MXPA05002816A

MXPA05002816A - Use of saccharomyces cerevisiae erg4 mutants for the expression of glucose transporters from mammals.

Info

Publication number: MXPA05002816A
Application number: MXPA05002816A
Authority: MX
Inventors: Eckhard Boles
Original assignee: Aventis Pharma Gmbh
Priority date: 2002-09-14
Filing date: 2003-09-04
Publication date: 2005-05-27
Also published as: EP1539958A2; WO2004026907A2; ZA200501871B; CN1694960A; AU2003264257B2; KR20050056206A; BR0314115A; RU2005110955A; KR101233998B1; CA2498636A1; CN1694960B; CA2498636C; WO2004026907A3; IL197621A0; JP4520854B2; DE10242763A1; RU2345136C2; JP2006517088A; IL197621A; IL167321A

Abstract

The invention relates to yeast strains in which a human GLUT4 transporter or a human GLUT1 transporter can be functionally expressed and in particular GLUT4 transport proteins which can be particularly easily functionally expressed in yeast strains.

Description

USE OF M LITANTES ERG4 OF SACCHAROMYCES CEREVISIAE FOR THE EXPRESSION OF TRANSPORTERS OF MAMMALS GLUCOSE. The invention relates to strains of yeast in which the human Glut 4 and Glut 1 transporters can be expressed functionally. Most heterotropic cells transport glucose via special transport proteins inside the cell. Several organisms have developed different mechanisms that mediate glucose transport, such as, in particular, parallel proton cotransport systems, Na + glucose transporters, binding protein dependent systems, phosphotransferase systems, and systems for facilitated diffusion. In eukaryotes, a family of glucose transporters that are encoded in mammals by the GLUT (GLUT = glucose transporter) and Saccharomyces cerevisiae genes by the HXT (HXT = hexose transporter) genes mediates glucose consumption via facilitated diffusion . These 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 role in disorders associated with defective glucose homeostasis, such as, for example, diabetes mellitus or Fanconi-Bickel syndrome. The transport of glucose in mammals has therefore been the subject of numerous studies. So far, thirteen proteins of the glucose transporter type have been identified (from GLUT1 to GLUT12, HMIT - H-myo-inositol transporter)). These transporters play key roles that include the consumption of glucose in various tissues, their storage in the liver, their insulin-dependent consumption in muscle cells and adipocytes and the measurement of glucose by the ß cells of the pancreas. GLUT1 mediates glucose transport in erythrocytes and across the blood-brain barrier, but is also expressed in many other tissues, while GLUT4 is limited to insulin-dependent tissues, mainly muscle and adipose tissue. In said insulin-dependent tissues, control of the direction of GLUT4 transporters by intracellular compartments or cell membrane compartments represent an important mechanism for regulating glucose consumption. In the presence of insulin, intracellular GLUT4 is redistributed through the cell membrane to facilitate glucose consumption. GLUT1 is expressed in the same way in said insulin-dependent tissues, and its distribution in the cell is under the influence of insulin in the same way, although not as strongly. In addition, the relative efficiency with which GLUT1 or GLUT4 catalyze sugar transport is determined not only by the degree of direction of each transporter to the cell surface, but also by its kinetic properties. The fact that different isoforms of the glucose transporter and rapid glucose metabolism are coexpressed has yielded studies on the role and exact properties of each isoform of the glucose transporter in these complicated insulin-dependent tissues. 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 arose in connection with these systems: too weak activity of the heterologously expressed transporters, intrinsic glucose transporters in such systems, intracellular retention of a considerable proportion of the transporters or even production of inactive transporters. The GLUT4 protein that occurs naturally in mammals, in particular, in humans, can be expressed in a functional manner in strains of Saccharomyces cerevisiae under particular conditions. Yeast cells are unicellular eukaryotic organisms. They are, therefore, for some proteins, more suitable for expression than bacterial systems, in particular with respect to carrying out screening tests to identify pharmaceutically active substances. The present invention relates to a purified and isolated polynucleotide comprising a DNA sequence encoding the GLUT4V85M protein. Said protein contains at position 85 of the amino acid chain of the human GLUT4 protein a change of amino acids from valine to methionine. This altered GLUT4V85M protein provides other alternatives for expressing a functional GLUT4 protein. A GLUT4 protein should be considered functional in relation to Saccharomyces cerevisiae if glucose consumption can be observed in a strain of Saccharomyces cerevisiae whose glucose transporters are inactive in their entirety (= hxt (-)) after the expression of said GLUT4 protein. Glucose consumption can be determined by measurements of transport by radioactively labeled glucose or by growth in glucose medium as an exclusive source of carbon. In a preferred embodiment, the purified and isolated polynucleotide comprising a DNA sequence encoding a GLUT4V85M protein can include or comprise a sequence of the following groups: a) a nucleotide sequence according to the sequence ID N °. 1, b) a nucleotide sequence that hybridizes with a sequence of the sequence ID N °. 1 under restrictive conditions and coding for a GLUT4V85M protein. The purified and isolated polynucleotide preferably encodes a GLUT4V85M protein having an amino acid sequence of the sequence ID N °. 2. The purified and isolated polynucleotide comprising a DNA sequence encoding, as mentioned above, for a GLUT4V85M protein, can be operably linked to a promoter. Suitable promoters are, in particular, prokaryotic or eukaryotic promoters such as, for example, the Lac, trp, ADH or HXT7 promoters. The part of the polynucleotides encoding the GLUT4V85M protein is operably linked to a promoter with precision if a bacterial or eukaryotic organism produces, by means of said promoter with the aid of a vector, an mRNA that can be translated into the GLUT4V85M protein. An example of such a vector is the vector p4H7GLUT4V85M (sequence ID No. 3). The GLUT4V85M protein can be expressed in yeast cells by said vector. The polynucleotide described above comprising a DNA sequence encoding a GLUT4V85M protein is, in a preferred embodiment, suitable for replicating said polynucleotide in a yeast cell or for expressing the part of the polynucleotide, encoding the GLUT4V85M protein, in a cell of yeast to give the GLUT4V85M protein. A yeast cell of Saccharomyces cerevisiae is particularly suitable. For replication and expression in a yeast cell, the polynucleotide comprising a DNA sequence encoding a GLUT4V85M protein is present in the form of a yeast vector. The polynucleotide region encoding the GLUT4V85M protein can be operably linked to a yeast cell-specific promoter such as, for example, the ADH promoter (alcohol dehydrogenase promoter) or the HXT7 promoter (the hexose transporter promoter). The yeast sectors are a group of vectors that were developed for the cloning of DNA in yeast. The invention further relates to a yeast cell of Saccharomyces cerevisiae in which all glucose transporters are no longer functional (= hxt (-)) and which do not contain any functional protein Erg4. Such a yeast cell is preferably a deposited yeast cell such as DSM 15187 of Saccharomyces cerevisiae in the DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen 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 do not contain any functional Fgy1 and no functional Erg4 protein. The lack of an Erg4 protein or a Fgy1 protein can be attributed in particular to an interruption of the corresponding coding genome sections or to a partial or complete deletion of said coding genome sections. Preference is given to using, as a yeast cell containing no functional glucose transporters, any functional Fgy1 protein and no functional Erg4 protein, to a yeast cell as deposited in the DSMZ such as DSM 15184 of Saccharomyces cerevisiae. A yeast cell as described above is preferably used to express 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 to express a human GLUT4 or GLUT1 protein. A yeast cell of Saccharomyces cerevisiae whose whole glucose transporters and also the Erg4 protein are no longer functional may contain a polynucleotide of the present invention, which comprises a DNA sequence encoding a GLUT4V85M protein. Said yeast cell can also express the GLUT4V85M protein and thus contain said protein. A yeast strain of this kind, which contains a polynucleotide comprising a DNA sequence encoding the GLUT4V85M protein, is preferably the yeast strain DSM 15185 of Saccharomyces cerevisiae which has been deposited in 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 encoding a GLUT4V85M protein can 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) provide an isolated and purified polynucleotide comprising a DNA sequence encoding the GLUT4V85M protein and capable of replicating in the yeast cell, c) transforming the yeast cell of a) with the polynucleotide of b), d) selecting a transformed yeast cell, e) where appropriate, expressing the GLUT4V85M protein. An isolated and purified polynucleotide comprising a DNA sequence encoding the GLUT4V85M protein is preferably a vector that can be replicated in a yeast cell and wherein said DNA sequence was cloned. An example of such vector is p4H7GLUT4V85M (sequence 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 contain a polynucieotide comprising a DNA sequence encoding the GLUT4V85M protein. Said yeast cell can also express the GLUT4V85M protein and thus contain said protein. A yeast strain of this class is preferably DSM 15186 of Saccharomyces cerevisiae deposited in the DS Z. A yeast cell whose glucose transporters in their entirety and also the Fgy1 and Erg4 proteins are no longer functional and which contains a polynucieotide comprising a DNA sequence encoding the GLUT4V85M protein can be prepared, for example, by a) providing a yeast cell whose glucose transporters in their entirety and also the Fgy1 and Erg4 proteins are no longer functional, b) providing an isolated and purified polynucleotide which comprises a DNA sequence encoding the GLUT4V65M protein and which can be replicated in the yeast cell, c) transforming the yeast cell of a) with the polynucleotide of b), d) selecting a transformed yeast cell, e) when appropriate, express the GLUT4V85M protein. The above isolated and purified polynucleotide comprising a DNA sequence encoding the GLUT4V85M protein is preferably a vector that can be replicated in a yeast cell and wherein said DNA sequence was cloned. An example of such vector is p4H7GLUT4V85M (sequence ID No. 3). The invention also relates to a yeast cell whose glucose transporters as a whole are no longer functional and which contains a polynucleotide comprising a DNA sequence encoding the GLUT4V85M protein. Said yeast cell can also express the GLUT4V85M protein and thus contain said protein. A preferred yeast strain of this class is the yeast strain 15188 of Saccharomyces cerevlsiae deposited in the DSMZ. A yeast cell whose glucose transporters as a whole are no longer functional and which contains a polynucleotide comprising a DNA sequence encoding the GLUT4 V85M protein can be prepared, for example, by a) providing a yeast cell whose glucose in its entirety are no longer functional, b) provide an isolated and purified polynucleotide comprising a DNA sequence encoding the GLUT4V85M protein and capable of replicating in the yeast cell, c) transforming the yeast cell of a) with the polynucleotide of b), d) select a transformed yeast cell, e) where appropriate, express the GLUT4V85M protein. An isolated and purified polynucleotide comprising a DNA sequence encoding the GLUT4V85 protein is preferably a vector that can be replicated in a yeast cell and wherein said DNA sequence was cloned. An example of such vector is P4H7GLUT4V85M (sequence ID No. 3). The invention also relates to a protein having the amino acid sequence according to the sequence ID N °. 2. Said protein is a human GLUT4 protein in which a valine has been replaced by a methionine at position 85 of the amino acid chain. The invention also relates to a method for identifying a compound that stimulates the activity of a GLUT4 protein, which method comprises the steps of: a) providing a yeast cell whose glucose transporters in their entirety and also the Erg4 protein are no longer functional and containing a polynucleotide comprising a DNA sequence encoding a GLUT4V85 protein, b) providing a chemical compound, c) contacting the yeast of a) with the chemical compound of b), d) determining the consumption of glucose by the yeast of c), e) relating the detected value of the glucose consumption of d) to the detected value of glucose consumption in a yeast cell as claimed in a), which has been contacted with a compound chemical as claimed in b), with a compound that causes an increase in the amount of glucose captured in the yeast as claimed in d) by stimulating the activity of said protein na GLUT4. It can be assumed that the compounds that stimulate the activity of the GLUT4V85M protein also stimulate the activity of GLUT4. The invention also relates to a pharmaceutical agent containing a compound that has been identified by the method described above and in addition to additives and excipients for formulating a pharmaceutical agent. In addition, the invention relates to the use of a compound that has been identified by the method described above to produce a pharmaceutical agent for the treatment of type I and / or II diabetes.

