A Glucose Transporter Chimera Confers a Dominant Negative Glucose Starvation Phenotype in Saccharomyces cerevisiae

Corresponding author: Marian Carlson, Columbia University, 701 W. 168th St., HHSC 922, New York, NY 10032., mbc1{at}columbia.edu (E-mail)

Communicating editor: M. JOHNSTON

	ABSTRACT

TOP ABSTRACT LITERATURE CITED

A family of glucose transporters mediates glucose uptake in Saccharomyces cerevisiae. We show that the dominant mutation GSF4-1, which impairs glucose repression of SUC2, results in a nonfunctional chimera of the transporters Hxt1p and Hxt4p. Hxt1/4p inhibits the function of wild-type glucose transporters. Similar mutations may facilitate analysis of the major facilitator superfamily.

THE extracellular concentration of glucose strongly influences gene expression and cell physiology in many organisms. In eukaryotes, a family of glucose transporters with 12 transmembrane domains mediates glucose uptake via facilitated diffusion. These transporters, encoded by the GLUT genes in mammals and by the HXT genes in Saccharomyces cerevisiae (MUECKLER et al. 1985 ; BISSON et al. 1993 ; MUECKLER 1994 ; KRUCKEBERG 1996 ; BOLES and HOLLENBERG 1997 ; CHARRON et al. 1999 ; OZCAN and JOHNSTON 1999 ), are members of the sugar permease family within the major facilitator superfamily (MARGER and SAIER 1993 ; NELISSEN et al. 1997 ).

The yeast hexose transporters have diverse kinetic properties and patterns of expression (BISSON et al. 1993 ; KRUCKEBERG 1996 ; BOLES and HOLLENBERG 1997 ; REIFENBERGER et al. 1997 ; OZCAN and JOHNSTON 1999 ). Six genes (HXT1–HXT4, HXT6, HXT7) encode the major proteins responsible for glucose transport. Their transcription is regulated by the concentration of glucose in the environment, and the different genes are induced and/or repressed by different levels of glucose (OZCAN and JOHNSTON 1995 ).

We previously isolated mutants of S. cerevisiae, designated gsf (glucose signaling factor), that are defective in glucose repression of SUC2 transcription (SHERWOOD and CARLSON 1997 ). Defects in glucose transport can confer this phenotype (SHERWOOD and CARLSON 1999 ). Here we have characterized the dominant mutation GSF4-1. During growth on high concentrations of glucose, the GSF4-1 mutant expresses SUC2 at levels 20-fold higher than the wild type and exhibits a His⁺ phenotype when bearing the reporter SUC2::HIS3, a fusion of the SUC2 promoter to HIS3 (SHERWOOD and CARLSON 1997 ). We show here that GSF4-1 results in synthesis of a nonfunctional chimera of the transporters Hxt1p and Hxt4p (Hxt1/4p).

We cloned the GSF4-1 locus from a library of genomic DNA of the mutant PS1450-2B (Table 1). The library contained partial Sau3AI DNA fragments in the centromeric vector pRS316 (SIKORSKI and HIETER 1989 ). We selected a plasmid that conferred a His⁺ phenotype to a glucose-grown wild-type strain (W303-1A) containing the SUC2::HIS3 reporter (pSUC2::HIS3; TU and CARLSON 1994 ). The recovered plasmid (pC1) also conferred glucose-insensitive SUC2 expression.

Plasmid pC1 bears a region of the genome that contains two glucose transporter genes, HXT1 and HXT4 (Fig 1A). However, in pC1 the 5' two-thirds of HXT1 is joined to the 3' end of HXT4. Southern blot and genetic linkage analysis confirmed that this gene fusion is tightly linked to the GSF4-1 mutation (data not shown).

View larger version (30K): In this window In a new window Download PPT slide   Figure 1. Structure of the GSF4-1 locus and the Hxt1/4p chimera. (a) Structure of the wild-type HXT1-HXT4 locus (top) and the corresponding region of the HXT1/4 (GSF4-1) locus in pC1 (bottom). Striped and solid arrows represent HXT1 and HXT4 sequences, respectively, and open bar represents an adjacent open reading frame. (b) Alignment of the HXT1, HXT4, and HXT1/4 sequences in the region corresponding to the HXT1/4 junction. Positions of diagnostic nucleotide similarities (boldface) and codon 433 of HXT1 and HXT1/4 (arrowhead) are indicated. (c) Alignment of the C-terminal 140 residues of Hxt1p and the Hxt1/4p chimera indicating identical residues (-) and amino acid substitutions (single letter code). Positions of residue 433 of Hxt1p and Hxt1/4p (arrowhead) and predicted transmembrane (TM) domains 10 to 12 (thick underline) are indicated. (d) Predicted membrane topology of the Hxt1/4p chimera. Striped and solid regions represent Hxt1p and Hxt4p sequences, respectively.

