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Dominant and Recessive Suppressors That Restore Glucose Transportin a Yeast Snf3 Mutant

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Dominant and Recessive Suppressors That Restore Glucose Transportin a Yeast Snf3 Mutant Copyright 0 1991 by the Genetics Society of America Dominant and Recessive Suppressors That Restore Glucose Transportin a Yeast snf3 Mutant Linda Marshall-Cadson*, Lenore Neigeborn*, David Coons?, Linda Bisson? and Marian Carlson* *Department of Genetics and Development and Institute of Cancer Research, Columbia University College of Physicians and Surgeons, New York, New York 10032, and tDepartment of Viticulture and Enology, University of Calijornia, Davis, Calvornia 95616 Manuscript received October 29, 1990 Accepted for publication March 23, 1991 ABSTRACT The SNF3 gene of Saccharomycescereuisiae encodes a high-affinity glucose transporter that is homologous to mammalian glucose transporters. To identify genes that are functionally related to SNF3, we selected for suppressorsthat remedy the growth defect ofsnf3 mutants on low concentrations of glucose or fructose. We recovered 38 recessive mutations that fall into a single complementation group, designated rgtl (restores glucose transport).The rgtl mutations suppressa snf3 null mutation and are not linked to snf3. A naturally occurring rgtl allele was identified in a laboratory strain. We also selected five dominant suppressors. At least two are tightly linked to one another and are designated RGT2. The RGT2 locus was mapped 38 cM from SNF3 on chromosome N.Kinetic analysis of glucose uptake showedthat the rgtl and RGT2 suppressors restore glucose-repressible high-affinity glucose- transport in a snf3 mutant. These mutations identify genes that may regulate or encode additional glucose transport proteins. HE transport of glucose into eukaryotic cells is gene, was first identified by isolating mutants defec- T mediated by specific carrier proteins. The genes tive in growth on sucrose or raffinose (NEIGEBORN encoding a variety of glucosetransporters from mam- and CARLSON1984). These sugars are hydrolyzed malian cells have been sequenced, and many of the extracellularly, and the resulting glucose and/or fruc- proteins are closely related, containing 12 putative tose, released at low concentration, must be trans- membrane-spanning regions and conserved sequence ported into the cell. The mutants are also defectivein motifs (MUECKLERet al. 1985; BIRNBAUM,HASPEL growth on medium containing glucose at low concen- and ROSEN1986; THORENSet al. 1988; FUKUMOTOet tration (NEIGEBORNand CARLSON1984; NEIGEBORN al. 1988; BIRNBAUM1989; CHARRONet al. 1989; et al. 1986). Kineticanalysis showed thatthe snf3 JAMES,STRUBE and MUECKLER1989). Different mam- mutants lack high-affinity glucose uptake, but exhibit malian transport systems are subject to different reg- normal low-affinity uptake (BISON et al. 1987). The ulation; for example, some transporters are regulated defect in high-affinity transport accounts for the in response to insulin (CHARRONet al. 1989; JAMES, growth phenotypes of snf3 mutants. STRUBEand MUECKLER1989). The SNF3 gene was cloned (NEIGEBORNet al. 1986) We have studied glucose transport in Saccharomyces and encodes a 97-kilodalton protein, containing 12 cerevisiae with the view that genetic analysis should putative membrane-spanning regions, that is homol- prove useful in studying a complex, highly regulated ogous to mammalian glucose