Two Glucose Transporters in Saccharomyces Cerevisiae

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 12428-12432, October 1996 Genetics

Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression (yeast/glucose induction/glucose signaling/BXT/SNF3) SABIRE OZCAN*, JIM DOVER*, ANNE G. ROSENWALDt, STEFAN WOLFLt, AND MARK JOHNSTON*§ *Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110; tDepartment of Health and Human Services, National Institutes of Health, National Cancer Institute, Bethesda, MD 20892; and tHans-Knoll-Institut, Beutenbergstrasse 11, D-07745 Jena, Germany Communicated by David Botstein, Stanford University School of Medicine, Stanford, CA, August 12, 1996 (received for review May 13, 1996)

ABSTRACT Glucose is the preferred carbon source for inhibited in glucose-grown cells, leading to derepression of the most eukaryotic cells and has profound effects on many HXT genes. cellular functions. How cells sense glucose and transduce a Inhibition of Rgtlp by low levels of glucose, and hence signal into the cell is a fundamental, unanswered question. induction of HXT2 and HXT4 expression, requires Snf3p, Here we describe evidence that two unusual glucose trans- which encodes an apparent high-affinity glucose transporter porters in the yeast Saccharomyces cerevisiae serve as glucose (1, 2, 6-8). This suggests that Snf3p may be a sensor of low sensors that generate an intracellular glucose signal. The levels of glucose. Snf3p is highly similar to mammalian and Snf3p high-affinity glucose transporter appears to function as yeast glucose transporters, but differs from them in having a a low glucose sensor, since it is required for induction of long C-terminal extension of 303 amino acids that is predicted expression of several hexose transporter (HAT genes, encod- to be in the cytoplasm and may serve as a signaling domain (2, ing glucose transporters, by low levels of glucose. We have 8, 9). Induction of HXTI expression by high levels of glucose identified another apparent glucose transporter, Rgt2p, that is normal in a snf3A mutant, suggesting the existence of a is strikingly similar to Snf3p and is required for maximal separate high glucose sensor. induction of gene expression in response to high levels of As a consequence of their apparent signaling defect, snf3A glucose. This suggests that Rgt2p is a high glucose-sensing mutants fail to express glucose transporters and are therefore counterpart to Snf3p. We identified a dominant mutation in unable to grow on low levels of glucose. Dominant mutations RGT2 that causes constitutive expression of several ILT in the RGT2 gene, which bypasses the requirement of Snf3p for genes, even in the absence of the inducer glucose. This same growth on low levels of glucose, were identified by Marshall- mutation introduced into SNF3 also causes glucose- Carlson et al. (10). Here we show that RGT2 encodes a putative independent expression of ILT genes. Thus, the Rgt2p and glucose transporter highly similar to Snf3p, which affects Snf3p glucose transporters appear to act as glucose receptors expression of the HXT genes. Our results suggest that Rgt2p that generate an intracellular glucose signal, suggesting that and Snf3p act as glucose receptors, which generate an intra- glucose signaling in yeast is a receptor-mediated process. cellular signal in response to the effector glucose.