The invention also relates to a pharmaceutical agent comprising a compound that has been identified by the method described above and to additives and excipients for formulating a pharmaceutical agent. In addition, the invention relates to the use of a compound identified by the method described above to produce a pharmaceutical agent for the treatment of diabetes. The invention further relates to the use of a compound identified by a method described above to produce a pharmaceutical agent for the treatment of diabetes. The present invention also comprises a method for identifying a compound that inhibits the protein encoded by the Erg4 gene, which method comprises the steps of: a) providing a yeast cell whose glucose transporters as a whole no longer function and which contains a polynucleotide comprising a DNA sequence encoding the GLUT4V85M protein and capable of replicating in a yeast cell, b) providing a chemical compound c) contacting the yeast of a) with the chemical compound of b), d) determining the glucose consumption by the yeast of c), e) relating the detected value of the glucose consumption of d) with respect to the detected value of glucose consumption in a yeast cell as claimed in a), which has not been put into contact with a chemical compound as claimed in b), with a compound that causes an increase in the amount of glucose captured in the yeast as claimed in d) by stimulating the activity of an Erg4 protein. The invention further relates to a method for identifying a compound that inhibits the corresponding protein of the Fgy1 gene, comprising the steps of: a) providing a yeast cell whose glucose transporters in their entirety and whose Erg4 protein is no longer functional and containing a GLUT4 protein, b) providing a chemical compound c) contacting the yeast of a) with the chemical compound of b), d) determining the glucose consumption by the yeast of c), e) relating the detected value of the glucose consumption of d) with respect to the detected value of the glucose consumption in a yeast cell as claimed in a) with which a chemical compound as claimed in b) is not contacted, with a compound causing a increase in the amount of glucose captured in the yeast as claimed in d) by stimulating the activity of a Fgy1 protein. The invention also relates to a pharmaceutical agent comprising a compound that has been identified by the method described above and to additives and excipients for formulating a pharmaceutical agent. The invention can be illustrated in more detail below with regard to technical details.