transporters (CELENZA, process that is essential to all eukaryotic cells. Like MARSHALL-CARLSONand CARLSON1988). The SNF3 higher organisms, S. cerevisiae also appears to express protein differs from the mammalian transporters in multiple, differently regulated glucose transport sys- having additional sequences at the N and C termini. tems. Kinetic analysis of glucose uptake in yeast has The large C-terminal extension (303 amino acids) revealed at least two components, a high affinity com- contributes to, but is not essential for, SNF3 function ponent (K, - 1-2 mM) that is dependent on the pres- (MARSHALL-CARLSONet al. 1990). SNF3 is alsoho- ence of a cognate hexose kinase and a low affinity mologous to other yeast and bacterial sugar trans- component (K, -20-50 mM) (BISON and FRAENKEL porters (MAIDENet al. 1987; SZKUTNICKAet al. 1989; 1983; LANG and CIRILLO1987). Bothsystems also CHENGand MICHELS1989; NEHLIN,CARLBERG and transport fructose. The two components are differ- RONNE1989). The SNF3 product is associated with ently regulated: the high-affinity system is repressed membranes and is localized at the cell surface (CE- by glucose, and the low-affinity system is expressed LENZA, MARSHALL-CARLSONand CARLSON 1988). constitutively (BISSONand FRAENKEL1984). Taken together, these data indicate that SNF3 en- A glucose transporter gene of S. cerevisiae, the SNF3 codes a high-affinity glucosetransporter. Genetics 128: 505-512 uuly, 1991) 506 Marshall-Carlson L. et al. Previousstudies have identified additional genes TABLE 1 that appear functionally related to SNF3. A selection List of S. cerarisiae strains for multicopy plasmids that complement the growth defect of a snf3 mutant yielded at least five different Strain" Genotype genes (BISSONet al. 1987). One of these, named HXT2, MCY657 MATa snf3-72 ura3-52 lys2-801SUC2 encodes a protein that resembles other glucose trans- (SUC7?) porters, and mutations in HXT2 affect high-affinity MCY659 MATa snf3-72 ura3-52 lys2-801 ade2-101 hexose transport, although not as severely as muta- suc2 (SUC7?) tions in SNF3 (KRUCKEBERGand BISON 1990). Low MCY7 14 MATa snf3-217 ura3-52SUC2 MCY 1093 MATa ura3-52 lys2-801 his4-539SUC2 stringencyblot hybridization analysis of genomic MCY 1094 MATa ade2-I01 ura3-52SUC2 DNA suggested thatthe yeast genome contains a MCY 1408 MATa snf3-A4::HIS3 his3-A200 ura3-52 family of sequences homologous to HXT2, probably lys2-801 ade2-101 SUC2 including additional glucose transporter genes.At MCY 1409 MATa snf3-A4::HIS3 his3-A200 ura3-52 least one additional transporter gene must exist, as 1~~2-801SUC2 MCY1410 MATa snf3-A4::HIS3 his3-A200 ade2-101 neither SNF3 nor HXT2 is responsible for low-affinity 1~~2-801SUC2 transport. MCY1471 MATa rgtl-1 ade2-I01SUC2 In this study, we have used a different approach to MCY1516 MATa rgtl-1 snf3-A4::HIS3 ura3-52 identify genes that are functionally related to SNF3. ade2-I01 (his3-A200?)SUC2 We sought to identifygenes that could mutate to MCY 1520 MATa rgtl-1 snf3-A4::HIS3 ade2-I01 (his3-A200?)SUC2 suppress the transport defect caused by a snf3 muta- MCY1710 MATa RGT2-I snf3-A4::HIS3 his3-A200 tion. We therefore selected for suppressors that re- ura3-52 lys2-801 SUC2 store growth of mutants on raffinose, which requires MCY1711 MAT@ RGT2-1snf?