 % SDS at 68°C for another hour.

A 2 x SSC, 1 x SSC or 0.2 x SSC solution is prepared by diluting a 20 x SSC 20 solution appropriately. A 20 x SSC solution contains 3 mol/l NaCI and 0.3 mol/l Na citrate. The pH is 7.0. The skilled worker is familiar with the methods for hybridizations of polynucleotides under stringent conditions. Appropriate instructions can be found in specialist books such as, in particular, Current Protocols in . Molecular Biology (Wiley Interscience; editors: Frederich M. Ausubel, Roger Brant, 25 Robert E. Kingston, David J. Moore, J. G. Seidmann, Kevin Struhl; ISBN: 0-471- 50338-X).

The yeast vectors can be divided into different subgroups. Yip vectors (yeast integrating plasmids) essentially correspond to the vectors used in bacteria for 30 clonings, but contain a selectable yeast gene (e.g. URA3, LEU2).

Only when the foreign DNA integrates into a yeast chromosome after introduction of said vector, are these sequences replicated together with the chromosome and, with the formation of a clone, stably transferred to all daughter cells.

Based on this method, plasmids have been derived which can replicate autonomously owing to eukaryotic ORIs (origins of replication). Such yeast vectors are referred to as YRp vectors (yeast replicating plasmids) or ARS vectors (autonomously replicating sequence). Furthermore, there are YEp vectors (yeast episomal plasmids) which are derived from the yeast 2μΓη plasmid and which contain a selective marker gene. The class of the YAC vectors (yeast artificial chromosome) behave like independent chromosomes.

A yeast vector containing a gene to be expressed is introduced into the yeast by means of transformation in order for said gene to be able to be expressed. Examples of methods suitable for this purpose are electroporation or incubation of competent cells with vector DNA. Suitable yeast expression promoters are known to the skilled worker, examples being the SOD1 promotor (superoxide dismutase), ADH promotor (alcohol dehydrogenase), the promotor of the gene for acidic phosphatase, HXT2 promotor (glucose transporter 2), HXT7 promotor (glucose transporter 7), GAL2 promotor (galactose transporter) and others. The construct comprising a yeast expression promotor and a gene to be expressed (e.g. GLUT4V85 ) is, for the purpose of expression, part of a yeast vector. To carry out expression, said yeast vector may be a self-replicating particle which is independent of the yeast genome or may be stably integrated into the yeast genome. A suitable yeast vector is in principle any polynucleotide sequence which can be propagated in a yeast. Yeast vectors which may be used are in particular yeast plasmids or yeast artificial chromosomes. Yeast vectors usually contain an origin of replication (2μ, ars) or starting the replication process and a selection marker which usually comprises an auxotrophy marker or an antibiotic resistance gene. Examples of yeast vectors known to the skilled worker are pB 272, pCS19, pEMBCYe23, pFL26, pG6, pNN414, pTV3, p426MET25, p4H7 and others.

In accordance with the present invention, selection of a cell means the specific concentration thereof, owing to a selection marker such as, for example, resistance to an antibiotic or the ability to grow on a particular minimal medium, and furthermore the isolation and subsequent cultivation thereof on an agar plate or in submerged culture.

Cultivation, transformation and selection of a transformed yeast cell and also expression of a protein in a yeast cell are among the methods commonly used by the skilled worker. Instructions regarding said methods can be found in standard text books, for example in Walker Graeme M.: Yeast Physiology and Biotechnology, Wiley and Sons, ISBN: 0-471-9446-8 or in Protein Synthesis and Targeting in Yeast, Ed. Alistair J. P. Brown, Mick F. Fruite and John E. G. Mc Cartly; Springer Berlin; ISBN: 3-540-56521-3 or in "Methods in Yeast Genetics, 1997: A Cold Spring Harbor Laboratory Course Manual; Adams Alison (Edt.); Cold Spring Harbor Laboratory; ISBN: 0-87969-508-0".

The yeast Saccharomyces cerevisiae has 17 known hexose transporters and additionally three known maltose transporters, which are capable of transporting hexoses into said yeast, provided that their expression is sufficiently high. In one known strain all transporters suitable for hexose uptake were removed by deletion. Said strain contains merely just the two genes MPH2 and MPH3 which are homologous to maltose transport proteins. The two genes MPH2 and MPH3 are repressed in the presence of glucose in the medium. Wieczorke et al., FEBS Lett. 464, 123 - 128 (1999) describe the preparation and characterization of this yeast strain. Said strain is not able to propagate on a substrate containing glucose as sole carbon source. It is possible to select from said strain mutants which functionally express GLUT1 , starting from a corresponding vector (hxt fgy1-1 strain).

If the yeast strain hxt fgy1-1 is transformed with a plasmid vector which carries a GLUT4 gene under control of a yeast promoter, still only very little glucose is transported. Functional GLUT4 expression requires further adjustments to this yeast strain in order to make possible a significant glucose transport by means of GLUT4. Such yeast strains whose cells take up glucose by means of a single glucose transporter GLUT4 can be isolated on substrates having glucose as sole carbon source. For this purpose, a yeast hxt fgy1-1 strain carrying a GLUT4 gene under the functional control of a yeast promotor is transformed. These yeast cells transformed in this way are applied to a nutrient medium containing glucose as sole carbon source and are incubated thereon. After a few days of incubation at, for example 30 °C, the growth of individual colonies is observed. One of these colonies is isolated. The removal of the yeast plasmid from said colony prevents propagation on the nutrient medium containing glucose as sole carbon source, if this strain which no longer contains a vector'plasmid is again transformed with a yeast vector carrying a GLUT4 gene under the functional control of a yeast promotor, said strain is then again able to propagate on a medium containing glucose as sole carbon source. The respective mutation of this strain is called fgy4-X and allows propagation of the strain on a medium with glucose as sole carbon source.

The abovementioned yeast strains are the subject matter of International Application PCT/EP02/01373 (corresponding to WO 02/064784), filed on February 9, 2002, which claims the priority of DE 10106718.6 of February 14, 2002. Moreover, this document discloses yeast strains comprising mutants of the GLUT1 protein, transforming the GLUT1 protein to a functional glucose transporter.

Yeast strains whose indigenous transporters for hexoses (glucose transporters) in their entirety are no longer functional have already been deposited at an earlier date in connection with International Application PCT/EP02/01373 (corresponding to WO 02/064784) with the Deutsche Sarnmlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the number DSM 14035, DSM 14036 or DSM 14037.

