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. USA 85 (1988) 2131 -853 ~~~~~~~~~~~~~~~~~~~~~~~~~~GTCGACTTCTTrGA -840AAAGATG'CGTAACTGCCAAGA'ATkACTTAGATTAAAGTAAA'CTTTCA''GAAACGATATAC -720 ACACACATTTCTACCTGATAGAGAAAAAkAAGTTGCTC'GTAArGTC--kC'G'CtGGGG''ATG Met Asp Pro Asn Ser Asn Ser Ser Ser Gin Thr Leo Arg Gin Gin Lys Gin Gly Phe Leu Asp Lys Ala Leo Gin Arg Val Lvs Cv Ilie 30 I ATG CAT CCT AAT AGT NAG ACT TCT AGC GA.A ACA TTA CCC CAA GAG AMA CAG ATA GAC GAG AAC TCA ATT TIA TTT TCA GAG CCT CCt CAC Ala Leu Ar gAr g Asn Asn Set Asn Lys As p His Thr Thr As pAs Thr Thr GIy Ser Ilie Arg TFr Pro Thr Ser Leol Gin Arg Gln Asn 60 91 AAA CMA TCT ATG ATG ATG TGT MAC AIA CTCAT ACA ACA CAT CAT AGC ACA CCI AGC ATA CGA ACG CCI ACC AGC TTG GAG CCC CMA MT 5cr As p Gin Ser -Asn Met Thr Set Val Phe Thr As p As pIlie Ser Thr Ilie As As p sn Set Ilie Leo Phe Set Gin Pro Pro Gin 90 181 TCT GAC ArmAGC CM TCT MAT AIG ACA TCG GTG TTT ACG CAT GAC ATT TCT ACC ATA GAC CACMCI TCA AlT TTA TTT TCA GAG CCI CCT GAG Lys Gin Set Met Met Met Set tIle Cys Vat ClyV Val The Val l a l l Te.tensThelytrM ItGy eUlejAsn Set 12u 271 AAA CMATCTATG AIGATG TCTIATAITUO GTA CIGil TT GT MCA CIT CCC IMG !TTTAXIIGGT AUCT ACA' GTt GTGATCMAC ACT lie Thr Set Met Asn It- Val Lys Set His Val Ala Pro Asn His As pSet Phe Thr Ala Gin Gin Met Set'l eTru Val erPhe Leu 150 3o1 ATT ACA TCT ATG MAC iAr CTG MAG TCA GACGTCA GCA CCT MAT CAC CAT TCA TTT ACC CCC CMA CMA ATC TCC TTflGTOGTATTTT2 G SerLeuGlyThrPhePheGI laLen Thr Ala Pro Phe Ile -Set As p Set lyr Gly ArgLys Pro Thr leT IlePhe Ser Thr-lieiPh 180 451 C TGGGA ACT TTT TIT GC OCT TA ACT OUCAOA ITT AlATICGAT TCC IAT CCC AGG MkG CCI ACT C5 2TTIGCAGTACA ATIT T IlePheSerIlGl As Se Lu G-n al lyAla Gly Gly Ilie Thr Leo Le Ilie Vai Gly Arg a Il~e Sex Gly Ile GI Ile GIy 213 541 AT T C T G A TTACGGAGT=G ATC ACA TTA TIC ATT GIG CCA AG GT ATTA GGT ATOC CC AlA. CC AlaIle Set Al# Vat Vat Pro Len Ty-r Gin Ala Gin AlaiThr His Lys Set Leu Argr ati leSlet ThrT Gh IrAe i 24 631 GCA TOTTC TTGTOCA TTA TAC CAA GGA GAA GC1 AGA CAT AMA TGA TTA AGA 001 MT AnT ATT ITT ACT TAG CAA TOXGCCC ATT iThr:TIrQy ten tan, Vat Set- Set Ala: Set Gin Gly STh His Aia Ar g Ason As 1)Ala Set Set Tyr Arg-7le Pr Ol Le Gin 270 721 AO CCCCIr,T=TGCTGI GTG [CA ACT OGA GTj TCG CAA CCC AGCA CAL GCA AGAAAMG GAL GGA TCT TCG PATlG CCCA±TACCCITRG CA t Vat Itr Set Set Phe Len Ala Ile Gly Met Phe Phe Len Pro 'in Set Pro Arg Ivr Tvr Val Leo Lys Asp Lys Leo As GIn Aia 3100) 811 T1T1Ir TOG10 QTC1 TT CCG CC ATC GGC AIG TTG TIT GIG CCT 'AGAT CGA CCC T'AT TAG GTT TTG AMA GAG MAG CTA CAT GMA GGA Aia Lvs Set Leo Set Phe Leo Arg Civ Val Pro Vai His As p Set Gly Leo Len Gin Glu Leo Val Gin TIle Lvs Ala Thr Tyr As p Tvr 330 901 GGT AAA TGT TTA TGG TTT TTA AGA CCI GTA GGA GTG CAT CAT TGT CCC TTA CTC GMA GM- GTA GTT CMA ATA ACG GGA ACA TAT CAT T.AG Ciu Ala Set Phe Clv Set Set Asn Phe Iie Asp Cys Phe Iie Set Set Lvs Set Atg Pro Lys Gin Thr Leo Arg et Ph ITt Gly 13$ 360 991 GAG CGA TCT ITT CCi TGT TGC AAG TTG ATT CAT TCT ITT ATT TGA ACT AAA ACT AGA GGA MAG CMA ACT GTA AG6CATG TTT AGO GGA AIT Ala Len Gin Ala Phe Gin Gin Phe Se' 01 Ilie Asan he tIle Pha Iyr Tsr Civ Val As n Phe Phe [K T7Yhr Ciy Val 5cr Asn Set 390 1081 CCC CTI CIA OGA TTT CAA CAA TTT ICA COT ITO 1A0 ITI ATA ITTT Tk k ~ TC MAT TITC TIrC MAT MGAGA GCA GTC AGT MAT AGT TreVat Set Pa tIle Thr yr Ate Vat IAn Vai Vat The IAn Vat Pro Gly Lau Phe Phe Val-lGin Phe