Hybridization means the assembly of two single strands of nucleic acids that have complementary base sequences in double strands. Hybridization can occur between two strands of DNA, one strand of DNA and one strand of RNA and between two strands of RNA. In principle, it is possible to prepare hybrid molecules by heating the nucleic acids involved which at first may be in the double-stranded form, and boiling, for example, in a water bath for 10 minutes, until they disintegrate into single-stranded molecules without the secondary structure. Subsequently, they can be cooled slowly. During the cooling phase, the complementary strands are paired to give hybrid double-stranded molecules. In laboratory conditions, hybridizations are usually carried out with the help of hybridization filters to which the single-stranded or denaturing polynucleotide molecules are applied by transfer or electrophoresis. Hybridization can be visualized using appropriate complementary polynucleotide molecules by providing said polynucleotide molecules that hybridize with a fluorescent radioactive label. The restriction describes the degree of complementarity or alignment in particular conditions. High restriction has higher demands on complementarity than low restriction. Depending on the application and the objective, particular conditions are established with different restriction for the hybridization of nucleic acids. At high restriction, the reaction conditions for hybridization are set up such that only complementary molecules that complement each other very well can hybridize to each other. The low restriction allows the molecules to also hybridize partially with relatively large sections of unpaired or unpaired bases. Hybridization conditions should be understood as being restrictive, in particular, if the hybridization is carried out in an aqueous solution containing 2 x SSC at 68 ° C for at least 2 hours, followed by washing first in 2 x 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. The 2 x SSC, 1 x SSC or 0.2 x SSC solution is prepared by diluting a 20 x SSC solution appropriately. A 20 x SSC solution contains 3 mol / l of NaCl and 0.3 mol / l of Na citrate. The pH is 7.0. The person skilled in the art is familiar with the methods for the 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, 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. The Yip vectors (plasmids that make up the yeast) correspond essentially to the vectors used in bacteria for cloning, but contain a yeast selection gene (for example, URA3, LEU2).

Only when the foreign DNA is integrated into a yeast chromosome after the introduction of said vector, are these sequences replicated together with the chromosome and, with the formation of a clone, they are transferred stably in all the daughter cells. Based on this method, plasmids that can be replicated autonomously belonging to eukaryotic ORI (origins of replication) have been derived. Such yeast vectors are referred to as YRp vectors (yeast replicating plasmids) or ARS vectors (autonomously replicating sequence). In addition, there are YEp vectors (yeast episomal plasmids) that are derived from the yeast 2 μm plasmid and that contain a selective marker gene. The class of YAC vectors (artificial yeast chromosome) behaves as independent chromosomes. A yeast vector containing a gene that is expressed is introduced into the yeast by transformation so that said gene is capable of being expressed. Examples of suitable methods for this purpose are electroporation or incubation of competent cells with vector DNA. Yeast expression promoters suitable for the person skilled in the art are known, examples being the SOD1 promoter (superoxide dismutase), the ADH promoter (alcohol dehydrogenase), the gene promoter for acid phosphatase, the HXT2 promoter (transporter 2 glucose), the HXT7 promoter (the glucose transporter 7), the GAL2 promoter (galactose transporter) and others. The construct comprises a yeast expression promoter and a gene that is expressed (e.g., GLUT4V85M) is, for the purpose of expression, part of a yeast vector. To carry out the expression, said yeast vector can be a self-replicating particle that is independent of the yeast genome or that can be stably integrated into the yeast genome. A suitable yeast vector is, in principle, any sequence of polynucleotides that can be propagated in a yeast. Yeast vectors that can be used are, in particular, yeast plasmids or artificial yeast chromosomes. Yeast vectors usually contain an origin of replication (2μ, ars) or, they start the replication procedure and a selection marker which usually comprises an auxotrophic marker or an antibiotic resistance gene. Examples of yeast vectors known to the person skilled in the art are ?? 272, pCS19, pEMBCYe23, pFL26, pG6, pNN414, pTV3, p426MET25, p4H7 and others. According to the present invention, the selection of a cell means its specific concentration, having a selection marker such as, for example, resistance to an antibiotic or culture capacity in a particular minimum medium, and in addition its isolation and subsequent culture in a agar plate or in submerged culture. The cultivation, transformation and selection of a transformed yeast cell and also the expression of a protein in a yeast cell are among the methods commonly used by the person skilled in the art. Instructions regarding such methods can be found in standard textbooks, 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 JP Brown, Mick F. Fruite and John EG Me Cartly; Springer Berlin; ISBN: 3-540-56521-3 or in "Methods in Yeast Genetics", 1997: A Cold Spring Harbor Laboratory Course Manual; Adams Alison (Ed); Cold Spring Harbor Laboratory; ISBN: 0-87969-508-0. The yeast Saccharomyces cerevisiae has 17 known hexose transporters and also three known maltose transporters, which are capable of transporting hexoses in said yeast, provided that their expression is sufficiently high. In a known strain, all transporters suitable for hexose consumption were deleted by deletion. This strain simply contains just the two MPH2 and MPH3 genes that are homologous to the 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 capable of propagating in a substrate containing glucose as an exclusive source of carbon. It is possible to select from the mutants of said strain that functionally express GLUT1, starting from a corresponding vector (strain hxt fgy1-1). If the yeast strain hxt fgy1-1 is transformed with a plasmid vector carrying a GLUT4 gene with the control of a yeast promoter, only very little glucose is still transported.