The polynucleotide and amino acid sequences of GLUT4 are accessible, for example, via the following entries in gene banks: M20747 (cDNA; human), EMBL: D28561 (cDNA; rat), EMBL: M23382 (cDNA; mouse), Swissprot P14672 (protein; human), Swissprot: P19357 (protein; rat) and Swissprot: P 142 (protein; mouse), Polynucleotide sequences and amino acid sequences of GLUT1 are disclosed under the following code numbers of the databases indicated: EMBL: M20653 (cDNA; human), EMBL: M13979 (cDNA; rat), EMBL: M23384 (cDNA; mouse), Swissprot: P1 1166 (protein; human), Swissprot: P1 1167 (protein; rat) and Swissprot: P17809 (protein; mouse).

Pharmaceuticals are dosage forms of pharmacologically active substances for the therapy of diseases or bodily malfunctions in humans and animals. Examples of dosage forms for oral therapy are powders, granules, tablets, pills, lozenges, sugar-coated tablets, capsules, liquid extracts, tinctures and syrups. Examples which are used for external application are aerosols, sprays, gels, ointments or powders. Injectable or infusible solutions allow parenteral administration, using vials, bottles or prefilled syringes. These and other pharmaceuticals are known to the skilled worker in the field of pharmaceutical technology.

Excipients for formulating a pharmaceutical made possible the preparation of the active substance with the purpose of optimizing the application, distribution and development of action of the active ingredient for the particular application.

Examples of such excipients are fillers, binders, disintegrants or glidants, such as lactose, sucrose, mannitol, sorbitol, cellulose, starch, dicalcium phosphate, polyglycols, alginates, polyvinylpyrrolidone, carboxymethylcellulose, talc or silicon dioxide.

Diabetes manifests itself by the excretion of glucose together with the urine with an abnormal increase in the blood glucose level (hyperglycaemia), owing to a chronic metabolic condition due to insulin deficiency or reduced insulin action. The lack of, or reduced, insulin action leads to insufficient absorption and conversion by the cells of the glucose taken up into the blood. In fatty tissue, insulin-antagonistic hormones have the effect of increasing lypolysis accompanied by an increase in the free fatty acid levels in the blood.

Adiposity (obesity) is the abnormal weight gain owing to an energy imbalance due to excessive intake of calories, which constitutes a health risk.

The amount of a hexose which is taken up by a provided yeast strain as described just above can be determined by means of uptake studies using radioactively labeled glucose. For this purpose, a particular concentration of the yeast cells is suspended in, for example, 100 μΙ of a buffer, for example at a concentration of 60 mg (wet weight) per ml, and admixed with a defined amount of ^c- or ^H-labeled glucose as sole carbon source. The cells are incubated, and defined amounts thereof are removed at specific times. The amount of glucose taken up is determined with the aid of LSC (Liquid Scintillation Counting). The amount of a hexose which is taken up by a yeast strain provided and as just described above may, however, also be determined by means of a growth assay on media containing glucose as sole carbon source. For this purpose, the rate of growth of the strain is determined, after addition of the compound, for example by measuring the optical density of the culture at 600 nm at regular intervals, and this value is compared with the rate of growth of a control strain {e.g. yeast wild-type strain).

A compound is provided, in particular, by chemical synthesis or by isolating chemical substances from biological organisms. It is also possible to carry out chemical synthesis in an automated manner. The compounds obtained by synthesis or isolation can be dissolved in a suitable solvent. Suitable solvents are in particular aqueous solutions which contain a specific proportion of an organic solvent such as, for example, DMSO (dimethylsulfoxide).

Conducting a strain of the yeast with a compound for identifying a compound in accordance with an invention mentioned above is done in particular in automated laboratory systems provided therefor. Such systems may comprise specifically prepared chambers with depressions, or microtiter plates, Eppendorf tubes or laboratory glassware. Automated laboratory systems are usually designed for high throughput rates. A method such as the one mentioned above, carried out with the aid of an automated laboratory system, is therefore also referred to as HTS (High Throughput Screening).

Seq ID No. 1 discloses a polynucleotide sequence comprising the coding region of the GLUT4V85 protein, Seq ID No. 2 discloses the amino acid sequence of the GLUT4V85M protein. Seq ID No. 3 discloses the polynucleotide sequence of the p4H7GLUT4V85M vector.

Examples Use of yeast strains All of the yeast strains described herein were derived from strain CEN-PK2-1C (MATa leu2-3, 112 ura3-52 trp1-289 his3-A1MAL2-8c SUC2). The preparation of a yeast strain having deletions in the hexose transporter genes (HXT) has been described by Wieczorke et al., FEBS Lett. 464, 123 - 128 (1999): EBY-18ga (MATa Δηχί1-17 Agal2 Aagtl AstH leu2-3, 112 ura3-52 trp 1-289 his3-A1 MAL2-8C SUC2), EBY.VW4000 (MATa Ahxt1-17 Agal2 Aagtl Amph2 Amph3 AstH leu2-3, 112 ura3-52 trp1-289 his3-Al MAL2-8C SUC2). The media were based on 1 % yeast extract and 2% peptone (YP), while the minimal media were composed of 0.67% Difco yeast nitrogen base without amino acids (YNB) and contained additives required for auxotrophy and different carbon sources. The yeast cells were grown under aerobic conditions at 30°C on a rotary shaker or on agar plates. Cell growth was monitored by measuring the optical density at 600 nm (OD6oo) or by determining the diameter of yeast colonies.

Determination of glucose uptake Glucose transport was measured as uptake of D-[U- 4C]-glucoses (Amersham) and the kinetic parameters were determined from Eadie-Hofstee plots. The celts were removed by centrifugation, washed with phosphate-buffer and resuspended in phosphate buffer at a concentration of 60 mg (wet weight) per ml. Glucose uptake was determined for glucose concentrations between 0 2 and 100 mM, and the specific activity of the substrate was between 0.1 and 55.5 kBq μητιοΓ1. The cells and the glucose solutions were prelncubated at 30°C for 5 minutes, Glucose uptake was started by adding radioactive glucose to the cells. After incubation for 5 seconds, 10 ml of ice-cold stop buffer (0.1 M KiPO,,, pH 6.5, 500 mM glucose) were added and the cells were removed quickly by filtering on glass fiber filters (0 = 24 mm, Whatman). The filters were quickly washed three times with ice-cold buffer and the radioactivity incorporated was measured using a liquid scintillation counter. An addition by cytochalasin B (final concentration 20μΜ, dissolved in ethanol) was measured in a 15-second uptake assay with 50 mM or 100 mM radioactive glucose, after the cells had been incubated in the presence of the inhibitor or of only the solvent for 15 minutes.