-A4::HIS3 his3-A200 high-affinity fructose uptake. We anticipated that this lys2-801 ade2-101 ura3-52 SUC2 selection could yield mutations that alter other trans- MCY1713 MATa RGT2-2snf3-A4::HIS3 his3-A200 ura3-52 lys2-801 ade2-101 SUC2 porters so that they can bind and transport fructose MCY1714 MATa Rgt#3 snf3-A4::HIS3 his3-A200 with high affinity. Alternatively, the selection could ura3-52 lys2-801 ade2-I01SUC2 yield mutations that increaseexpression of other MCY1717 MATa Rgt#4 snf3-A4::HlS3 his3-A200 transporters or allow expression of normally cryptic ura3-52 lys2-801 ade2-101 SUC2 transporters. We describe here the isolation of two MCY1719 MATa Rgt#5 snf3-A4::HIS3 his3-A200 ura3-52 lys2-801 ade2-I01SUC2 classes of suppressors that restore high-affinity uptake MCY 1807 MATa ccsl snf3-A4::HIS3 (his3-A200?) in snf3 mutants: recessive rgtl mutations and domi- ura3-52 SUC2 nant RGT2 mutations. MCY2035 MATa rgtl-2snf3-72 lys2-801 his4-539 ura3-52::pLSI 1 SUC2 MCY2 157 MATa RGT2-1 his3-A200 lys2-801 SUC2 MATERIALS AND METHODS MCY2 160 MATa cdc9 snf3-A4::HIS3 (his3-A200?) suc2 Strains and general genetic methods: Strains of S. cere- MCY2 162 MATa leu2-3 SUF25-1 ura3-52 his4- uisiae used in this study are listed in Table 1. pLS 1 1carries 519R SUC2 the URA3 gene and a SUCZ-LEU2-lacZ fusion (SAROKINand MCY2166 MATa cdc9 snf3-A4::HIS3 (his3-A200?) CARLSON1985) that is irrelevant to this study. Genetic lys2-801 ura3-52 SUC2 analysis was carried out by standard methods (SHERMAN, MCRY 168 MATa snf3-72 lys2-801 his4-539 ura3- FINKand LAWRENCE1978). Growth phenotypes were de- 52::PLSl I suc2 termined by spotting cell suspensionsonto plates usinga 32- LBY415 MATa hxt2::LEU2 snf3-A4::HIS3 hid- point inoculator and incubating the plates at 30" under A200 ura3-52 lys2-801 ade2-101 trpl- anaerobic conditions in a GasPak disposable anaerobic sys- A43 leu2-AI SUC2 tem (BBL). Growth ofsingle colonies was examined as 1629' MATa leu2-3 described in the legend to Figure 1. Unless otherwise noted, 1695b MATa leu2-3 his4-519R1 ura3-52 plates contained rich medium (YEP) and 2% of the indicated SUF25-1 carbon source. Glucose uptake assays: Cells were grown in yeast nitro- a MCY strains are from the CARLSONlaboratory, and the LBY gen base (0.67%) containing casamino acids (0.2%), auxo- strain is from the BISON laboratory. * Obtained from MICHAELCULBERTSON. trophic requirements, and the indicated carbon source. Cul- tures were harvested in early or mid log phase, and glucose uptake assays were performed by measuring uptake of D- antimycin A (1 rg/ml). Cells were then exposed to 100 J/ [U-'4C]glucose(New England Nuclear) over the concentra- m' of UV radiation. In control experiments, 30% of the tion range of 0.2 to 200 mM, as described previously cells remained viable. The plates were incubated at 30" for (KRUCKEBERGand BISON 1990). Each strain was assayed at 5 days. Revertants arose at frequencies of 1 to 5 X least twice after growth under the specified conditions. Revertants derived from three single coloniesof each strain Isolation of revertants of haploid sn.mutants: Strains (10 from MCY657,8 from MCY659,7 from MCY714, and MCY657,MCY659, MCY714 and MCRY168 were sub- 13 from MCRY 168) were colony purified and retested. jected to UV mutagenesis. Single colonies were suspended Complementationanalysis: Mutations were tested for in water and spread on a YEP-2% raffinose plate containing dominance by crossing each revertant to asnf3 null mutant. in YeastGlucose Transport in 507 To test for complementation, we constructed snf3/snf3 dip- port the low amounts of fructose released by extra- loids that were heterozygous for the suppressor mutations cellularhydrolysis of the trisaccharide.
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