Functional expression of GLUT4 requires further adjustments in this yeast strain to enable significant glucose transport by GLUT4. Such yeast strains whose cells consume glucose via a single GLUT4 glucose transporter can be isolated on substrates having glucose as the exclusive carbon source. For this purpose, a yeast strain hxt fgy1-1 carrying a GLUT4 gene with the functional control of a yeast promoter is transformed. These yeast cells transformed in this way are applied to a nutrient medium containing glucose as exclusive carbon source and are incubated therein. After a few days of incubation at, for example 30 ° C, the growth of individual colonies is observed. One of these colonies is isolated. Removal of the yeast plasmid from said colony prevents propagation in the nutrient medium containing the glucose as the exclusive carbon source. If this strain that no longer contains a vector plasmid is transformed again with a yeast vector carrying the GLUT4 gene with the functional control of a yeast promoter, said strain is then again able to propagate in a medium containing glucose as an exclusive source of carbon. The aforementioned yeast strains are the subject of International Application PCT / EP02 / 01373, filed on February 9, 2002, which claims the priority of document DE 10106718.6 of February 14, Yeast strains whose autogenous transporters for hexoses (transporters glucose) as a whole are no longer functional, they have been previously deposited in connection with the International Application PCT / EP02 / 01373 of the Deutsche Sammiung von Mikroorganismen and Zelikulturen GmbH (DSMZ) with the number DSM 14035, DSM 14036 or DSM 14037. GLUT4 polynucleotide and amino acid sequences are accessible, for example, via the following entries in the gene banks: M20747 (cDNA; human), EMBL: D28561 (cDNA; rat), EMBL: M23382 (cDNA; mouse), Swissprot: P14672 (protein: human), Swissprot: P19357 (protein, rat) and Swissprot: P14142 (protein, mouse). The polynucleotide sequences and the amino acid sequences of GLUT1 are described with the following codes of the indicated databases: EMBL: M20653 (cDNA: human), EMBL: M13979 (cDNA, rat), EMBL: M23384 (cDNA, mouse) , Swissprot: P 166 (protein, human), Swissprot: P 1167 (protein, rat) and Swissprot: P17809 (protein, mouse). Pharmaceutical products are dosage forms of pharmacologically active substances for the therapy of diseases or physical dysfunctions 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. The examples that are used for the external application are aerosols, sprays, gels, ointments or powders. Injectable or infusible solutions allow parenteral administration, using pre-filled bottles, bottles or syringes. These and other pharmaceuticals are known to the person skilled in the art in the field of pharmaceutical technology. The excipients for formulating a pharmaceutical agent make it possible to prepare the active substance with the aim of optimizing the application, distribution and action development of the active ingredient for the particular application. Examples of such excipients are fillers, binders, disintegrating or sliding agents, such as lactose, sucrose, mannitol, sorbitol, cellulose, starch, dicalcium phosphate, polyglycols, alginates, polyvinylpyrrolidone, carboxymethylcellulose, talc or silicon dioxide. The very manifestations of diabetes by the excretion of glucose together with the urine with an abnormal increase in blood glucose levels (hyperglycemia), are due to a chronic metabolic condition due to an insulin deficiency or a reduced insulin action . The lack, or the reduced action, of insulin leads to insufficient absorption and to the conversion by the cells of the glucose captured in the blood. In fat tissue, insulin antagonist hormones have the effect of increasing lipolysis accompanied by an increase in the levels of free fatty acids in the blood. The adiposity (obesity) is the abnormal weight gain that results from an energy imbalance due to an excessive supply of calories, which constitutes a danger to health.

The amount of a hexose that is taken up by a yeast strain provided as described just above can be determined by consumption studies using radiolabeled glucose. For this purpose, a particular concentration of yeast cells is suspended in, for example, 100 μ? of a buffer, for example at a concentration of 60 mg per ml (wet weight), and mixed with a defined amount of glucose marked with C or 3H as the exclusive carbon source. The cells are incubated, and defined amounts of them are removed at specific times. The amount of a glucose captured is determined with the help of LSC (Liquid Scintillation Counter). The amount of a hexose that is taken up by a yeast strain provided and described just above, however, can be further determined by a growth assay in a medium containing glucose as the exclusive carbon source. For this purpose, the growth rate 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 to the growth rate of a control strain. (for example, the wild type strain of yeast). 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 containing a specific proportion of an organic solvent such as, for example, DMSO (dimethyl sulfoxide). The conduction of a strain of the yeast with a compound for identifying a compound according to the invention mentioned above is done in particular in automated laboratory systems provided for this purpose. Such systems may comprise specifically prepared chambers with depressions, or microtiter plates, Eppendorf tubes or laboratory material. Automated laboratory systems are usually designed for high performance speeds. Such a method as mentioned above, is carried out with the help of an automated laboratory system, is consequently called HTS (High Performance Selection). The sequence ID N °. 1 describes a polynucleotide sequence comprising the coding region of the GLUT4V85 protein. The sequence ID N °. 2 describes the amino acid sequence of the GLUT4V85M protein. The sequence ID N °. 3 describes the polynucleotide sequence of vector p4H7GLUT4V85M. EXAMPLES Use of yeast strains All the yeast strains described herein were derived from strain CEN-PK2-1C (MATa leu2-3, 112 ura3-52 trpl-289 his3-A1 MAL2-80 SUC2). The preparation of a yeast strain having deviations in hexose transporter genes (HXT) has been described by Wieczorke et al., FEBS Lett. 464, 123-128 (1999): EBY-18ga (MATa Ahxt1-17 Agal2 Aagtl Astil leu2-3, 1 12 ura3-52 trp1-289 his3-A1 MAL2-8C SUC2), EBY.VW4000 (MATa Ahxt1-17 Agal2 Aagtl Amph2 Amph3 AstH leu2-3, 112 ura3-52 trp1-289 his3-A1 MAL2-80 SUC2). The medium was based on yeast extract of 1% and peptone of 2% (YP), while the minimum medium was composed of Difco yeast nitrogen base of 0.67% without amino acids (YNB) and contained additives required for auxotrophy and different carbon sources. The yeast cells were cultured under aerobic conditions at 30 ° C on a rotary shaker or on agar plates. Cell growth was controlled by measuring the optical density at 600 nm (OD600) or determining the diameter of the yeast colonies. Determination of glucose consumption Glucose transport was measured as consumption of D- [U-14C] -glucose (Amersham) and the kinetic parameters were determined from representations of Eadie-Hofstee. The cells were removed by centrifugation, washed with phosphate buffer and resuspended in phosphate buffer at a concentration of 60 mg per ml (wet weight). The glucose consumption 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 μ ??? G1. Cells and glucose solutions were preincubated at 30 ° C for 5 minutes. Glucose consumption was started by adding radioactive glucose to the cells. After incubation for 5 seconds, 10 ml of ice-cold stop buffer (0.1 M K1PO4, pH 6.5, 500 mM glucose) were added and the cells were rapidly recovered by filtering on glass fiber filters (0 = 24). mm, Whatman). The filters were washed rapidly three times with ice cold buffer and the incorporated radioactivity was measured using a liquid scintillation counter. An addition by cytochalasin B (final concentration 20 μ ?, dissolved in ethanol) was measured in a 15-second consumption test with radioactive glucose of 50 mM or 100 mM, after the cells had been incubated in the presence of the inhibitor or only the solvent for 15 minutes. A new heterologous expression system for glucose transporters of mammalian cells has been developed. The system is based on a strain of S. cerevisiae from which all endogenous glucose transporters have been eliminated by destroying the coding genes. This strain is not able to consume more glucose via the cell membrane and to grow with glucose as an exclusive carbon source. To integrate heterologous glucose transporters from humans or from other mammals, GLUT1 and GLUT4 into an active form in the yeast cell membrane, additional mutations had to be introduced into the yeast strain. GLUT1 is active in only one strain of the mutant fgy1-1 and GLUT4 only in the double mutants fgy1-1 fgy4-X. The FGY1 gene has been cloned. It is the ORF YMR212c of S. cerevisiae. As far as the function is concerned, the results indicate that both Fgy1 and the product generated by Fgy1 inhibit the activity of human glucose transporters or is involved in the fusion of the vesicles that transport GLUT to the cell membrane. In contrast to GLUT1 and similarly to cells in mammals, a large proportion of GLUT4 proteins in yeast are located in intracellular structures. A total of nine recessive mutants were isolated (from fgy4-1 to fgy4-9) in which GLUT4 is now also directed to the cell membrane and, in the case of a concomitant fgyl-1 mutation, becomes active there. The insertion gene bank described by Bruns et al. . { Genes Dev. 1994; 8: 1087-105) was used for the complementation analysis. The strain of hxt fgy1-1 was first transformed with a GLUT4 plasmid and then with the mobilized insertion gene bank. This was followed by (a selection of transformants that were able to grow in the glucose medium.) It turned out that in one of the studied mutants the ERG4 gene had been destroyed, ERG4 codes for an enzyme (oxidoreductase) of ergosterol biosynthesis. This enzyme, the esteral C-24 (28) -reductase catalyses the last stage of ergosterol biosynthesis and converts ergosta-5,7,22,24, (28) -tetraenol into the final product ergosterol. this moment contains eight transmembrane domains and is located in the endoplasmic reticulum.A erg4 mutant is viable, since the incorporation of ergosterol precursors in the yeast membranes compensates for the loss of ergosterol.