A heterologous expression system for glucose transporters from mammalian cells has been developed. The system is based on an S, cerevisiae strain from which all endogenous glucose transporters have been removed destroying the encoding genes. Said strain is no longer able to take up glucose via the plasma membrane and to grow with glucose as sole carbon source. In order to integrate the heterologous glucose transporters of humans or of other mammals, GLUT1 and GLUT4 in an active form into the yeast plasma membrane, additional mutations had to be introduced into the yeast strain. GLUT1 is active only in an fgy1-1 mutant strain and GLUT4 only in fgy1-1 fgy4-X double mutants.

The FGY1 gene has been cloned. It is the S. cerevisiae ORF YMR212c. With respect to the function, the results indicate that either Fgy1 or a product generated by Fgy1 inhibits the activity of human glucose transporters or is involved in fusing the GLUT-transporting vesicles to the plasma membrane.

In contrast to GLUT1 and similarly to mammalian cells, a large proportion of the GLUT4 proteins in the yeast is located in intracellular structures. A total of nine recessive mutants were isolated (fgy4-1 to fgy4-9) in which GLUT4 is now directed further to the plasma membrane and, in the case of a concomitant fgy1-1 mutation, becomes active there.

The insertion gene bank described by Bruns et al. (Genes Dev. 1994; 8: 1087-105) was used for complementation analysis. The hxt fgy1-1 strain was transformed first with a GLUT4 plasmid and then with the mobilized insertion gene bank. This was followed by screening for transformants which were able to grow on glucose medium. It turned out that in one of the mutants studied the ERG4 gene had been destroyed. ERG4 codes for an enzyme (oxidoreductase) of ergosterol biosynthesis. This enzyme, sterol C-24(28)-reductase catalyzes the last step of ergosterolbiosynthesis and converts ergosta-5,7,22,24,{28)-tetraenol to the final product ergosterol. The Erg4 protein presently contains eight transmembrane domains and is located in the endoplasmic reticulum. An erg4 mutant is viable, since incorporation of the ergosterol precursors into the yeast membranes compensates for the loss of ergosterol.

The inhibiting influence of Erg4 on GLUT4 functionality was confirmed by specific deletion of erg4 in the hxt fgy1-1 strain. The resulting strain (hxt fgy1-1 Aerg4) was referred to as SDY022.

Protein interaction assays with the aid of the split-ubiquitin system showed that human GLUT4 directly interacts with yeast Erg4. It can therefore be assumed that the yeast Erg4 protein in the endoplasmic reticulum either directly prevents further translocation of GLUT4 or modifies GLUT4 in some way which is important for translocation and/or function.

Likewise, it was shown that deletion of ERG4 in the hxt null strain alone, i.e. despite functional FGY1 , activates GLUT1 , but not GLUT4. The results of the growth assay are summarized in Table 1.

In order to rule out that Ergosterol itself exerts a negative influence on GLUT4, growth assays were carried out on agar plates containing Ergosterol under aerobic conditions. Any yeast strains transformed with GLUT4 were unable to grow under these conditions (Table 2). The GLUT1 transformants in the hxt fgy1-1 strain showed, in contrast to aerobic growth, no growth on glucose under anaerobic conditions. GLUT1 transformants were able to grow only after deletion of ERG4.

The exchange of Val85 for Met by in vitro mutagenesis rendered GLUT4 independent of the fgy1-1 mutation and resulted in GLUT4V85M being functional even in an hxt erg4 strain. This observation indicates that Fgy1 acts directly or indirectly on this position which is located within the second transmembrane helix of GLUT transporters.

Table 3 displays the descriptions of the yeast strains deposited in connection with the present patent application with the Deutsche Sammlung von Mikroorganismen und Ze!lkulturen (DS Z) - Mascheroder Weg 1b 38124 Brunswick, Germany.

Table 1 : Growth of GLUT1 and GLUT4 transformants on glucose medium.

Table 2: Growth of GLUT1 and GLUT4 transformants on glucose medium with or without ergosterol under anaerobic conditions Table 3: Features of the deposited yeast strains (Saccharomyces cerevisiae) Basic medium: 0.67% Yeast Nitrogen Base without amino acids (Difco); pH 6.2. Auxotrophy supplementation: Leucine (0.44 mM), tryptophan (0.19 mM), histidine (0.25 mM9, uracil (0.44 mM). Maltose may be between 1 -2%.

Material described in the specification, which is not within the ambit of the claims is not covered by the claimed invention. The scope of protection is as defined in the claims, and as stipulated in the Patent Law (5727-1967).

BUDAPEST TREATY ON THE INTERNATIONAL DSMZ RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlung FOR THE PURPOSES OF PATENT PROCEDURE von Mikroorganismen und Zellkulturen GmbH INTERNATIONAL FORM Aventis Pharma Deutschland GmbH Industriepark Hochst RECEIPT IN THE EVENT OF AN ORIGINAL DEPOSIT D-65926 Frankfurt Issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY Identified at the bottom of this page I. IDENTIFICATION OF THE MICROORGANISM Identification reference given by the ACCESSION NUMBER issued by DEPOSITOR: the INTERNATIONAL DEPOSITARY AUTHORITY: DSMefg DSM 15185 II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION The microorganism identified in section I was accompanied by: 13 a scientific description El a proposed taxonomic designation (Indicate as applicable) RECEIPT AND ACCEPTANCE The present International Depositary Authority accepts the microorganism identified in section I, which it received on 2002-09-03 (date of the original deposit)1 IV. RECEIPT OF A REQUEST FOR CONVERSION The present International Depositary Authority received the microorganism identified under section I on (date of the original deposit) and received a request for conversion of the original deposit to a deposit conforming to the Budapest Treaty on (date of receipt of the request for conversion) V. INTERNATIONAL DEPOSITARY AUTHORITY Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of the person(s) having the power to represent MIKROORGANISMEN UND ZELLKULTUREN GmbH the International Depositary Authority, or of authorized official(s) Address: Mascheroder Weg 1b (illegible signature) D- 38124 Brunswick Date: 2002-09-10 If Rule 6.4.d) applies, this date shall be the date on which the status of the international depositary authority was acquired.