Glucose, the preferred carbon and energy source for most MATERIALS AND METHODS eukaryotic cells, has wide-ranging effects on cellular function. Among these are its ability to stimulate the first step of its Yeast Strains and Growth Media. The yeast strains used in metabolism-transport across the plasma membrane-by in- this study are listed in Table 1. Yeast cells were grown on fluencing the quantity, types, and activity of glucose trans- standard media as described (1, 11). Plasmids were transporters, both at the transcriptional and post-translational formed into yeast cells using frozen competent cells (12). levels. How eukaryotic cells sense glucose and transduce the Escherichia coli DH5aF' was used as a host for all plasmids. signal into the cell to affect these processes is a fundamental Construction of Plasmids and of Disruption Strains. The question. construction of the SNF3::lacZ and of the HXT::lacZ fusions There are two general ideas for how a glucose signal could [pBM2982, pBM2636 (HXT1) and pBM2717 (HXT2)] have be generated. First, a signal could be produced by metabolism been described (1). The plasmids pBM3212 and pBM3213 of glucose: it could be intracellular glucose or one of its contain the HXT1 and HXT2 promoter lacZ fusions of metabolites. Alternatively, extracellular glucose could be pBM2636 and pBM2717, respectively, in the LEU2 vector sensed by a receptor, and a signal generated in the absence of YEp367R (13). Fusion of the RGT2 promoter to lacZ was glucose metabolism (or transport), much like certain peptide accomplished by cloning PCR products (primers: OM1035- or hormone receptors generate a signal upon agonist binding. OM1034; + 1 to -1200) with an EcoRI site (starting at ATG) Bakers yeast provides an excellent experimental system for and a BamHI site into the vector YEp357R (13). The SNF3 studying glucose sensing and signaling in a eukaryote. Expres- deletion plasmid (pBM3103) was constructed in two steps: first sion of several yeast HXT genes encoding hexose transporters by inserting the 3.3-kb EcoRI/SalI fragment of the SNF3 gene is induced by different levels of glucose: transcription ofHXT1 into the vector YIp5 (yielding pBM3096) and then by replacing is induced solely at high concentrations of glucose; expression the 1.6-kb BamHI/BglII fragment of the coding region of of HXT2 and HXT4 is induced only by low levels of glucose SNF3 by the 3.8-kb BamHI/BglII fragment of pNKY51 that (1-5). This regulation is due to repression by Rgtlp, a zinc finger-containing repressor of the HXT genes, which represses Abbreviation: ,B-gal, ,B-galactosidase. HXT expression in the absence of glucose. Rgtlp function is Data deposition: The sequence reported in this paper has been deposited in the GenBank data base [accession no. Z74186 for RGT2 (YDL138w)]. The publication costs of this article were defrayed in part by page charge gDepartment of Genetics, Box 8232, Washington University School of payment. This article must therefore be hereby marked "advertisement" in Medicine, 4566 Scott Avenue, St. Louis, MO 63110. e-mail: accordance with 18 U.S.C. §1734 solely to indicate this fact. [email protected]. 12428 Downloaded by guest on September 28, 2021 Genetics: bzcan et al. Proc. Natl. Acad. Sci. USA 93 (1996) 12429

Table 1. List of Saccharomyces cerevisiae strains ing the complete open reading frame of SNF3) and was used a in YM4127 MAT a ura3-52 his3A200 ade2-101 lys2-801 leu2-3,2-112 to repair gap generated the SNF3-containing plasmid trpl-901 tyrl-501 (pBM3111) by digestion with BsaAI [which cleaves at nucle- YM4714 MAT a ura3-52 his3A200 ade2-101 lys2-801 leu2-3,2-112 otides 391 and 2067 (relative to the ATG) of SNF3]. This was trpl-901 tyrl-501 snf3A::hisG accomplished by cotransforming strain YM4714 to Ura+ and MCY1710 MAT a ura3-52 his3A200 lys2-801 RGT2-1 snf3A4::HIS3 Leu+ with BsaAI-digested pBM3111 (CEN-URA3-SNF3) and YM4767 MAT a ura3-52 his3A200 ade2-101 lys2-801 leu2-3,2-112 the PCR product (along with pBM3213, an HXT2-lacZ- RGT2-1 containing