The influence of Erg4 inhibition on GLUT4 functionality was confirmed by the specific deletion of erg4 in the strain of hxt fgy1-1. The resulting strain (hxt fgy1-1 Aerg4) is called SDY022. Protein interaction assays with the help of the ubiquitin cleavage system showed that human GLUT4 acts directly with the yeast Erg4. Therefore, it can be assumed that the yeast Erg4 protein in the endoplasmic reticulum directly prevents the translocation of GLUT4 or modifies GLUT4 in some way that is important for translocation and / or function. In the same way, it was shown that the deletion of ERG4 in the strain of hxt nuil alone, that is, despite functional FGY1, activates GLUT1, but not GLUT4. The results of the growth assay are summarized in Table 1. To exclude that ergosterol itself exerts a negative influence on GLUT4, growth assays were carried out on agar plates containing ergosterol under aerobic conditions. Any yeast strain transformed with GLUT4 was unable to grow under these conditions (Table 2). The GLUT1 transformants in the hxt fgy1-1 strain did not show, in contrast to aerobic growth, no growth in glucose under anaerobic conditions. The GLUT1 transformants were able to grow only after deletion of ERG4. The change of Val85 by Met through in vitro mutagenesis gave GLUT4 independent of the mutation of fgy1-1 and caused GLUT4V85M that was functional even in a strain of hxt erg4. This observation indicates that Fgy1 acts directly or indirectly on this position which is located within the second transmembrane helix of the GLUT transporters. Table 3 shows the descriptions of the yeast strains deposited in connection with the present patent application with the Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ) - ascheroder Weg 1b 38124 Brunswick, Germany.

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

Genotype Glucose of 1% Glucose of 1% Ahxt fgy1-1 GLUT4 - GLUT1 ++ Ahxt fgy1-1 Aerg4 GLUT4 ++ GLUT1 ++ Ahxt fgyl -1 Aerg4 Vector - Vector - Ahxt fgy1-1 Aerg5 GLUT4 - GLUT1 ++ Ahxt fgy1 -1 Aerg4 GLUT4 + GLUT1 ++ Ahxt Aerg4 GLUT4 - GLUT1 + Ahxt Aerg5 GLUT4 - GLUT1 - Table 2: Growth of GLUT1 and GLUT4 transformants in glucose medium with or without erqosterol under anaerobic conditions Glucose of 1% + Genotype Glucose of 1% 33 mg / l of ergosterol Ahxt fgy1-1 GLUT1 GLUT4 - - Ahxt fgyl -1 Aerg4 GLUT1 ++ GLUT4 - Ahxt fgy1-1 Aerg5 GLUT1 GLUT4 - - A xt fgy1-í Aer4 Aerg5 ++ GLUT4 - Ahxt Aerg4 GLUT1 (+) GLUT4 - Ahxt Aerg5 GLUT1 GLUT4 - - Table 3: Properties of the deposited yeast strains (Saccharomvces cerevisiae) Growth of strain with ATa Ahxtl-17 Agal2 P4H7GLLIT4V85M maltose 1% as Aagtl Astil Amph2 (carbon source marker; DSM 15188 Amph3 leu2-3, 112 auxotrophic selection for glucose, ura3-52 trp1-289 his3-URA3) leucine, tryptophan and MAL2-8C AI-SUC2 = Sec. ID N °. 3 histidine Basic medium: Base of Yeast Nitrogen of 0.67% 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%.