Form DSMZ-BP/4 (single page) 12/2001 BUDAPEST TREATY ON THE INTERNATIONAL DS Z RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlung FOR THE PURPOSES OF PATENT PROCEDURE von Mikroorganismen und Zellkulturen GmbH INTERNATIONAL FORM VIABILITY STATEMENT issued pursuant to Rule 10.2 by trie INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page The viabiiity of the microorganism identified under section II above was tested on 2002-09-062. On that date, the said microorganism was viable no longer viable IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED4 V. INTERNATIONAL DEPOSITARY AUTHORITY Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of the person(s) having the power to represent MIKROORGANISMEN UND ZELLKULTUREN GmbH the International Depositary Authority, or of authorized officials ) Address: Mascheroder Weg 1b D-38124 Brunswick (illegible signature) Date: 2002-09-10 Indicate the date of the original deposit or, where a new deposit or a transfer has been made, the date of the most recent relevant new deposit or transfer.

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Form DSMZ-BP/9 (single page) 12/2001 BUDAPEST TREATY ON THE INTERNATIONAL DSMZ RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlung FOR THE PURPOSES OF PATENT PROCEDURE von Mikroorganismen und Zellkulturen GmbH INTERNATIONAL FORM Aventis Pharma Deutschland GmbH Industriepark Hochst RECEIPT IN THE EVENT OF AN ORIGINAL DEPOSIT D-65926 Frankfurt issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page I. IDENTIFICATION OF THE MICROORGANISM Identification reference given by the ACCESSION NUMBER issued by DEPOSITOR: the INTERNATIONAL DEPOSITARY AUTHORITY: DSMhij DSM 15186 II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION The microorganism identified in section I was accompanied by: B a scientific description B a proposed taxonomic designation (Indicate as applicable) RECEIPT AND ACCEPTANCE The present International Depositary Authority accepts the microorganism identified in section I, which it received on 2002-09-03 (date of the original deposit)1 IV. RECEIPT OF A REQUEST FOR CONVERSION The present International Depositary Authority received the microorganism identified under section I on (date of the original deposit) and received a request for conversion of the original deposit to a deposit conforming to the Budapest Treaty on (date of receipt of the request for conversion) V. INTERNATIONAL DEPOSITARY AUTHORITY Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of the person(s) having the power to represent MIKROORGANISMEN UND ZELLKULTUREN the International Depositary Authority, or of authorized GmbH official(s) Address: Mascheroder Weg 1b (illegible signature) D-38124 Brunswick Date: 2002-09-10 If Rule 6.4.d) applies, this date shall be the date on which the status of the international depositary authority was acquired.

Form DSMZ-BP/4 (single page) 12/2001 BUDAPEST TREATY ON THE INTERNATIONAL DSMZ RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlung FOR THE PURPOSES OF PATENT PROCEDURE von ikroorganismen und Ze!ikulturen GmbH INTERNATIONAL FORM VIABILITY STATEMENT Issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page The viability of the microorganism identified under section II above was tested on 2002-09-062. On that date, the said microorganism was viable no longer viable IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED4 Indicate the date of the original deposit or, where a new deposit or a transfer has been made, the date of the most recent relevant new deposit or transfer.

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Form DSMZ-BP/9 (single page) 12/2001 BUDAPEST TREATY ON THE INTERNATIONAL DSMZ RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlung FOR THE PURPOSES OF PATENT PROCEDURE von Mikroorganismen und Zellkulturen GmbH INTERNATIONAL FORM Aventis Pharma Deutschland GmbH Industriepark Hochst RECEIPT IN THE EVENT OF AN ORIGINAL DEPOSIT D-65926 Frankfurt issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page IDENTIFICATION OF THE MICROORGANISM Identification reference given by the ACCESSION NUMBER issued by DEPOSITOR: the INTERNATIONAL DEPOSITARY AUTHORITY: DSMsyz DSM 15187 II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION The microorganism identified in section I was accompanied by: I3 a scientific description H a proposed taxonomic designation (Indicate as applicable) RECEIPT AND ACCEPTANCE The present international Depositary Authority accepts the microorganism identified in section I, which it received on 2002-09-03 (date of the original deposit)1 IV. RECEIPT OF A REQUEST FOR CONVERSION The present International Depositary Authority received the microorganism identified under section I on (date of the original deposit) and received a request for conversion of the original deposit to a deposit conforming to the Budapest Treaty on (date of receipt of the request for conversion) V. INTERNATIONAL DEPOSITARY AUTHORITY Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of the person(s) having the power to represent MIKROORGANISMEN UND ZELLKULTUREN the International Depositary Authority, or of authorized GmbH officials} Address: Mascheroder Weg 1b (illegible signature) D-38124 Brunswick Date: 2002-09-10 If Rule 6.4.d) applies, this date shall be the date on which the status of the international depositary authority was acquired.

Form DSMZ-BP/4 (single page) 12/2001 BUDAPEST TREATY ON THE INTERNATIONAL DSMZ RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlung FOR THE PURPOSES OF PATENT PROCEDURE von Mikroorganismen und Zellkulturen GmbH INTERNATIONAL FORM VIABILITY STATEMENT issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page The viability of the microorganism identified under section II above was tested on 2002-Q9-062. On that date, the said microorganism was viable no longer viable IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED4 Indicate the date of the original deposit or, where a new deposit or a transfer has been made, the date of the most recent relevant new deposit or transfer. !n the cases referred to in Rule 10.2(a)(ii) and (iii), refer to the most recent viability test.

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Form DSMZ-BP/9 (single page) 12/2001 BUDAPEST TREATY ON THE INTERNATIONAL DSM2 RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlung FOR THE PURPOSES OF PATENT PROCEDURE von ikroorganismen iind Zellkulturen GmbH INTERNATIONAL FORM Aventis Pharma Deutschland GmbH Industriepark HOchst RECEIPT IN THE EVENT OF AN ORIGINAL DEPOSIT D-65926 Frankfurt issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page I. IDENTIFICATION OF THE MICROORGANISM Identification reference given by the ACCESSION NUMBER issued by DEPOSITOR: the INTERNATIONAL DEPOSITARY AUTHORITY: DSMuvw DSM 15188 II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION The microorganism identified in section I was accompanied by: H a scientific description EJ a proposed taxonomic designation (Indicate as applicable) I. RECEIPT AND ACCEPTANCE The present International Depositary Authority accepts the microorganism identified in section I, which it received on 2002-09-03 (date of the original deposit)1 IV. RECEIPT OF A REQUEST FOR CONVERSION The present International Depositary Authority received the microorganism identified under section I on (date of the original deposit) and received a request for conversion of the original deposit to a deposit conforming to the Budapest Treaty on (date of receipt of the request for conversion) V. INTERNATIONAL DEPOSITARY AUTHORITY Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of the person(s) having the power to represent MIKROORGANISMEN UND ZELLKULTUREN the International Depositary Authority, or of authorized GmbH official(s) Address: Mascheroder Weg 1 b (illegible signature) D-38124 Brunswick Date: 2002-09-10 If Rule 6.4.d) applies, this date shall be the date on which the status of the international depositary authority acquired.