Hxt1p and Hxt4p are 73% identical and 81% similar, but their kinetic properties and patterns of expression are distinct. Hxt1p is a major low-affinity (K_m = 100 mM), high-capacity transporter that is expressed exclusively when glucose is abundant (>200 mM); Hxt4p is an intermediate-affinity transporter that is expressed when glucose levels are low (LEWIS and BISSON 1991 ; KO et al. 1993 ; OZCAN and JOHNSTON 1995 ; REIFENBERGER et al. 1997 ). Hxt1p, together with the low-affinity transporter Hxt3p, is responsible for glucose uptake in cells growing on high concentrations of glucose. The fusion of Hxt1p and Hxt4p sequences occurs at the beginning of the tenth transmembrane segment, on the basis of the predicted membrane topology of glucose transporters (MARGER and SAIER 1993 ). The sequence of the chimera differs from that of Hxt1p at 27 positions in the 140-residue C-terminal region where Hxt4p residues replace those of Hxt1p (Fig 1).

We next tested whether pC1, renamed pHXT1/4, restores glucose transport to a strain deleted for the six HXT genes that encode the major glucose transporters and SNF3 (hxt; LIANG and GABER 1996 ). pHXT1/4 did not support growth of the hxt strain on medium containing 5% glucose, suggesting that the Hxt1/4p chimera is nonfunctional for glucose transport (Fig 2A). The dominant GSF4-1 mutant phenotype is not due to loss of Hxt1p function because deletion of HXT1 does not confer a Gsf^- phenotype: SUC2 expression in hxt1 and hxt1/HXT1 strains is properly glucose repressed (~1 unit of invertase activity in these and wild-type strains). Moreover, a C-terminal truncation of Hxt1p (without fusion to Hxt4p) also did not confer a Gsf^- phenotype: deletion of codons 428 through 570 in pHXT1/4 did not affect SUC2::HIS3 expression (data not shown). Thus, the presence of the nonfunctional Hxt1/4p fusion protein is required for the dominant negative mutant phenotype.

View larger version (73K): In this window In a new window Download PPT slide   Figure 2. Effect of mutation and altered dosage of hexose transporter genes on glucose utilization and SUC2::HIS3 expression. (a) A strain lacking all major glucose transporter genes (hxt) was transformed with the vector pRS316 or its derivatives pHXT1 and pHXT1/4, expressing Hxt1p or Hxt1/4p from the HXT1 promoter, respectively. To assess the ability of Hxt1p and Hxt1/4p to restore glucose transporter function, 10-fold serial dilutions of cells were spotted onto selective synthetic media containing 5% glycerol/2% ethanol or 5% glucose. (b) Wild-type and GSF4-1 strains containing pSUC2::HIS3 were transformed with the indicated plasmids. To monitor expression of the SUC2::HIS3 reporter, 10-fold serial dilutions of cells were spotted onto selective synthetic medium plus 2% glucose containing (+His) or lacking (-His) histidine. (c) Wild-type and GSF4-1 strains containing pSUC2::HIS3 were transformed with the vector pSK134 or pADH1::HXT1, which expresses Hxt1p from the ADH1 promoter. SUC2::HIS3 expression was monitored as in b. Plates were incubated aerobically at 30°, except that the plate containing 5% glucose in a was incubated anaerobically.

To determine whether this dominant negative phenotype reflects a competition between nonfunctional Hxt1/4p and wild-type glucose transporters, we altered the relative dosage of the HXT1 and HXT1/4 (GSF4-1) genes. Expression of Hxt1p from the centromeric plasmid pHXT1 partially suppressed the His⁺ phenotype of the GSF4-1 strain bearing SUC2::HIS3 (Fig 2B, rows 2 and 4). Conversely, the additional expression of Hxt1/4p from plasmid pHXT1/4 enhanced the mutant phenotype (Fig 2B, rows 2 and 6). Moreover, expression of Hxt1p from the strong ADH1 promoter on multicopy plasmid pADH1-HXT1 completely suppressed the mutant phenotype of a GSF4-1 strain (Fig 2C, rows 3 and 4). Thus, there is a correlation between the relative dosage of the mutant and wild-type alleles and the severity of the mutant phenotype. These findings suggest that the nonfunctional Hxt1/4p chimera interferes with the synthesis or function of wild-type glucose transporters. At least one other transporter besides Hxt1p must be affected because the GSF4-1 mutant lacks Hxt1p.