DSMZ DEPOSIT OF MICROORGANISMS FOR THE PURPOSES Deutsche Sammlung FOR PATENT PROCESSING von Mikroorganismen and Zellkulturen GmbH INTERNATIONAL FORMAT Aventis Phacma Deutschland GmbH Industriepark Hechst CONFIRMATION OF RECEPTION IN THE CASE OF A D 65926 Frankfurt FIRST PRESENTATION Issued according to Rule 7.1 by the INTERNATIONAL DEPOSITORY AUTHORITY identified at the end of this page I. CHARACTERIZATION OF THE MICROORGANISM Reference sign attributed by the DEPOSITANT: ENTRY NUMBER attributed by the INTERNATIONAL DEPOSITORY AUTHORITY: DSMefg DSM 15185 ? SCIENTIFIC DESCRIPTION AND / OR PROPOSAL TAXONOMIC DESIGNATION With the microorganism designated in section I, the following was presented: T a scientific description (The one proposed taxonomic designation (Mark with a cross what is relevant) HL ENTRY AND ACCEPTANCE This International Depositary Authority accepts the microorganism designated by I, which has entered on 03-09-2002 (date of the first presentation) 'IV. ENTRY OF THE REQUEST FOR TRANSFORMATION The microorganism designated by I has entered this International Depositary Authority on (the date of the first presentation) and a request for transformation of this first presentation has been submitted in a presentation according to the Budapest Treaty (entry date of the transformation request) V. INTERNATIONAL DEPOSITORY AUTHORITY Name DSMZ-DEUTSCHE SAMMLUNG VON Signature of the authorized person (s) to represent MIKROORGANISMEN UND ZELLKULTUREN GmbH the International Depositary Authority, or of the Address: MascherWeg Ib fiincionario (s) empowered (s) for it (illegible signature) D-38124 Brunswick Date: 09-10-2002 'If Rule 6.4.d is relevant, this is the date on which the Status of an International Depositary Authority has been acquired. Form DSMZ-BP / 4 (one page only) 12/2001 BUDAPEST TREATY ON INTERNATIONAL RECOGNITION DSMZ DEPOSIT OF MICROORGANISMS FOR THE PURPOSES Deutsche Sammlung FOR PATENT PROCESSING von Mikroorganismen and Zellkulíuren GmbH INTERNATIONAL FORMAT Aventis Pharma Deutschland GmbH Industriepark HOchst EXPOSURE OF VIABILITY D 65926 Frankfurt issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITORY AUTHORITY identified at the end of this page The viability of the microorganism identified in the section? previous was tested on 09-06-20022. On that date, the microorganism was _3 viable? 3 no longer viable IV. CONDITIONS IN WHICH THE FEASIBILITY TEST HAS BEEN CARRIED OUT4 V. INTERNATIONAL DEPOSITORY AUTHORITY Name DSMZ-DEUTSCHE SAMMLUNG VON Signature (s) of the person (s) having the power to represent the MIKROORGANISMEN UND ZELLKULTUREN GmbH International Depositary Authority, or of the authorized official (s) Address: Mascher Weg Ib D-38124 Brunswick (illegible signature) I Date: 10-09-2002 Enter the date of the first filing or, when a new deposit or a transfer has been made, the most recent relevant date (date of the new deposit or transfer). In the cases cited in Rule 10.2 letter a, figures (ii) and (iii), refer to the most recent feasibility test. Mark the relevant box with a cross. Fill in if the information has been requested and if the test results were negative.

Form DSMZ-BP / 9 (single page) 12/2001 BUDAPEST TREATY ON INTERNATIONAL RECOGNITION DSMZ DEPOSIT OF MICROORGANISMS FOR THE PURPOSES Deutsche Sammlung FOR PATENT PROCESSING von Mikroorganismen and Zellkulturen GmbH INTERNATIONAL FORMAT Aventis Pharma Deutschland GmbH Industriepark Hochst CONFIRMATION OF RECEPTION IN THE CASE OF A D 65926 Frankfurt FIRST PRESENTATION Issued according to Rule 7.1 by the INTERNATIONAL DEPOSITORY AUTHORITY identified at the end of this page I. IDENTIFICATION OF THE MICROORGANISM Reference sign attributed by the DEPOSENT: ENTRY NUMBER attributed by the INTERNATIONAL DEPOSITORY AUTHORITY: DSMhij DSM 15186 ? SCIENTIFIC DESCRIPTION AND / OR PROPOSAL TAXONOMIC DESIGNATION With the microorganism designated in I, the following was presented: A scientific description [YES a proposed taxonomic designation (Indicate with a cross what is relevant) IH. ENTRY AND ACCEPTANCE This International Depositary Authority accepts the microorganism designated by L that entered on 03-09-2002 (date of the first presentation) 1 IV. ENTRY OF THE REQUEST FOR TRANSFORMATION The microorganism designated by I has entered this International Depositary Authority on (the date of the first presentation) and a request for transformation of this first presentation has been submitted in a presentation according to the Budapest Treaty on (date of entry of the request for transformation) V. INTERNATIONAL DEPOSITORY AUTHORITY Name DSMZ-DEUTSCHE SAMMLUNG VON Signature (s) of the person (s) having the power to represent the MIKROORGANISMEN UND ZELLKULTUREN the International Depositary Authority, or the (the) official (s) GmbH authorized (s) Address: Mascher eg Ib (illegible signature) D-38124 Brunswick Date: 09-10-2002 If Rule 6.4.d) is applied, this is the date on which the Status of the international depository authority has been acquired. Form DSMZ-BP / 4 (one page only) 12/2001 BUDAPEST TREATY ON INTERNATIONAL RECOGNITION DSMZ DEPOSIT OF MICROORGANISMS FOR THE PURPOSES Deutsche Sammlung FOR PATENT PROCESSING von Mikroorganismen and Zellkulturen GmbH INTERNATIONAL FORMAT Aventis Pharma Deutschland GmbH Industriepark Hochst EXPOSURE OF VIABILITY D 65926 Frankfurt issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITORY AUTHORITY identified at the end of this page Enter the date of the first presentation or, when a new deposit or a transfer has been made, the most relevant relevant date (date of the new deposit or transfer). In the cases cited in Rule 10.2 letter a, figures ii and iii, refer to the most recent feasibility test. Mark the relevant box with a cross. Fill in if the information has been requested and if the test results were negative.

Form DSMZ-BP / 9 (one page only) 12/2001 BUDAPEST TREATY ON INTERNATIONAL RECOGNITION DSMZ DEPOSIT OF MICROORGANISMS FOR THE PURPOSES Deutsche Sammlung FOR PATENT PROCESSING von Mikroorganismen and Zellkulturen GmbH INTERNATIONAL FORMAT Aventis Pharma Deutschland GmbH Industriepark HiSchst CONFIRMATION OF RECEPTION IN THE CASE OF A D 65926 Frankfurt FIRST PRESENTATION issued according to Rule 7.1 by the INTERNATIONAL DEPOSITORY AUTHORITY identified at the end of this page I. IDENTIFICATION OF THE MICROORGANISM Reference sign attributed by the DEPOSITANT: ENTRY NUMBER attributed by the INTERNATIONAL DEPOSITORY AUTHORITY: DSMsyz DS 15187 H. SCIENTIFIC DESCRIPTION AND / OR PROPOSAL TAXONOMIC DESIGNATION With the microorganism presented in I was presented: The a scientific description HO a proposed taxonomic designation (Point to a cross what is relevant) ?? ENTRY AND ACCEPTANCE This International Depositary Authority accepts the microorganism designated by L that has entered on 03-09-2002 (date of the first presentation) 1 TV. ENTRY OF THE TRANSFORMATION REQUEST This International Depositary Authority received the microorganism identified in section I on (date of the first presentation) and a request for the transformation of this first presentation has been submitted in a presentation according to the Budapest Treaty on (date of the entry of the request for transformation) V. INTERNATIONAL DEPOSITORY AUTHORITY Name DSMZ-DEUTSCHE SAMMLUNG VON Signature (s) of the person (s) with the power to represent the MU ROORGANISMEN UND ZELLKULTUREN International Depositary Authority, or the (of the) authorized officer (s) GmbH Address: Mascheroder Weg Ib (illegible signature) D-38124 Brunswick Date:! O-09-20O2 If Rule 6.4.d is relevant, this is the date on which the Status of the International Depositary Authority has been acquired. Form DSMZ-BP / 4 (one page only) 12/2001 BUDAPEST TREATY ON INTERNATIONAL RECOGNITION DSMZ DEPOSIT OF MICROORGANISMS FOR THE PURPOSES Deutsche Sammlung FOR PATENT PROCESSING von Mikroorganismen and Zellkulturen GmbH INTERNATIONAL FORMAT Aventis Pharma Deutschland GmbH Industriepark Hochst EXPOSURE OF VIABILITY D 65926 Frankfurt issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITORY AUTHORITY identified at the end of this page Indicate the date of the first presentation or, when a new deposit or a transfer has been made, the most relevant relevant date (date of the new deposit or transfer). In the cases cited in Rule 10.2 letter a, figures ii and iii, refer to the most recent feasibility test. Mark the relevant box with a cross. Fill in if the information has been requested and if the test results were negative.