Form DSMZ-BP/4 (single page) 12/2001 BUDAPEST TREATY ON THE INTERNATIONAL DSMZ RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlung FOR THE PURPOSES OF PATENT PROCEDURE von Mikroorganismen und Zellkulturen GmbH INTERNATIONAL FORM VIABILITY STATEMENT issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page The viability of the microorganism identified under section II above was tested on 2002-09-06 . On that date, the said microorganism was viable no longer viable IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED4 V. INTERNATIONAL DEPOSITARY AUTHORITY Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of the person(s) having the power to represent MIKROORGANISMEN UND ZELLKULTUREN the International Depositary Authority, or of authorized GmbH official(s) Address: Mascheroder Weg 1 b (illegible signature) D-38124 Brunswick Date: 2002-09-10 Indicate the date of the original deposit or, where a new deposit or a transfer has been made, the date of the most recent relevant new deposit or transfer.

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Form DSMZ-BP/9 (single page) 12/2001 BUDAPEST TREATY ON THE INTERNATIONAL DSMZ RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlung FOR THE PURPOSES OF PATENT PROCEDURE von Mikroorganismen und Zellkulturen GmbH INTERNATIONAL FORM Aventis Pharma Deutschland GmbH Industriepark Hiichst RECEIPT IN THE EVENT OF AN ORIGINAL DEPOSIT D-65926 Frankfurt Issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY Identified at the bottom of this page IDENTIFICATION OF THE MICROORGANISM Identification reference given by the ACCESSION NUMBER issued by DEPOSITOR: the INTERNATIONAL DEPOSITARY AUTHORITY: DSMdef DSM 15184 II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOM!C DESIGNATION The microorganism identified in section I was accompanied by: El a scientific description E] a proposed taxonomic designation (Indicate as applicable) RECEIPT AND ACCEPTANCE The present International Depositary Authority accepts the microorganism identified in section I, which it received on 2002-09-03 (date of the original deposit)1 IV. RECEIPT OF A REQUEST FOR CONVERSION The present International Depositary Authority received the microorganism identified under section I on (date of the original deposit) and received a request for conversion of the original deposit to a deposit conforming to the Budapest Treaty on (date of receipt of the request for conversion) V, INTERNATIONAL DEPOSITARY AUTHORITY Name: DSMZ-DEUTSCHE SAMMLUNG VON. Signature(s) of the person(s) having the power to represent MIKROORGANISMEN UND ZELLKULTUREN the International Depositary Authority, or of authorized GmbH official(s) Address: Mascheroder Weg 1 b (illegible signature) D-38124 Brunswick Date: 2002-09-10 If Rule 64, d) applies, this date shall be the date on which the status of the international depositary authority was acquired.

Form DSMZ-BP/4 (single page) 12/2001 BUDAPEST TREATY ON THE INTERNATIONAL DSMZ RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlune FOR THE PURPOSES OF PATENT PROCEDURE von Mikroorganismen und Zellkulturen GmbH INTERNATIONAL FORM VIABILITY STATEMENT issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page The viability of the microorganism identified under section II above was tested on 2002-09-062. On that date, the said microorganism was viable no longer viable IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED4 V. INTERNATIONAL DEPOSITARY AUTHORITY Name: DS Z-DEUTSCHE SAMMLUNG VON Signature(s) of the person(s) having the power to represent MIKROORGANISMEN UND ZELLKULTUREN the International Depositary Authority, or of authorized GmbH official(s) Address: Mascheroder Weg 1b (illegible signature) D-38124 Brunswick Date: 2002-09-10 Indicate the date of the original deposit or, where a new deposit or a transfer has been made, the date of the most recent relevant new deposit or transfer.