plasmid). Transformants were screened for consti- YM4817 MAT a ura3-52 his3A200 ade2-101 lys2-801 leu2-3,2-112 tutive HXT2 expression on galactose plus 5-bromo-4-chloro- trpl-901 tyrl-501 rgt2A::HIS3 3-indolyl ,B-D-galactoside plates (i.e., for blue colonies). The YM4576 MAT a ura3-52 his3A200 ade2-101 leu2-3,2-112 gal80 gap-repaired SNF3-containing plasmid (pBM3259) was iso- grrlA::hisG lated in E. coli from several such transformants (19), and the YM4825 MAT a ura3-52 his3A200 lys2-801 leu2-3,2-112 RGT2-1 presence of the SNF3-1 mutation was verified by sequencing. grrl1A::hisG-URA3-hisG Three independent plasmids that contain the mutation were obtained: all three behaved essentially identically, providing All strains have the S288C genetic background. similar levels of constitutive HXT2 expression (Table 3). contains hisG-URA3-hisG (14). pBM2101 contains the 7-kb BamHI fragment with the GRR1 gene in pUC18, with the RESULTS coding region of GRR1 (from +68 to +1801) replaced by hisG::URA3::hisG of pNKY51. The RGT2-1 Mutation Restores the Glucose Signaling De- The complete coding region of RGT2 was deleted and fect to a snJ3A Mutant. The growth and high-affinity glucose replaced with GFP-HIS3 in the wild-type strain YM4127 using uptake defects of a snf3A mutant are likely due to impaired the PCR disruption technique (primers: OM1031 and induction of hexose transporter genes (HXT2, HXT3, and OM1032) as described (15, 16). Correct disruption was verified HXT4) by low levels of glucose (1, 2, 20, 21). The dominant by PCR analysis. The snf3A::hisG strain (YM4717) was created RGT2-1 mutation restores growth of a snf3A mutant on low by transformingyeast cells to Ura+ with the 5.6-kb EcoRI/SalI levels of glucose by restoring high-affinity glucose transport fragment of pBM3103, followed by selection for uracil auxot- (10). To test whether RGT2-1 does this by restoring the rophy. YM4825 (RGT2-1 grrlA) was constructed by transform- signaling defect caused by the lack of Snf3p, we assayed ing the RGT2-1 strain YM4767 to uracil prototrophy with the expression of the HXT genes in RGT2-1 mutants (Table 2). 9-kb BamHI fragment of pBM2101 (grrl::URA3). Low glucose induced expression of HXT2 is impaired in j8-Galactosidase (f3-Gal) Assays. Cells were pregrown to snf3A mutants (Table 2, compare lines 1 and 2, column E). This mid-log phase (OD600 1-2) on yeast nitrogen base (YNB)/5% induction defect is fully restored in the snf3A RGT2-1 double glycerol with 0.5% galactose and transferred to YNB medium mutant (line 3, column E). Furthermore, HXT2 expression is containing either 4% glucose or 5% glycerol, or 5% glycerol constitutive in this strain (line 3, column D). [Note that HXT2 plus 0.5% galactose, or 5% glycerol plus 0.1% glucose. After expression remains low in RGT2-1 mutants growing on high incubation at 30°C for 4-5 h, the cultures were assayed for levels of glucose. This is because HXT2 expression is subject to 3-gal activity in permeabilized cells as described (17). Activ- glucose repression mediated by the Miglp repressor (1, 5), and ities are given in Miller units (U), and are the average of three this is unaffected by RGT2 mutations (Table 2). The RGT2-1 to six assays of three independent transformants. mutation also causes constitutive expression of the high glu- DNA Sequencing. To determine the sequence change of the cose-induced HXT1 gene (compare lines 1 and 4, column B) RGT2-1 mutation, a 3297-bp region of the yeast genome that [though the level of constitutive expression is somewhat lower includes the RGT2 gene (795 bp upstream of the ATG codon in the absence of glucose (column A)]. The ability of the to 210 bp downstream of the stop codon) was amplified from RGT2-1 mutation to induce HXT expression even in the RGT2-1 (MCY1710; ref. 10) and RGT2 (YM4127) by PCR. absence of glucose is independent of Snf3p function (line 4, The sequence of the PCR products was determined by "shot- columns A and D). The RGT2-1 mutation mediates constitu- gun" sequencing, as