Form DSMZ-BP / 9 (single page) 12/2001 BUDAPEST TREATY ON INTERNATIONAL RECOGNITION DSMZ DEPOSIT OF MICROORGANISMS FOR THE PURPOSES Deutsche Sammlung FOR PATENT PROCESSING von Mikroorganismen and Zellkulturen GmbH INTERNATIONAL FORMAT Aventis Pharma Deutschland GmbH Industriepark Hochst CONFIRMATION OF RECEPTION IN THE CASE OF A D 65926 Frankfurt FIRST PRESENTATION issued according to Rule 7.1 by the INTERNATIONAL DEPOSITORY AUTHORITY identified at the end of this page I. IDENTIFICATION OF THE MICROORGANISM Reference sign attributed by the DEPOSITANT: ENTRY NUMBER attributed by THE INTERNATIONAL DEPOSITORY AUTHORITY: DSMuvw DSM 15188 ? SCIENTIFIC DESCRIPTION AND / OR PROPOSAL TAXONOMIC DESIGNATION With the microorganism presented in I was presented: Dü a scientific description _U a proposed taxonomic designation (Mark with a cross what is relevant) ??. ENTRY AND ACCEPTANCE This International Depositary Authority accepts the microorganism designated by L that entered on 03-09-2002 (date of the first presentation) 1 IV. ENTRY OF THE REQUEST FOR TRANSFORMATION This International Depositary Authority received the microorganism identified in section I on (the date of the first presentation) and a request for the transformation of this first presentation has been submitted in a presentation according to the Budapest Treaty on (date of the entry of the request for transformation) V. INTERNATIONAL DEPOSITORY AUTHORITY Name DSMZ-DEUTSCHE SAMMLUNG VON Signature (s) of the person (s) having the power to represent the MIKROORGANISMEN UND ZELLKULTUREN International Depositary Authority, or ( of) authorized official (s) GmbH Address: Mascheroder Weg Ib (illegible signature) D-38124 Brunswick Date: 09-10-2002 1 If Rule 6.4.d is relevant, this is the date on which the Status of the international depository authority has been acquired. Form DSMZ-BP / 4 (one page only) 12/2001 BUDAPEST TREATY ON INTERNATIONAL RECOGNITION DSMZ DEPOSIT OF MICROORGANISMS FOR THE PURPOSES Deutsche Sammlung FOR PATENT PROCESSING von Mikroorganismen and Zellkulturen GmbH INTERNATIONAL FORMAT Aventis Pharma Deutschland GmbH Industriepark Hóchst EXPOSURE OF VIABILITY D 65926 Frankfurt issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITORY AUTHORITY identified at the end of this page Indicate the date of the first presentation or, when a new deposit or a transfer has been made, the most relevant relevant date (date of the new deposit or transfer). In the cases cited in Rule 10.2 letter a, figures ii and iii, refer to the most recent feasibility test. Mark the relevant box with a cross. Fill in if the information has been requested and if the test results were negative.

Form DSMZ-BP / 9 (one page only) 12/2001 BUDAPEST TREATY ON INTERNATIONAL RECOGNITION DSMZ DEPOSIT OF MICROORGANISMS FOR THE PURPOSES Deutsche Sammlung FOR PATENT PROCESSING von Mikroorganismen and Zellkulturen GmbH INTERNATIONAL FORMAT Aventis Pharma Deutsehland GmbH Industriepark Hochst CONFIRMATION OF RECEPTION IN THE CASE OF A D 65926 Frankfurt FIRST PRESENTATION issued according to Rule 7.1 by the INTERNATIONAL DEPOSITORY AUTHORITY identified at the end of this page L IDENTIFICATION OF THE MICROORGANISM Reference sign attributed by the DEPOSITANT: ENTRY NUMBER attributed by THE INTERNATIONAL DEPOSITORY AUTHORITY: DSMdef DSM 15184 E. SCIENTIFIC DESCRIPTION AND / OR PROPOSAL TAXONOMIC DESIGNATION With the microorganism presented in I, the following was presented: The scientific description The proposed taxonomic designation (To mark with a cross the pertinent thing) THE ENTRY AND ACCEPTANCE This International Depositary Authority accepts the microorganism designated by I, who has entered on 03-09-2002 (date of the first presentation) 1 IV. ENTRY OF THE TRANSFORMATION REQUEST This International Depositary Authority received the microorganism identified in section I on (date of the first presentation) and a request for transformation of this first presentation has been submitted in a presentation under the Budapest Treaty on (date of the entry of the request for transformation) V. INTERNATIONAL DEPOSITORY AUTHORITY Name DSMZ-DEUTSCHE SAMMLUNG VON Signature (s) of the person (s) having the power to represent the MIKROORGANISMEN UND ZELLKULTUREN International Depositary Authority, or ( of) authorized official (s) ® GmbH Address: MascheroderWeg Ib (illegible signature) D-38124 Brunswick Date: 09-10-2002 If Rule 6.4.d is relevant, this is the date on which the Status of the international depository authority has been acquired.

Form DSMZ-BP / 4 (one page only) 12/2001 BUDAPEST TREATY ON INTERNATIONAL RECOGNITION DSMZ DEPOSIT OF MICROORGANISMS FOR THE PURPOSES Deutsche Sammlung FOR PATENT PROCESSING von Mikroorganismen and Zellkulturen GmbH INTERNATIONAL FORMAT Aventis Pharma Deutschland GmbH Industriepark Hochst EXPOSURE OF VIABILITY D 65926 Frankfurt issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITORY AUTHORITY identified at the end of this page Hydrate the date of the first presentation or, when a new deposit or a transfer has been made, the most relevant relevant date (date of the new deposit or transfer). In the cases cited in Rule 10.2 letter a, figures ii and iii, refer to the most recent feasibility test. Mark the relevant box with a cross. Fill in if the information has been requested and if the test results were negative. Form DSMZ-BP / 9 (one page only) 12/2001