We considered the possibility that the expression of Hxt1/4p interferes with the localization of glucose transporters to the plasma membrane because gsf2 mutations cause a defect in protein trafficking of Hxt1p (SHERWOOD and CARLSON 1999 ). We examined an Hxt1p-green fluorescent protein (GFP) fusion protein expressed from the HXT1 promoter. Hxt1-GFP is efficiently localized to the plasma membrane in wild-type cells and restores glucose transport as effectively as native Hxt1p to an hxt strain (SHERWOOD and CARLSON 1999 ). The Hxt1/4p mutant protein did not appear to affect the localization of Hxt1-GFP to the cell periphery (Fig 3).

View larger version (105K): In this window In a new window Download PPT slide   Figure 3. Localization of Hxt1-GFP in wild-type and GSF4-1 strains. Strains PS1451-5A and PS1451-5D were transformed with the centromeric plasmid pHXT1-GFP, which expresses Hxt1-GFP from the HXT1 promoter (SHERWOOD and CARLSON 1999 ). Transformants were grown to midlogarithmic phase in SC-Leu + 5% glycerol/2% ethanol medium, glucose was added to 4% to induce Hxt1-GFP synthesis (OZCAN and JOHNSTON 1995 ; SHERWOOD and CARLSON 1999 ), and aliquots were harvested 90 min after glucose addition. GFP autofluorescence was visualized in unfixed cells using a Nikon Eclipse E800 microscope. Images were captured using a digital camera (Orca-100, Inovision, Durham, NC) and imaging system (Openlab 2.0.5, Improvision) and were converted to Adobe Photoshop files for processing. Exposure time was 0.7 sec. Bar, 2.5 µm.

Together, these results suggest that Hxt1/4p actively interferes with the function of other members of the glucose transporter family, including Hxt1p and most likely Hxt3p, which also contributes to glucose uptake during growth on high glucose. The direct relationship between the relative dosage of the mutant and wild-type alleles and the severity of the mutant phenotype suggests that the nonfunctional Hxt1/4p chimera competes with wild-type glucose transporters.

A likely explanation for the dominant negative effect of GSF4-1 is that Hxt1/4p directly inhibits the function of glucose transport proteins. Dominant negative mutations often inhibit the function of oligomeric complexes (HERSKOWITZ 1987 ), and studies indicate an oligomeric structure for the glucose transporter Glut1p, which is 47% similar and 26% identical to Hxt1p. Glut1p is capable of forming functional dimers and tetramers (HEBERT and CARRUTHERS 1991 , HEBERT and CARRUTHERS 1992 ; PESSINO et al. 1991 ; ZOTTOLA et al. 1995 ). We propose that, in a GSF4-1 mutant, the Hxt1/4p chimeric protein interacts with wild-type glucose transporter subunits to create nonfunctional complexes. The number of functional complexes in the plasma membrane is thus decreased, leading to inhibition of glucose transport and glucose starvation phenotypes.

The conservation of structure and function between yeast and mammalian glucose transporters suggests that similar dominant negative chimeras will inform investigations of glucose transport in other eukaryotes. Moreover, glucose transporters belong to the major facilitator superfamily, which includes proteins with 12 transmembrane domains that function as sugar, amino acid, ammonia, phosphate, calcium, sulfate, purine, and multidrug transporters (MARGER and SAIER 1993 ; NELISSEN et al. 1997 ). Thus, chimeric proteins like the one characterized here may prove useful for the study, and possibly the genetic manipulation, of a wide variety of transport systems.

	FOOTNOTES

¹ Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.
² Present address: Instituto de Biomedicina de Valencia (CSIC), Valencia 46010, Spain.

	ACKNOWLEDGMENTS

We thank F. Chang, K. Ferenbacher, and P. Tran for assistance with microscopy and A. Mitchell, R. Rothstein, and L. Symington for valuable discussions. This work was supported by National Institutes of Health (NIH) grant GM34095 to M.C. P.W.S. was supported by a Damon Runyon-Walter Winchell Postdoctoral Fellowship and NIH/NIAID training grant 2T32A107161.

Manuscript received January 5, 2000; Accepted for publication February 24, 2000.

	LITERATURE CITED

TOP ABSTRACT LITERATURE CITED

BISSON, L. F., D. M. COONS, A. L. KRUCKEBERG, and D. A. LEWIS, 1993  Yeast sugar transporters. Crit. Rev. Biochem. Mol. Biol. 28:259-308[Medline].

BOLES, E. and C. P. HOLLENBERG, 1997  The molecular genetics of hexose transport in yeasts. FEMS Microbiol. Rev. 21:85-111[Medline].