described (18). The entire sequence of the tive expression of the HXT genes in the presence of the coding region was determined on both strands. Because of the wild-type RGT2 gene, indicating that the constitutive glucose depth of sequence coverage (average of 4.2 reads for RGT2-1, signaling caused by this mutation is dominant (compare lines 8.8 for RGT2; 9 independent reads of the mutant site), 4, 5, and 6). sequence changes generated by PCR (1 mistake in 3000 bp) Identification of the RGT2 Gene. The RGT2-1 mutation is were obvious. Only one sequence difference between RGT2-1 located 38 centimorgans from SNF3 on chromosome IV (10). and wild type (G691 -> A, numbering relative to the ATG The sequence of a portion of this region of the genome codon) was found. revealed the existence of an open reading frame encoding a Introduction of the Arg-229 to Lys Change into Snf3p. The protein very similar to Snf3p. To determine whether this open change in SNF3 analogous to that caused by the RGT2-1 reading frame encodes RGT2, we replaced its coding region mutation (G686 > A; nucleotide 1 is the A of the SNF3 ATG with HIS3 in the dominant RGT2-1 strain. Deletion of this codon) was introduced by in vitro mutagenesis. Two primers open reading frame in the RGT2-1 mutant results in a loss of containing the SNF3-1 mutation (OM1094, pointing upstream: constitutive expression of the HXT genes (data not shown), tTTAATGATTTATGTGTAGCTTCTGC; OM1090, point- consistent with-the idea that it is RGT2. In addition, a cloned ing downstream: CCAAGCAGAAGCTACACATAAATCA- copy of the RGT2-1 gene causes constitutive expression of the TTAAaAGGTGCTATTATTTCTACT TACC; mutated nu- HXT genes in a wild-type strain (data not shown). cleotide in lowercase letters) were used in a series of PCRs to Rgt2p has 12 predicted transmembrane domains with 60% generate a 2714-bp fragment containing the mutation, the overall similarity to Snf3p (Fig. 1). Rgt2p, like Snf3p, is highly ends of which are derived from primers OM939 (upstream of similar to mammalian and yeast glucose transporters, but both the mutation, ggcgaattcgaagcATGGATCCTAATAGTAAC) proteins differ from all other glucose transporters in having and OM957 (downstream of the mutation, cgcggatccCCGCT- unusually long C-terminal extensions that are predicted to be TAATTAATACATCG); SNF3 sequences are in uppercase cytoplasmic. The sequences of these C-terminal regions of letters, restriction sites added to the primers in lowercase Rgt2p (218 aa) and Snf3p (303 aa) are dissimilar, except for a letters. This PCR product was digested with BamHI (contain- sequence of 17 amino acids that is nearly identical (15 of 17 Downloaded by guest on September 28, 2021 12430 Genetics: Ozcan et al. Proc. Natl. Acad. Sci. USA 93 (1996)

Table 2. RGT2-1 restores the glucose signaling defect to snf3A mutants Mean ,-gal activity, U, ± SD HXT1 HX72 A B C D E F Genotype gly gly + 0.1% glu 4% glu gly gly + 0.1% glu 4% glu 1 WT 0.9 ± 0.2 4 ± 0.9 303 ± 35 22 ± 4 305 ± 12 18 ± 4 2 snf3A 0.7 ± 0.09 1.2 ± 0.27 227 ± 30 16 ± 1.4 23 ± 1.7 7 ± 0.9 3 snf3ARGT2-1 48 ± 10 185 ± 15 454 ± 101 236 ± 18 371 ± 36 41 ± 8 4 RGT2-1 30t 4 223 ± 27 393 ± 59 213 ± 76 415 ± 29 48 ± 7 5 WT dipl.* 1.3 0.3 4.7 ± 0.87 295 ± 9 23 ± 6 281 ± 43 18 ± 1.3 6 RGT2-J/RGT2t 12 2 78 ± 11 280 ± 34 152 ± 31 330 ± 36 22 ± 4 7 rgt2A1::HIS3 0.8 0.13 4.6 ± 0.7 62 ± 11 19 ± 4 397 ± 83 34 ± 9 gly, 5% glycerol + 0.5% galactose; gly + 0.1% glu, 5% glycerol + 0.1% glucose; 4% glu, 4% glucose. *Diploid strain homozygous for wild-type RGT2 gene. tExpression of HXTJ is induced about 300-fold by high levels of glucose. This is due to two independent regulatory mechanisms (1). In addition to the Grrlp-Rgtlp mechanism, maximal induction of HXTI requires a second pathway whose components have not been identified, which is required for full induction of HXTI expression at high concentrations of glucose. Thus, maximal induction of HXTJ in the RGT2-1 strain still requires high levels of glucose. tDiploid strain heterozygous for RGT2-1 mutation.

