The Yeast SNF3 Gene Encodes a Glucose Transporter Homologous to the Mammalian Protein (1Acz Fusions/Membrane Proteins/Immunofluorescence/Glucose Repression) JOHN L

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The Yeast SNF3 Gene Encodes a Glucose Transporter Homologous to the Mammalian Protein (1Acz Fusions/Membrane Proteins/Immunofluorescence/Glucose Repression) JOHN L Proc. Nail. Acad. Sci. USA Vol. 85, pp. 2130-2134, April 1988 Biochemistry The yeast SNF3 gene encodes a glucose transporter homologous to the mammalian protein (1acZ fusions/membrane proteins/immunofluorescence/glucose repression) JOHN L. CELENZA, LINDA MARSHALL-CARLSON, AND MARIAN CARLSON* Department of Genetics and Development and Institute for Cancer Research, Columbia University College of Physicians and Surgeons, New York, NY 10032 Communicated by David Botstein, December 23, 1987 ABSTRACT The SNF3 gene is required for high-affinity Construction of SNF3-lacZ Fusions. SNP'3-lacZ gene fu- glucose transport in the yeast Saccharomyces cerevisiae and sions were constructed by inserting SNF3 DNA fragments has also been implicated in control of gene expression by containing at least 0.8 kilobases of 5' noncoding sequence glucose repression. We report here the nucleotide sequence of into the 2-Am plasmid vectors YEp353 and YEp357R (11). the cloned SNF3 gene. The predicted amino acid sequence The SNF3(3)-lacZ fusion was constructed by inserting an shows that SNF3 encodes a 97-kilodalton protein that is EcoRI-BamHI fragment into YEp353, SNF3(321)-lacZ by homologous to mammalian glucose transporters and has 12 inserting a Sal I-Spe I fragment into YEp357R, and SNF3- putative membrane-spanning regions. We also show that a (797)-lacZ by inserting a Sal I-EcoRI fragment into YEp- functional SNF3-lacZ gene-fusion product cofractionates with 357R. membrane proteins and is localized to the cell surface, as Preparation of Glucose-Derepressed Cells. Wild-type judged by indirect immunofluorescence microscopy. Expres- (SNF3) yeast cells carrying each of the SNF3-lacZ gene sion of the fusion protein is regulated by glucose repression. fusions were grown to midlogarithmic phase in synthetic complete medium containing 2% glucose, with selection for the plasmid. Cells were derepressed by shifting to medium Transport of glucose in the yeast Saccharomyces cerevisiae, containing 0.05% glucose for 3 hr. as in most mammalian cell types, occurs by carrier-mediated Protein Fractionation and Enzyme Assays. Crude lysates facilitated diffusion (1-3). Bisson and Fraenkel (4, 5) showed were prepared from glucose-derepressed cells as described by kinetic analysis of glucose uptake that yeast has a (12), except that protease inhibitor was omitted, and were constitutive low-affinity transport system (Km 20 mM for fractionated by centrifugation at 13,000 x g in a microcen- glucose) and a glucose-repressible high-afflinity system (Km trifuge (Fisher) at 4°C. Protein concentrations were deter- 1 mnM). Previously, Neigeborn and Carlson (6) described mined with the Bio-Rad protein-assay kit. p-Galactosidase the isolation of mutations in the SNF3 (sucrose nonferment- (13), dipeptidyl aminopeptidase (14), and glucose-6-phos- ing) gene that caused defects in regulation of glucose-repres- phate dehydrogenase (15) were assayed as described. sible genes, and Neigeborn et al. (7) reported the cloning of Indirect Immunofluorescence. Cells were fixed with form- SNF3 and the construction of snJ3 null mutations. Mutations aldehyde, treated with Glusulase (DuPont) and Zymolyase in SNF3 cause defects in growth on glucose at low concen- lOOT (Seikagaku Kogyo, Tokyo), immobilized on polyly- tration (0.1%) and on sugars such as sucrose, which is sine-coated slides, and stained with antibody and 4',6- hydrolyzed extracellularly by invertase to yield glucose and diamidino-2-phenylindole (DAPI), essentially as described fructose. Subsequent studies showed that snJ3 mutants lack (16). Mouse monoclonal antibody to f3-galactosidase was the high-affinity glucose-uptake system, whereas low- purchased from Promega Biotec (Madison, WI), and fluo- affinity uptake appears to be unimpaired (8). These findings rescein isothiocyanate (FITC)-conjugated sheep antibody to suggested the possibility that SNF3 encodes a structural mouse immunoglobulin G was purchased from Sigma. Cells component of the high-affinity glucose-uptake system. We were examined with a Zeiss photomicroscope III equipped therefore determined the sequence of the cloned SNF3 for epi-illumination fluorescence with standard FITC and gene,t and we report here that SNF3 encodes a protein that DAPI filter sets. Cells were photographed with Kodak is homologous to mammalian glucose transporters. We also T-MAX 400 film and a x 40 objective. Exposure times were show that a functional SNF3-lacZ gene-fusion product cof- 3 min for FITC fluorescence and 2 min for DAPI fluores- ractionates with membrane proteins and is localized to the cence. cell surface. Expression of the gene fusion is regulated by glucose repression. RESULTS Sequence Homology Between the SNF3-Encoded Protein and Mammalian Glucose Transporters. We determined the MATERIALS AND METHODS nucleotide sequence of a 3690-base-pair region of the previ- Nucleotide Sequence Analysis. Intact and BAL-31 nucle- ously cloned (7) SNF3 gene. Fig. 1 shows the nucleotide ase-treated restriction fragments from the SNF3 gene were sequence and the inferred amino acid sequence. The se- cloned in the vectors M13mpl8 and M13mpl9 (9). The quence of the predicted 96,713-dalton protein (884 amino sequence of both strands was determined by the method of acids) was compared to the sequences of the glucose trans- Sanger et al. (10) with the 17-nucleotide sequencing primer porters from human HepG2 hepatoma cells (17) and rat brain (Amersham). (18). Significant homology (28% identity) was found between Computer Methods. Amino acid sequences were aligned with the assistance of the program ALIGN (30). Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; FITC, fluo- rescein isothiocyanate. *To whom reprint requests should be addressed. The publication costs of this article were defrayed in part by page charge tThis sequence is being deposited in the EMBL/GenBank data base payment. This article must therefore be hereby marked "advertisement" (Bolt, Beranek, and Newman Laboratories, Cambridge, MA, and in accordance with 18 U.S.C. §1734 solely to indicate this fact. Eur. Mol. Biol. Lab., Heidelberg) (accession no. J03246). Downloaded by guest on September 26, 2021 2130 Biochemistry: Celenza et al. Proc. Natl. Acad. Sci. 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