Claims

CLAIMS 1. - A purified and isolated polynucleotide comprising a DNA sequence encoding a GLUT4V85M protein. 2. - The polynucleotide as claimed in claim 1, comprising a sequence of any of the following groups: a) a nucleotide sequence according to the sequence ID
N °. 1 b) a nucleotide sequence that hybridizes to a sequence of Sec. ID No. 1 under restrictive conditions and coding for a GLUT4V85M protein.
3. The polynucleotide as claimed in claim 1 or 2, wherein the GLUT4V85M protein has an amino acid sequence according to the sequence ID N °. 2.
4. - The polynucleotide as claimed in claims 1 to 3, wherein the coding region for the GLUT4V85M protein is operably linked to a promoter.
5. The polynucleotide as claimed in claim 1 to 4, which can be replicated in a yeast cell.
6. - The polynucleotide as claimed in claim 5, which can be used to express a protein in a yeast cell.
7. - A yeast cell of Saccharomyces cerevisiae, in which all glucose transporters are no longer functional and which does not contain any functional protein Erg4.
8. - A yeast cell of Saccharomyces cerevisiae, in which all glucose transporters are no longer functional and which does not contain any functional protein Fgy1 and no functional protein Erg4.
9. - The yeast cell as claimed in claim 7 or 8, wherein the ERG4 gene is completely or partially deleted.
10. Yeast cells as claimed in claim 7 which is deposited as DSM 15187 of Saccharomyces cerevisiae.
11. - The yeast cells as claimed in claim 8 or 9 which is deposited as DSM 15184 of Saccharomyces cerevisiae.
12. The use of a yeast cell as claimed in claims 15 to 18 to express a GLUT1 protein or a mammalian GLUT4 protein.
13. The use as claimed in claim 12 for expressing a human GLUT4 protein or a human GLUT1 protein.
14. The yeast cell as claimed in claim 7 comprising a polynucleotide as claimed in claims 1 to 6.
15. The yeast cell as claimed in claim 14 comprising a GLUT4V85M protein.
16. - The yeast cell as claimed in claim 14 and / or 15, which is deposited as DSM 15185 of Saccharomyces cerevisiae.
17. - A method for preparing a yeast cell as claimed in claims 14 to 16, comprising the steps of: a) providing a yeast cell as claimed in claim 7, b) providing a polynucleotide as claimed in claim 5 or 6, c) transforming the yeast cell as claimed in a) with the polynucleotide as claimed in b), d) selecting a transformed yeast cell, e) when appropriate, expressing a GLUT4V85M protein .
18. - The yeast cell as claimed in claim 8 or 9 comprising a polynucleotide as claimed in claims 1 to 6.
19. The yeast cells claimed in claim 8 comprising a GLUT4V85M protein.
20. - The yeast cell as claimed in claim 18 and / or 19, deposited as DSM 15186 of Saccharomyces cerevisiae.
21. - A method for preparing a yeast cell as claimed in claims 18 to 20, comprising the steps of: a) providing a yeast cell as claimed in claim 8 or 9, b) providing a polynucleotide as claimed in claim 5 or 6, c) transforming the yeast cell as claimed in a) with the polynucleotide as claimed in b), d) selecting a transformed yeast cell, e) when appropriate, expressing a GLUT4V85M protein.
22. - A yeast cell whose glucose transporters are no longer functional in their entirety comprising a polynucleotide as claimed in claims 1 to 6.
23. The yeast cell as claimed in claim 22 comprising a GLUT4V85 protein.
24. The yeast cell as claimed in the claim (s) 22 and / or 23, deposited as DSM 15188 of Saccharomyces cerevisiae.
25. A method for preparing a yeast cell as claimed in claims 22 to 24, comprising the steps of: a) producing a yeast cell whose glucose transporters are no longer functional in their entirety, b) providing a polynucleotide as claimed in claim 5 or 6, c) transforming the yeast cell as claimed in a) with the polynucleotide as claimed in b), d) selecting a transformed yeast cell, e) where appropriate, express a GLUT4V84M protein.
26. - A protein having the functional activity of a glucose transporter that is encoded by a polynucleotide sequence as claimed in any of claims 1 to 3.
27. The protein as claimed in claim 13 comprising an amino acid sequence according to Sec. ID No. 2.
28. - A method for identifying a compound that stimulates the activity of a GLUT4 protein, comprising the steps of: a) providing a yeast cell as claimed in one or more of claims 14 to 17, b) provide a chemical compound, c) contact the yeast of a) with the chemical compound of b), e) determine the glucose consumption by the yeast of c), f) relate the detected value of the glucose consumption of d) with respect to the detected value of glucose consumption in a yeast cell as claimed in a), which has not been contacted with a chemical compound as claimed in b), with a compound that causes an increase in the amount of glucose captured in the yeast as claimed in d) by stimulating the activity of said GLUT4 protein.
29. A pharmaceutical agent comprising a compound that is identified by a method as claimed in claim 28 and additives and excipients for formulating a pharmaceutical agent.
30. The use of a compound that has been identified by a method as claimed in claim 28 for preparing a pharmaceutical agent for the treatment of type I and / or II diabetes.
31. - A method for identifying a compound that inhibits the corresponding protein of the Fgy1 gene, comprising the steps of: a) providing a yeast cell as claimed in one or more of claims 7 to 10 containing a GLUT4 protein , b) provide a chemical compound, c) contact the yeast of a) with the chemical compound of b), d) determine the glucose consumption by the yeast of c), e) relate the detected value of glucose consumption d) with respect to the detected value of glucose consumption in a yeast cell as claimed in a), which is not contacted with a chemical compound as claimed in b), with a compound that causes an increase in the amount of glucose captured in the yeast as claimed in d) by stimulating the activity of a Fgy1 protein.
32. - A pharmaceutical agent comprising a compound that has been identified by a method as claimed in claim 31 and additives and excipients for formulating a pharmaceutical agent.
33. The use of a compound that has been identified by a method as claimed in claim 31 for preparing a pharmaceutical agent for the treatment of diabetes.
34.- A method for identifying a compound that inhibits the protein encoded by the ERG4 gene, which method comprises the steps of: a) providing a yeast cell as claimed in one or more of the claims from 22 to 25, b) provide a chemical compound, c) contact the yeast of a) with the chemical compound of b), d) determine the glucose consumption by the yeast of c), e) relate the detected value of the glucose consumption of d) with respect to the detected value of the glucose consumption in a yeast cell as claimed in a) that is not contacted with a chemical compound as claimed in b), with a compound that causes an increase in the amount of the glucose captured in yeast as claimed in d) by inhibiting the activity of an Erg4 protein.
35. - A pharmaceutical agent comprising a compound that has been identified by a method as claimed in claim 34, and additives and excipients for formulating a pharmaceutical agent.
36. The use of a compound that has been identified by a method as claimed in claim 34 for preparing a pharmaceutical agent for the treatment of diabetes.