amino acids are identical) in the two proteins. Snf3p contains (Arg-231 -- Lys) predicted to be located in a cytoplasmic loop two of these sequences; Rgt2p contains only one (Fig. 1). just preceding the fifth transmembrane domain (Fig. 1). It is RGT2 Is Required for High Glucose Induction of Gene significant that this arginine residue is conserved among all Expression. Deletion of RGT2 causes a 5-fold decrease in known sugar transporters in yeast, mammals, and other or- HXTI expression on high levels of glucose (Table 2, compare ganisms. lines 1 and 7, column C) but has no effect on low glucose- Since Snf3p and Rgt2p are strikingly similar and seem to induced HXT2 expression (line 7, column E). Thus, in contrast function as sensors of glucose, we introduced the same change to Snf3p, which is required for low glucose induction, Rgt2p into SNF3. Changing the same arginine residue of Snf3p appears to be involved only in the response to high levels of (Arg-229) to lysine causes constitutive expression of HXT2 in glucose. This result suggests that Rgt2p is a high-glucose the absence of glucose (Table 3). Like RGT2-1, this mutation sensing counterpart to Snf3p. The RGT2 gene is not required in SNF3 also causes constitutive expression of HXTJ on to maintain glucose repression, because expression of SUC2 glycerol (data not shown). Thus, this same mutation in SNF3 and GALI is still repressible by high levels of glucose in a rgt2A (SNF3-1), also converts Snf3p into a dominant, constitutively and in the dominant RGT2-1 strain (data not shown). signaling glucose receptor. The RGT2-1 Mutation Alters a Highly Conserved Amino RGT2 Encodes the Most Upstream Component of the Acid. Determination of the sequence change of the dominant Glucose Signaling Pathway. In the absence of glucose, expres- RGT2-1 mutation revealed that it alters an arginine residue sion of the HXT genes is inhibited by the Rgtlp repressor. Rgt2p/Snf3p Outside rx\ /1>

COOH FIG. 1. A two-dimensional model of the transmembrane topology of the Rgt2p and Snf3p glucose transporters in the plasma membrane, based on the model of Mueckler et al. (22) for Glutlp. The predicted transmembrane domains are numbered 1-12 and shown as rectangles, with the numbers of the N- and C-terminal residues for each domain of Rgt2p indicated. *, Position of the Arg-231 (in Rgt2p) and Arg-229 (in Snf3p) that is mutated to a lysine in RGT2-1 and SNF3-1, respectively; black box indicates the repeated sequence of 17 amino acids in the carboxyl-terminal part of Rgt2p and Snf3p, which is also present in two copies in the Snf3p tail (the second repeat of Snf3p is indicated by the shaded box). Downloaded by guest on September 28, 2021 Genetics: bzcan et al. Proc. Natl. Acad. Sci. USA 93 (1996) 12431

Table 3. The Arg-229 to Lys change converts Snf3p into a Table 5. The RGT2 gene is expressed constitutively at low levels dominant, constitutively signaling receptor in wild-type cells Mean 13-gal activity, U, ± SD, HXT2 Mean ,3-gal activity (U) ± SD Genotype Plasmid gly gly + 0.1% glu 4% glu Reporter gly gly + 0.1% glu 4% glu 1 WT- Vector 19 ± 1.5 278 ± 42 20 ± 2 RGT2::lacZ 3 + 0.4 2.6 ± 0.4 1.8 + 0.33 2 SNF3 25 ± 4 448 ± 28 21 ± 2.3 SNF3::lacZ 0.48 ± 0.08 0.8 ± 0.09 <0.1 3 SNF3-1* 273 ± 39 470 ± 32 30 ± 4 HXTI::lacZ 0.9 ± 0.2 4 + 0.9 303 + 35 4 snf3A- Vector 11 ± 1 25 ± 4 46 ± 3 5 SNF3 26 ± 5 385 ± 27 6 ± 0.7 is the most upstream component of the glucose induction 6 SNF3-1* 378 ± 42 444 ± 98 65 ± 8 signaling pathway. *SNF3-1 contains the Arg-229 to Lys change. Our findings lead to three significant conclusions. First, because the dominant RGT2-1 and SNF3-1 mutations cause Function of Rgtlp is inhibited by glucose, and this requires glucose