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Form DSMZ-BP 9 (single page) 12/2001 31 167321/2 SEQUENCE LISTING <-110> Aventis P arma Deutschlancl GmbH Use of Saccaxocoyces cerevisiae Erg4 for the expression of * * glucose transporters from mammals <13Q> 2002/0065 <140> <141> <150> 10242763.1 <151> 2002-03-14 <160> 3 <170> Patentln Ver. 2.1 <210> 1 <211> 1530 <212> DNA <213> Homo sapiens <400> 1 atgeegtegg gcttccaaca gataggctcc gaagatgggg aaccccctca geagegagtg 60 actgggaccc tggtccttgc tgtgttctot gcggtgcttg gctccctgca gtttgggtac 120 aacattgggg teatcaatge ccctcagaag gtgattgaac agagctacaa tgagacgtgg 180 ctggggaggc aggggectga gggacccagc tccatccctc caggcaccct caccaccctc 240 tgggccctct ccatggccat ettttcegtg ggcggcatga tttcctcctt cctcattggt 300 atcatctctc agtggcCtgg aaggaaaagg gecatgetgg tcaacaatgt cctggcggtg 3S0 ctggggggca gcctcatggg cctggccaac gctgctgcct cctatgaaat gctcatcctt, 420 ggacgattcc teattggege ctactcaggg ctgacatcag ggctggtgcc catgtacgtg 4B0 ggggagattg ctcccactca cctgcggggc gccctgggga cgctcaacca actggecatt 540 gttateggea ttctgatege ccaggtgotg ggcttggagt ccctcctggg cactgccagc 600 ctgtggccac tgctcctggg cctcacagtg ctacctgccc tcctgcagct ggtcctgctg 660 cccttctgtc ccgagagccc ccgctacctc tacatcatcc agaatctcga ggggcctgcc 720 agaaagagtc tgaagegect gacaggctgg gccgatgttt ctggagtgct ggctgagctg 7B0 aaggatgaga ageggaaget ggagcgtgag cggccactgt ccctgctcca gctcctgggc Θ40 agccgtaccc accggcagcc cctgatcatt gcggtcgtgc tgcagctgag ccagcagctc 900 tctggcatca atgctgtttt ctattattcg accagcatct tcgagacagc aggggtaggc 960 cagcctgcct atgccaccat aggagctggt gtggtcaaca cagtcttcac cttggtctcg 1020 gtgttgttgg tggagcgggc ggggcgccgg acgctccatc tcctgggcct ggcgggcatg 1080 tgtggctgtg ccatcctgat gactgtggct ctgctcctgc tggagcgagt tccagccatg 1140 agetaegtet ccattgtggc catctttggc ttcgtggcat tttttgagat tggccctggc 1200 cccattcctt ggttcatcgt ggccgagctc ttcagccagg gaccccgccc ggcagccatg 1260 gctgtggctg gtttctccaa ctggacgagc aacttcatca ttggcatggg tttccagtat 1320 gttgcggagg ctatggggcc ctacgtcttc cttctatttg cggtcctcct gctgggcttc 1380 32 167321/2 ttcatcttca ccttcttaag agtacctgaa actcgaggcc ggacgtttga ccagatctca 1440 gctgccttcc accggacacc ctctctttta gagcaggagg tgaaacccag cacagaactt 1500 gagtatttag ggccagatga gaacgactga 1530 <210> 2 <211> 509 <212> P&T <213> Homo sapiens <400> 2 Met Pro Ser Gly Phe Gin Gin He Gly Ser Glu Asp Gly Glu Pro Pro 1 5 10 15 Gin Gin Arg Val Thr Gly Thr Leu Val Leu Ala Val Phe Ser Ala Val 20 25 30 Leu Gly Ser Leu Gin Phe Gly Tyr Asn He Gly Val He Asn Ala Pro 35 40 45 Gin Lys Val He Glu Gin Ser Tyr Asn Glu Thr Trp Leu Gly Arg Gin 50 55 60 Gly Pro Glu Gly Pro Ser Ser He Pro Pro Gly Thr Leu Thr Thr Leu 65 70 75 80 Trp Ala Leu Ser Met Ala He Phe Ser Val Gly Gly Met He Ser Ser 85 90 95 Phe Leu He Gly He He Ser Qln Trp Leu Gly Arg Lys Arg Ala Met 100 105 110 Leu Val Asn Asn Val Leu Ala Val Leu Gly Gly Ser Leu Met Gly Leu 115 120 125 Ala Asn Ala Ala Ala Ser Tyr Glu Met Leu He Leu Gly Arg Phe Leu 130 135 140 He Gly Ala Tyr Ser Gly Leu Thr Ser Gly Leu Val Pro Met Tyr Val 145 150 155 ISO Gly Glu He Ala Pro Thr His Leu Arg Gly Ala Leu Gly Thr Leu Asn 1G5 170 175 Gin Leu Ala He Val He Gly He Leu He Ala Gin Val Leu Gly Leu 180 1S5 190 Glu Ser Leu Leu Gly Thr Ala Ser Leu Trp Pro Leu Leu Leu Gly Leu 33 1S732 95 200 205 Thr Val Leu Pro Ala Leu Leu Gin Leu Val Leu Leu Pro Phe Cys Pro 210 215 220 Glu Ser Pro Arg Tyr Leu Tyr He He Gin Asn Leu Glu Gly Pro Ala 225 230 235 240 Arg Lya ser Leu Lys Arg Leu Thr Gly Trp Ala Asp Val Ser Gly Val 245 250 255 Leu Ala Glu Leu Lys Asp Glu Lya Arg Lys Leu Glu Arg Glu Arg Pro 260 2S5 270 Leu Ser Leu Leu Gin Leu Leu Gly Ser Arg Thr His Arg Gin Pro Leu 275 280 2B5 He He Ala Val Val Leu Gin Leu Ser Gin Gin Leu Ser Gly He Asn 290 295 300 Ala Val Phe Tyr Tyr Ser Thr Ser He Phe Glu Thr Ala Gly Val Gly 305 310 315 320 Gin Pro Ala Tyr Ala Thr He Gly Ala Gly Val Val Asn Thr val Phe 325 330 33S Thr Leu Val Ser Val Leu Leu Val Glu Arg Ala Gly Arg Arg Thr Leu 340 345 350 His Leu Leu Gly Leu Ala Gly Met Cys Gly Cys Ala He Leu Met Thr 355 3G0 3S5 Val Ala Leu Leu Leu Leu Glu Arg Val Pro Ala Met Ser Tyr val Ser 370 375 380 He Val Ala He Phe Gly Phe Val Ala Phe Phe Glu He Gly Pro Gly 3B5 390 395 400 Pro He Pro Trp Phe He Val Ala Glu Leu Phe Ser Gin Gly Pro Arg 405 410 415 Pro Ala Ala Met Ala Val Ala Gly Phe Ser Asn Trp Thr Ser Asn Phe 420 425 430 He He Gly Met Gly Phe Gin Tyr Val Ala Glu Ala Met Gly Pro Tyr 435 440 445 Val Phe Leu Leu Phe Ala Val Leu Leu Leu Gly Phe Phe He Phe Thr 34 16732 450 455 460 Phe Leu Arg Val Pro Glu Thr Arg Gly Arg Thr Phe Asp Gin He Ser 4GS 470 475 4B0 Ala Ala Phe His Arg Thr Pro Ser Leu Leu Glu Gin Glu Val Lys Pro 4B5 490 495 Thr Glu Leu Glu Tyr Leu Gly Pro Asp Glu Asn Asp 500 505 c210> 3 <211> 7803 c212> DNA <213> Saccharomyces cerevisiae <400> 3 cgtaggaaca atttcgggcc cctgcgtgtt cttctgaggt tcatctttta catttgcttc 60 tgctggataa ttttcagagg caacaaggaa aaattagatg gcaaaaagtc gtctttcaag 120 gaaaaatccc caccatcttt cgagatcccc tgtaacttat tggcflactga aagaatgaaa 100 aggaggaaaa tacaaaatat actagaactg aaaaaaaaaa agtataaata gagacgatat 240 atgccaatac ttcacaatgt tcgaatctat tcttcatttg cagc attgt aaaataataa 300 aacatcaaga acaaacaagc tcaacttgtc ttttctaaga acaaagaata aacacaaaaa 360 caaaaagttt ttttaatttt aatcaaaaaa tgccgtcggg cttccaacag ataggctccg 420 aagatgggga accccctcag cagcgagtga ctgggaccct ggtccttgct gtgttctctg 4B0 cggtgcttgg ctccctgc'ag tttgggtaca acattggggt catcaatgcc cctcagaagg 540 tgategaaca gagctacaat gagacgtggc tggggaggca ggggcctgag ggacccagct 600 ccatccctcc aggcacccfcc accaccctct gggccctctc catggccatc ttttccgtgg 660 gcggcatgat ttcctccttc ctcattggta tcatctctca gtggcttgga aggaaaaggg 720 ccatgctggt caacaatgtc ctggoggtgc tggggggcag cctcatgggc ctggccaacg 780 ctgctgcctc ctatgaaatg ctcatccttg gacgattcct cattggcgcc tactcagggc 840 tgacatcagg gctggtgccc atgtacgtgg gggagattgc tcccactcac ctgcggggcg 900 ccctggggac gctcaaccaa ctggccattg ttatcggcat tctgatcgcc caggtgctgg 960 gcttggagtc cctcctgggc actgccagcc tgtggccact gctcctgggc ctcacagtgc 1020 tacctgccct cctgcagctg gtcctgctgc ccttctgtcc cgagagcccc cgctacctct 10BO acatcatcca gaatctcgag gggcctgcca gaaagagtct gaagcgcctg acaggctggg 1140 ccga gtttc tggagtgctg gctgagctga aggatgagaa gcggaagctg gagcgtgagc 1200 ggccactgtc cctgctccag ctcctgggca gccgtaccca ccggcagccc ctgatcattg 1260 cggtcgtgct gcagctgagc cagcagctct ctggcatcaa tgctgttttc tattattcga 1320 ccagcatctt cgagacagca ggggtaggcc agcctgccta tgccaccata ggagctggtg 1380 tggtcaacac agtcttcacc ttggtctcgg tgttgttggt ggagcgggcg gggcgccgga 1440 cgctccatct cctgggcctg gcgggcatgt gtggctgtgc catcctgatg actgtggctc 1500 tgctcctgct ggagcgagtt ccagccatga gctacgtctc cattgtggcc atct ggct 1560 tcgtggcatt ttttgagatt ggccctggcc ccattccttg gttcatcgtg gccgagctct 1620 tcagccaggg accccgcccg gcagccatgg ctgtggctgg tttctccaac tggacgagca 16B0 acttcatcat tggcatgggt ttccagtatg ttgcggaggc tatggggccc tacgtcttcc 