The yeast SNF3 gene encodes a glucose transporter homologous to the mammalian protein.

Abstract

The SNF3 gene is required for high-affinity glucose transport in the yeast Saccharomyces cerevisiae and has also been implicated in control of gene expression by glucose repression. We report here the nucleotide sequence of the cloned SNF3 gene. The predicted amino acid sequence shows that SNF3 encodes a 97-kilodalton protein that is homologous to mammalian glucose transporters and has 12 putative membrane-spanning regions. We also show that a functional SNF3-lacZ gene-fusion product cofractionates with membrane proteins and is localized to the cell surface, as judged by indirect immunofluorescence microscopy. Expression of the fusion protein is regulated by glucose repression.Transport of glucose in the yeast Saccharomyces cerevisiae, as in most mammalian cell types, occurs by carrier-mediated facilitated diffusion (1)(2)(3) Protein Fractionation and Enzyme Assays. Crude lysates were prepared from glucose-derepressed cells as described (12), except that protease inhibitor was omitted, and were fractionated by centrifugation at 13,000 x g in a microcentrifuge (Fisher) at 4°C. Protein concentrations were determined with the Bio-Rad protein-assay kit. p-Galactosidase (13), dipeptidyl aminopeptidase (14), and glucose-6-phosphate dehydrogenase (15) were assayed as described.Indirect Immunofluorescence. Cells were fixed with formaldehyde, treated with Glusulase (DuPont) and Zymolyase lOOT (Seikagaku Kogyo, Tokyo), immobilized on polylysine-coated slides, and stained with antibody and 4',6-diamidino-2-phenylindole (DAPI), essentially as described (16). Mouse monoclonal antibody to f3-galactosidase was purchased from Promega Biotec (Madison, WI), and fluorescein isothiocyanate (FITC)-conjugated sheep antibody to mouse immunoglobulin G was purchased from Sigma. Cells were examined with a Zeiss photomicroscope III equipped for epi-illumination fluorescence with standard FITC and DAPI filter sets. Cells were photographed with Kodak T-MAX 400 film and a x 40 objective. Exposure times were 3 min for FITC fluorescence and 2 min for DAPI fluorescence. RESULTSSequence Homology Between the SNF3-Encoded Protein and Mammalian Glucose Transporters. We determined the nucleotide sequence of a 3690-base-pair region of the previously cloned (7) SNF3 gene. Fig. 1 shows the nucleotide sequence and the inferred amino acid sequence. The sequence of the predicted 96,713-dalton protein (884 amino acids) was compared to the sequences of the glucose transporters from human HepG2 hepatoma cells (17) and rat brain (18). Significant homology (28% identity) was found between Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.

Glucose Uptake and Metabolism in grr1/cat80 Mutants of Sacharomyces cerevisiae

“…DNA fragments as probes for Northern analysis were used as follows: HXTl, 2.8 kb EcoRI-HindIII; HXT3, 1.5-kb HindIII fragment; HXT4, 1.6-kb HindIII (for localization of the HXT sequences, see [21]); SNF3 [22] flanking sequence as a 0.8-kb HindIII fragment cloned into vector YEp352. A probe for the gene MALlI encoding maltose permease was obtained from plasmid pRM1.l as a 2-kb BglII fragment.…”

Rgt1p of Saccharomyces cerevisiae, a Key Regulator of Glucose-Induced Genes, Is both an Activator and a Repressor of Transcription

“…Glucose-stimulated inhibition of Rgt1p requires Grr1p, a leucine-rich repeat-containing protein, and Snf3p, a glucose transporter thought to be involved in sensing glucose and generating an intracellular signal (6,9,10,14,21,31,33). Thus, grr1⌬ and snf3⌬ mutants grow poorly on glucose and display severely impaired glucose transport because of reduced expression of glucose transporters (5,6,24,25,33,46).…”

“…In cells growing in the absence of glucose, Rgt1p represses HXT expression; addition of glucose causes Rgt1p function to be inhibited, leading to derepression of HXT expression. The different responses of HXT1 and HXT2 (and HXT4) to different levels of glucose are due to two additional regulatory mechanisms that act on these genes: HXT2 and HXT4 are also subject to glucose repression mediated by Mig1p, a repressor that is activated by high levels of glucose (32, 47), and HXT1 responds to an additional regulatory mechanism, whose components have not yet been identified, that requires high levels of glucose for function (31).Glucose-stimulated inhibition of Rgt1p requires Grr1p, a leucine-rich repeat-containing protein, and Snf3p, a glucose transporter thought to be involved in sensing glucose and generating an intracellular signal (6,9,10,14,21,31,33). Thus, grr1⌬ and snf3⌬ mutants grow poorly on glucose and display severely impaired glucose transport because of reduced expression of glucose transporters (5,6,24,25,33,46).…”

Functional expression of the Chlorella hexose transporter in Schizosaccharomyces pombe.

“…From its sequence the gene is predicted to code for a membrane protein 533 amino acids in length (5). The gene product shows a high degree of similarity (30% of the amino acids are identical) to bacterial (6), fungal (7), and mammalian (8) sugar transporters. Although the gene was not expressed in a Chlorella mutant defective in hexose uptake (5), direct proof that the gene product is the Chlorella hexose transporter had not been obtained.…”