signaling even in the absence of glucose, these mutants Grrlp (1, 23). Because RGT2 codes for a glucose transporter do not need to transport glucose into the cell to generate a and is required for high glucose induction of HXTI, it seemed glucose signal. We infer from.this that the primary signal for likely that it acts upstream of Grrlp in the glucose induction induction of HXT gene expression is neither intracellular pathway. To confirm this we disrupted GRR1 in the RGT2-1 glucose nor a glucosemetabolite. Rather, we propose that the strain (Table 4). The RGT2-1 grrl double mutant is impaired signal may be, for example, a protein whose function is in induction of HXTI and HXT2 expression, like the grrlA stimulated by binding of glucose to the receptor, or whose mutant (compare line 3 to line 4), indicating that the RGT2-1 modification (e.g., phosphorylation) is coupled to glucose mutation requires the function of Grrlp to mediate constitu- transport. Thus, generation of the glucose signal in yeast tive signaling. Thus, Rgt2p is the most upstream component of appears similar to the situation in bacteria, where synthesis of this glucose signaling pathway, consistent with its proposed the second messenger cAMP is coupled to glucose transport by role as a glucose receptor. a protein phosphorylation cascade (24). Second, our results RGT2 Is Constitutively Expressed at Low Levels. RGT2 suggest that the glucose signal is transmitted into the yeast cell expression, measured in a strain carrying an RGT2-lacZ by glucose transporters acting as glucose receptors that sense fusion, is weak, being about 100-fold less than fully induced extracellular glucose. In this view, the Rgt2p and Snf3p glucose HXT1 expression, and only about 3- to 5-fold higher than SNF3 transporters are working like other integral membrane recep- (Table 5). In contrast to SNF3, which is glucose repressible, tors, which bind an extracellular effector and tranduce a signal transcription of RGT2 is not regulated by glucose (Table 5). across the plasma membrane. Finally, our results support the Thus, the regulation of SNF3 and RGT2 expression is consis- idea that the components of the glucose signaling pathway are tent with their postulated roles as low and high glucose sensors, present in the cell even in the absence of glucose and that the respectively. Transcription of RGT2 is probably not autoregu- activity of the glucose sensor is the limiting factor for signaling. lated since there is no difference in expression of the RGT2- It is likely that Snf3p and Rgt2p have different affinities for lacZ fusion in wild-type and an rgt2A mutant (data not shown). glucose. Because RGT2 is only required for maximal induction of HXT gene expression in response to high levels of glucose, we imagine that it is a low affinity (high Km) glucose receptor. DISCUSSION Our observation that RGT2 is expressed in cells growing on RGT2 encodes a putative glucose transporter that is strikingly high levels of glucose is consistent with this idea. Snf3p, on the similar to Snf3p and to other glucose transporters of many other hand, seems likely to be a high-affinity (low Km) organisms. RGT2 is required for maximal expression of the receptor, since it is only required for induction of expression high glucose-induced HXTI gene, and SNF3 is required for low of the low glucose-induced HXT genes. The fact that SNF3 glucose-induced expression of HXT2 and HXT4, suggesting expression is repressed by high levels of glucose (Table 5; refs. that they play regulatory roles in glucose-induced gene ex- 1, 2, and 8) is consistent with this idea. The low levels of pression. We propose that these two transporters are glucose expression ofSNF3 