1740 35 167321/2 ttctatttgc ggtcctcctg ctgggcttct tcatcttcac cttcttaaga gtacctgaaa 1B0O ctcgaggccg gacgtttgac cagatctcag ctgccttcca ccggacaccc tctcttttag 1B60 agcaggaggt gaaacccagc acagaacttg agtatttagg gccagatgag aacgactgac 1920 tcgagtcatg taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct 1980 aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt 2040 tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg 23.00 cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa 2160 ggctttaatt tgcggccggt acccaattcg ccctatagtg agtcgtatta cgcgcgctca 2220 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 2280 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 2340 ccttcccaac agttgcgcag cctgaatggc gaatggcgcg acgcgccctg tagcggcgca 2400 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta 2460 gcgcccgctc ctttcgcttt ttcccttcc tttctcgcca cgttcgccgg ctttccccgt 2520 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac 25B0 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt 2640 tfctcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga 2700 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 2760 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata 2820 ttaacgttta caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 28Θ0 accgcatagg gtaataactg atataattaa attgaagctc taatttgtga gtttagtata 2940 catgcattta cttataatac agttttttag ttttgctggc cgcatcttct caaatatgct 3000 tcccagcctg cttttctgta acgttcaccc tctaccttag catcccttcc ctttgcaaat 3060 agtectctte caacaataat aatgtcagat cctgtagaga ccacatcatc cacggttcta 3120 tactgttgac ccaatgcgtc tcccttgtca tctaaaccca caccgggtgt cataatcaac 31B0 caatcgtaac cttcatctct tccacccatg tctctttgag caataaagcc gataacaaaa 3240 tctttgtcgc tcttcgcaat gtcaacagta cccttagtat attctccagt agatagggag 3300 cccttgcatg acaattctgc taacatcaaa aggcctctag gttcctttgt tacttcttct 3360 gccgcctgct tcaaaccgct aacaatacct gggcccacca caccgtgtgc attcgtaatg 3420 tctgcccatt ctgctattct gtatacaccc gcagagtact gcaatttgac tgtattacca 34Θ0 atgtcagcaa attttctgtc ttcgaagagt aaaaaattgt acttggcgga taatgccttt 3540 agcggcttaa ctgtgccctc catggaaaaa tcagtcaaga tatccacatg tgtttttagt 3600 aaacaaattt tgggacctaa tgcttcaact aactccagta attccttggt ggtacgaaca ,2660 tccaatgaag cacacaagtt tgtttgcttt tcgtgcatga tattaaatag cttggcagca 3720 acaggactag gatgagtagc agcacgt cc ttatatgtag ctttcgacat gatttatctt 3780 cgtttcctgc aggtttttgt tctgtgcagt tgggttaaga atactgggca atttcatgtt 3B40 tcttcaacac tacatatgcg tatatatacc aatctaagtc tgtgctcctt ccttcgttct 3900 tccttctgtt cggaga tac cgaatcaaaa aaatttcaaa gaaaccgaaa tcaaaaaaaa 3960 gaataaaaaa aaaatgatga attgaattga aaagctgtgg tatggtgcac tctcagtaca 4020 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg 40 BO ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg 4140 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc 4200 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gtatgatcca 4260 atatcaaagg aaatgatagc at gaaggat gagactaatc caattgagga gtggcagcat 4320 atagaacagc taaagggtag tgctgaagga agcatacgat accccgcatg gaatgggata 43 BO atatcacagg aggtactaga ctacctttca tcctacataa atagacgcat ataagtacgc 4440 aCttaagcat aaacacgcac tatgccgttc etctcatgta tatatatata caggcaacac 4500 gcagatatag gtgcgacgtg aacagtgagc tgtatgtgcg cagctcgcgt tgcattttcg 4560 gaagcgctcg ttttcggaaa cgctttgaag ttcctattcc gaagttccta ttctctagaa 4620 36 167321/2 agtataggaa cttcagagcg cttttgaaaa ccaaaagcgc tctgaagacg cactttcaaa 46BO aaaccaaaaa cgcaccggac tgtaacgagc tactaaaata ttgcgaatac cgcttccaca 4740 aacattgctc aaaagtatct ctttgctata tatctctgtg ctatatccct atataaccta 4B0D cccatccacc tttcgctcct tgaacttgca tctaaactcg acctctacat tttttatgtt 4B60 tatctctagt attactcttt agacaaaaaa attgtagtaa gaactattca tagagtgaat 4920 cgaaaacaat acgaaaatgt aaacatttcc tatacgtagt atatagagac aaaatagaag 49Θ0 aaaccgttca taattttctg accaatgaag aatcatcaac gctatcactt tctgttcaca 5040 aagtatgcgc aatccacatc ggtatagaat ataatcgggg atgcctttat cttgaaaaaa 5100 tgcacccgca gcttcgctag taatcagtaa acgcgggaag tggagtcagg ctttctttat 5160 ggaagagaaa atagacacca aagtagcctt cttctaacct taacggacct acagtgcaaa 5220 aagttatcaa gagactgcat tatagagcgc acaaaggaga aaaaaagbaa tctaagatgc 5260 tttgttagaa aaatagcgct ctcgggatgc atttttgtag aacaaaaaag aagtatagat 5340 tcttcgttgg taaaatagcg ctetcgcgtt gcatttctgt tctgtaaaaa tgcagctcag 5400 attctttgtt tgaaaaatta gcgctctcgc gttgcatttt tgttttacaa aaatgaagca 5460 cagattcttc gttggtaaaa tagcgctttc gcgttgcatt tctgttctgt aaaaatgcag 5520 ctcagattct ttgtttgaaa aattagcgct ctcgcgttgc atttttgttc tacaaaatga 55B0 agcacagatg cttcgttcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 5640 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 5700 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 5760 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 5820 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 5880 cggtaagatc ctcgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 5940 agbtctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 6000 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct G060 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 6120 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 61B0 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 5240 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 6300 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 6360 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 6420 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 64Θ0 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg, 6540 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 6600 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 6660 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 6720 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt cttttcegcg 67B0 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 6B40 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 6900 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 6960 tacafcacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 7020 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 70B0 ggggggctcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 7140 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 7200 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 7260 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 7320 ctogtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt bacggttcct 73 BO ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 7440 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 7500 167321/2 37 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc 7560 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga- aagcgggcag 7620 tgagcgcaac gcaattaafcg tgagttacct cactcattag gcaccccagg ctttacactt 7680 tatgcttccg gctcctatgt tgtgtggaat .tgtgagcgga taacaatttc acacaggaaa 7740 Cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct 7B00 ggagctttt 7B09