CHARRON, M. J., E. B. KATZ, and A. L. OLSON, 1999  GLUT4 gene regulation and manipulation. J. Biol. Chem. 274:3253-3256[Free Full Text].

HEBERT, D. N. and A. CARRUTHERS, 1991  Cholate-solubilized erythrocyte glucose transporters exist as a mixture of homodimers and homotetramers. Biochemistry 30:4654-4658[Medline].

HEBERT, D. N. and A. CARRUTHERS, 1992  Glucose transporter oligomeric structure determines transporter function. J. Biol. Chem. 267:23829-23838[Abstract/Free Full Text].

HERSKOWITZ, I., 1987  Functional inactivation of genes by dominant negative mutations. Nature 329:219-222[Medline].

KO, C. H., H. LIANG, and R. F. GABER, 1993  Roles of multiple glucose transporters in Saccharomyces cerevisiae.. Mol. Cell. Biol. 13:638-648[Abstract/Free Full Text].

KRUCKEBERG, A. L., 1996  The hexose transporter family of Saccharomyces cerevisiae.. Arch. Microbiol. 166:283-292[Medline].

LEWIS, D. A. and L. F. BISSON, 1991  The HXT1 gene product of Saccharomyces cerevisiae is a new member of the family of hexose transporters. Mol. Cell. Biol. 11:3804-3813[Abstract/Free Full Text].

LIANG, H. and R. F. GABER, 1996  A novel signal transduction pathway in Saccharomyces cerevisiae defined by Snf3-regulated expression of HXT6.. Mol. Biol. Cell 7:1953-1966[Abstract].

MARGER, M. D. and M. H. SAIER, 1993  A major superfamily of transmembrane facilitators that catalyse uniport, symport, and antiport. Trends Biochem. Sci. 18:13-20[Medline].

MUECKLER, M., 1994  Facilitative glucose transporters. Eur. J. Biochem. 219:713-725[Medline].

MUECKLER, M., C. CARUSO, S. A. BALDWIN, M. PANICO, and I. BLENCH et al., 1985  Sequence and structure of a human glucose transporter. Science 229:941-945[Abstract/Free Full Text].

NELISSEN, B., R. DE WACHTER, and A. GOFFEAU, 1997  Classification of all putative permeases and other membrane plurispanners of the major facilitator superfamily encoded by the complete genome of Saccharomyces cerevisiae.. FEMS Microbiol. Rev. 21:113-134[Medline].

ÖZCAN, S. and M. JOHNSTON, 1995  Three different regulatory mechanisms enable yeast hexose transporter (HXT) genes to be induced by different levels of glucose. Mol. Cell. Biol. 15:1564-1572[Abstract].

OZCAN, S. and M. JOHNSTON, 1999  Function and regulation of yeast hexose transporters. Microbiol. Mol. Biol. Rev. 63:554-569[Abstract/Free Full Text].

PESSINO, A., D. N. HEBERT, C. W. WOON, S. A. HARRISON, and B. M. CLANCY et al., 1991  Evidence that functional erythrocyte-type glucose transporters are oligomers. J. Biol. Chem. 266:20213-20217[Abstract/Free Full Text].

REIFENBERGER, E., E. BOLES, and M. CIRIACY, 1997  Kinetic characterization of individual hexose transporters of Saccharomyces cerevisiae and their relation to the triggering mechanisms of glucose repression. Eur. J. Biochem. 245:324-333[Medline].

SHERWOOD, P. W. and M. CARLSON, 1997  Mutations in GSF1 and GSF2 alter glucose signaling in Saccharomyces cerevisiae.. Genetics 147:557-566[Abstract].

SHERWOOD, P. W. and M. CARLSON, 1999  Efficient export of the glucose transporter Hxt1p from the endoplasmic reticulum requires Gsf2p. Proc. Natl. Acad. Sci. USA 96:7415-7420[Abstract/Free Full Text].

SIKORSKI, R. S. and P. HIETER, 1989  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.. Genetics 122:19-27[Abstract/Free Full Text].

THOMAS, B. J. and R. ROTHSTEIN, 1989  Elevated recombination rates in transcriptionally active DNA. Cell 56:619-630[Medline].

TU, J. and M. CARLSON, 1994  The GLC7 type 1 protein phosphatase is required for glucose repression in Saccharomyces cerevisiae.. Mol. Cell. Biol. 14:6789-6796[Abstract/Free Full Text].

ZOTTOLA, R. J., E. K. CLOHERTY, P. E. CODERRE, A. HANSEN, and D. N. HEBERT et al., 1995  Glucose transporter function is controlled by transporter oligomeric structure: a single, intramolecular disulfide promotes GLUT1 tetramerization. Biochemistry 34:9734-9747[Medline].

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