and RGT2 are consistent with the view that sensors involved in generating an intracellular glucose signal. they have regulatory rather than metabolic roles as glucose This idea is strengthened by the observation that dominant transporters. mutations in RGT2 (RGT2-1: Arg-231 -* Lys) and SNF3 The high degree of similarity between Snf3p and Rgt2p (SNF3-1; Arg-229 -- Lys) cause constitutive expression of suggests that both proteins may sense glucose in a similar way, several glucose-induced HXT genes. We imagine that this and may act with the same or similar protein(s) to transmit the mutation causes Rgt2p and Snf3p always to be in a "signaling glucose signal. Especially interesting is the finding that Rgt2p, competent" conformation, thus causing constitutive produc- like Snf3p, has a long C-terminal extension that is predicted to tion of an intracellular glucose signal that activates HXT gene be in the cytoplasm. In this respect, these two proteins are expression. Additional support for this view is the observation unlike all other glucose transporters, which have cytoplasmic that the ability of the RGT2-1 mutation to cause constitutive C terminii ofonly 30-50 amino acids. Thus, both proteins seem HXT expression is dependent on Grrlp, indicating that Rgt2p to consist of two different domains: a 12 transmembrane transporter domain and a cytoplasmic domain at the C termi- Table 4. Rgt2p acts upstream of Grrlp in the signaling pathway nus that could serve as a signaling domain that interacts with Mean 13-gal activity, U, ± SD the next component in the signal transduction pathway. The only portions of the C terminus of Rgt2p and Snf3p that HXTI HXT2 are similar are the 3 nearly identical 17 amino acid blocks of sequence they contain. Snf3p contains two of these sequences; Genotype gly 4% glu gly 0.1% glu Rgt2p contains one. Interestingly, Snf3p must possess only one of these repeats to function (2, 9). It is possible that the repeat 1 WT 1.2 ± 0.3 320 ± 44 19 ± 3.5 356 ± 37 mediates the interaction of the sensor with the next compo- 2 RGT2-1 34 ± 4 455 ± 72 182 ± 16 408 ± 83 nent(s) of the glucose signaling pathway. One candidate is 3 grrlA <1 1.1 ± 0.15 12 ± 2 15 ± 3 Hxk2p, the main glucose phosphorylating enzyme in yeast, 4 RGT2-l grrlA <1 2.1 ± 0.2 25 ± 6 17 ± 1.4 which catalyzes the first step of glucose metabolism. In addi- Downloaded by guest on September 28, 2021 12432 Genetics: 6zcan et al. Proc. Natl. Acad. Sci. USA 93 (1996)

tion to its catalytic role in hexose phosphorylation, Hxk2p 3. Wendell, D. L. & Bisson, L. F. (1994) J. Bacteriol. 176, 3730- appears to have a regulatory role in both induction and 3737. repression of gene expression by glucose (1, 25-27). 4. Theodoris, G., Fong, N. M., Coons, D. M. & Bisson, L. F. (1994) Genetics 137, 957-966. We speculate that binding of glucose to the transporter 5. Ozcan, S. & Johnston, M. (1996) Mo. Cell. Biol., 16, 5536-5545. domain of Rgt2p or Snf3p causes a conformational change that 6. Neigeborn, L., Schwartzberg, P., Reid, R. & Carlson, M. (1986) might be transmitted to the C-terminal signaling domain and Mol. Cell. Bio. 6, 3569-3574. affect its interaction with a component(s) of the glucose signal 7. Bisson, L. F., Neigeborn, .L., Carlson, M. & Fraenkel, D. G. transduction pathway. In this view, the RGT2-1 and SNF3-1 (1987) J. Bacteriol. 169, 1656-1662. mutations convert these proteins to the glucose-bound form, 8. Celenza, J. L., Marshall-Carlson, L. & Carlson, M. (1988) Proc. thus causing constitutive signal generation. The amino acid Natl. Acad. Sci. USA 85, 2130-2134. 