Claims (23)

38 167321/3 C L A I M S

1. A purified and isolated polynucleotide, comprising a DNA sequence encoding a Glucose Transporter 4 containing at position 85 an amino acid exchange from valine to methionine (GLUT4V85M) protein having a glucose transporting activity.

2. The polynucieotide of claim 1 , comprising a nucleotide sequence according to Seq ID No. 1.

3. The polynucieotide according to any one of claims 1 or 2, wherein said GLUT4V85M protein comprises an amino acid sequence according to Seq ID No. 2.

4. The polynucleotide according to any one of claims 1 to 3, wherein said DNA sequence encoding a GLUT4V85M protein is operatively linked to a promoter.

5. The polynucleotide according to any one of claims 1 to 4, replicable in a yeast cell.

6. The polynucleotide of claim 5, capable of expressing said protein in said yeast cell.

7. A yeast cell, comprising a polynucleotide as claimed in any one of claims 1 to 6.

8. The yeast cell of claim 7, comprising said GLUT4V85M protein.

9. The yeast cell according to any one of claims 7 or 8, deposited as Saccharomyces cerevisiae DSM 15185.

10. A process of preparing a yeast cell as claimed in any one of claims 7 to 9, comprising the steps: a) providing a yeast cell; b) providing a polynucleotide of claim 5 or 6; c) transforming said yeast cell with said polynucleotide; 39 167321/3 d) selecting a transformed yeast cell ; and e) expressing a GLUT4V85M protein in said transformed yeast cell.

11. 1 1. The transformed yeast cell of claim 10, deposited as Saccharomyces cerevisiae DSM 15186.

12. A yeast cell whose glucose transporters in their entirety are no longer functional, comprising a polynucleotide as claimed in any one of claims 1 to 6.

13. The yeast cell of claim 12, comprising a GLUT4V85M protein.

14. The yeast cell as claimed in any one of claims 12 or 13, deposited as Saccharomyces cerevisiae DSM 15188.

15. A process of preparing a yeast cell according to any one of claims 12 to 14, comprising the steps: a) producing a yeast cell whose glucose transporters in their entirety are no longer functional; b) providing a polynucleotide of claim 5 or 6; c) transforming said yeast cell whose glucose transporters in their entirety are no longer functional with said polynucleotide; d) selecting a transformed yeast cell; and e) expressing a GLUT4V84M protein in said transformed yeast cell.

16. A protein having the functional activity of a glucose transporter, said protein is encoded by a polynucleotide sequence as claimed in any one of claims 1 to 3.

17. A protein having the functional activity of a glucose transporter, said protein is encoded by Seq. ID No. 2. 40 167321/2

18. A method for identifying a compound which stimulates the activity of a GLUT4 protein, comprising the steps: a) providing a yeast cell as claimed in any one or more of claims 7 to 9; b) providing a compound; c) contacting said yeast cell with said compound; d) determining glucose uptake said yeast cell; e) relating the detected value of the glucose uptake of d) to the detected value of glucose uptake in a yeast cell as claimed in a) which is not contacted with a chemical compound as claimed in b), with a compound which causes an increase in the amount of glucose taken up in the yeast as claimed in d) stimulating the activity of said GLUT4 protein.

19. The polynucleotide according to any one of claims 1 -6 as described in the specification.

20. The yeast cell according to any one of claims 7-9, 1 1-1 as described in the specification.

21. The process according to any one of claims 10, 15 as described in the specification.

22. The protein according to any one of claims 16-17 as described in the specification.

23. The method according to claim 18 as described in the specification. For-t e Applicant, Pearl Cohen Zedek Latzer Advocates, Notaries & Patent Attorneys P-7764-IL