9. Marshall-Carlson, L., Celenza, J. L., Laurent, B. C. & Carlson, affected by this mutation, which is conserved in all known M. (1990) Mo. Cell. Biol. 10, 1105-1115. sugar transporters, could be involved in sugar binding and/or 10. Marshall-Carlson, L., Neigebom, L., Coons, D., Bisson, L. & translocation. In any case, the mutation uncouples the signal- Carlson, M. (1991) Genetics 128, 505-512. ing function of Rgt2p and Snf3p from their glucose transport 11. Sherman, F., Fink, G. R. & Hicks, J. B. (1986) Methods in Yeast activity, and strongly suggests that both glucose transporters Genetics. (Cold Spring Harbor Lab. Press, Plainview, NY). are involved in an initial step in glucose sensing. 12. Dohmen, R. J., Strasser, A. W. M., Honer, C. B. & Hollenberg, Rgt2p and Snf3p appear to have a function similar to that of C. P. (1991) Yeast 7, 691-692. 13. Myers, A. M., Tzagoloff, A., Kinney, D. M. & Lusty, C. J. (1986) Glut2p in mammalian cells, which is a low-affinity glucose Gene 45, 229-310. transporter required for glucose-induced expression of insulin 14. Alani, E., Cao, L. & Kleckner, N. (1987) Genetics 116, 541-545. in pancreatic (3-cells (28, 29). All three transporters seem to 15. Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F. function as sensors of glucose. However, in contrast to Rgt2p & Cullin, C. (1993) Nucleic Acids Res. 21, 3329-3330. and Snf3p, Glut2p does not have a large C-terminal extension. 16. Niedenthal, R. K., Riles, L., Johnston, M. & Hegemann, J. H. Perhaps a second protein associated with Glut2p provides the (1996) Yeast 12, 773-786. signaling domain that is functionally equivalent to the C- 17. Yocum, R. R., Hanley, S., West, R., Jr., & Ptashne, M. (1984) Mol. Cell. Biol. 4, 1985-1998. terminal part of Rgt2p. Alternatively, there might be an Rgt2p 18. Wilson, R., Ainscough, R., Anderson, K., Baynes, C., Berks, M., homologue in pancreatic cells that has not yet been identified et al. (1994) Nature (London) 368, 32-38. in cDNA libraries because of its low level expression. The 19. Hoffman, C. S. & Winston, F. (1987) Gene 57, 267-272. identification of glucose sensors in a simple eukaryotic cell 20. Ko, C. H., Liang, H. & Gaber, R. F. (1993) Moi. Cell. Bio. 13, might facilitate the discovery of similar proteins in higher 638-648. eukaryotes. 21. Coons, D. M., Boulton, R. B. & Bisson, L. F. (1995) J. Bacteriol. 177, 3251-3258. 22. Mueckler, M., Caruso, C., Baldwin, S. A., Panico, M., Blench, J., S.O. dedicates this publication to the memory of her colleague and Morris, H. R., Allard, W. J., Leinhard, G. E. & Lodish, H. F. valued mentor Michael Ciriacy. We thank Marian Carlson for the (1985) Science 229, 941-945. snf3A RGT2-1 strain and for encouraging us to pursue RGT2; Dr. 23. Vallier, L. G., Coons, D., Bisson, L. F. & Carlson, M. (1994) Richard Kahn and the intramural program of Developmental Ther- Genetics 136, 1279-1285. apeutics, Division of Cancer Treatment, National Cancer Institute for 24. Saier, M. H., Jr., Chauvaux, S., Deutscher, J., Reizer, J. & Ye, support of A.G.R.; Vera Hanemann and Hans-Peter Saluz for their J.-J. (1995) Trends Biochem. Sci. 20, 267-271. help in sequencing RGT2; and Ken Blumer, Marian Carlson, Ira 25. Johnston, M. & Carlson, M. (1992) in The Biology of the Yeast Herskowitz, Mike Mueckler, and Roland Stein for comments on the Saccharomyces, eds. Broach, J., Jones, E. W. & Pringle, J. (Cold manuscript. This work was supported by National Institutes of Health Spring Harbor Lab. Press, Plainview, NY), pp. 193-281. Grant GM32540 and funds provided to M.J. by the McDonnell 26. Rose, M., Albig, W. & Entian, K.-D. (1991) Eur. J. Biochem. 199, Foundation. S.0. was supported by a postdoctoral fellowship from the 511-518. Deutsche Forschungsgemeinschaft. 27. Bisson, L. F. & Fraenkel, D. G. 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