Std1 and Mth1 Proteins Interact with the Glucose Sensors To Control Glucose-Regulated Gene Expression in Saccharomyces cerevisiae

The Std1 protein modulates the expression of glucose-regulated genes, but its exact molecular role in this process is unclear. A two-hybrid screen for Std1-interacting proteins identified the hydrophilic C-terminal domains of the glucose sensors, Snf3 and Rgt2. The homologue of Std1, Mth1, behaves differently from Std1 in this assay by interacting with Snf3 but not Rgt2. Genetic interactions between STD1, MTH1, SNF3, and RGT2 suggest that the glucose signaling is mediated, at least in part, through interactions of the products of these four genes. Mutations in MTH1 can suppress the raffinose growth defect of a snf3 mutant as well as the glucose fermentation defect present in cells lacking both glucose sensors (snf3 rgt2). Genetic suppression by mutations in MTH1 is likely to be due to the increased and unregulated expression of hexose transporter genes. In media lacking glucose or with low levels of glucose, the hexose transporter genes are subject to repression by a mechanism that requires the Std1 and Mth1 proteins. An additional mechanism for glucose sensing must exist since a strain lacking all four genes (snf3 rgt2 std1 mth1) is still able to regulate SUC2 gene expression in response to changes in glucose concentration. Finally, studies with green fluorescent protein fusions indicate that Std1 is localized to the cell periphery and the cell nucleus, supporting the idea that it may transduce signals from the plasma membrane to the nucleus.     

Suppressors of snf2 Mutations Restore Invertase Derepression and Cause Temperature-Sensitive Lethality in Yeast

Mutations in the SNF2 gene of Saccharomyces cerevisiae prevent derepression of the SUC2 (invertase) gene, and other glucose-repressible genes, in response to glucose deprivation. We have isolated 25 partial phenotypic revertants of a snf2 mutant that are able to derepress secreted invertase. These revertants all carried suppressor mutations at a single locus, designated SSN20 (suppressor of snf2). Alleles with dominant, partially dominant and recessive suppressor phenotypes were recovered, but all were only partial suppressors of snf2, reversing the defect in invertase synthesis but not other defects. All alleles also caused recessive, temperature-sensitive lethality and a recessive defect in galactose utilization, regardless of the SNF2 genotype. No significant effect on SUC2 expression was detected in a wild-type (SNF2) genetic background. The ssn20 mutations also suppressed the defects in invertase derepression caused by snf5 and snf6 mutations, and selection for invertase-producing revertants of snf5 mutants yielded only additional ssn20 alleles. These findings suggest that the roles of the SNF2, SNF5 and SNF6 genes in regulation of SUC2 are functionally related and that SSN20 plays a role in expression of a variety of yeast genes.     

The SNF3 gene is required for high-affinity glucose transport in Saccharomyces cerevisiae.

Glucose uptake mutants have not been previously obtained in Saccharomyces cerevisiae, possibly because there seem to be at least two transport systems, of low and high affinities. We showed that snf3 (sucrose nonfermenting) mutants did not express high-affinity glucose uptake. Furthermore, their growth was completely impaired on low concentrations of glucose in the presence of antimycin A (which blocks respiration). Several genes which complemented the original snf3 gene were obtained on multicopy plasmids. Some of them, as well as plasmid-carried SNF3 itself, conferred a substantial increase in high-affinity glucose uptake in both snf3 and wild-type hosts. The effects of glucose on the expression of such a plasmid-determined high-affinity uptake resembled those in the wild type. Other genes complementing snf3 seemed to cause an increase in low-affinity glucose uptake. We suggest that SNF3 may function specifically in high-affinity glucose uptake, which is needed under some conditions of growth on low glucose concentrations. SNF3 itself or the other complementing genes may specify components of the glucose uptake system.     

Mth1 receives the signal given by the glucose sensors Snf3 and Rgt2 in Saccharomyces cerevisiae

We have determined that the mutant genes DGT1-1 and BPC1-1, which impair glucose transport and catabolite repression in Saccharomyces cerevisiae, are allelic forms of MTH1. Deletion of MTH1 had only slight effects on the expression of HXT1 or SNF3, but increased expression of HXT2 in the absence of glucose. A two-hybrid screen revealed that the Mth1 protein interacts with the cytoplasmic tails of the glucose sensors Snf3 and Rgt2. This interaction was affected by mutations in Mth1 and by the concentration of glucose in the medium. A double mutant, snf3 rgt2, recovered sensitivity to glucose when MTH1 was deleted, thus showing that glucose signalling may occur independently of Snf3 and Rgt2. A model for the possible mode of action of Snf3 and Rgt2 is presented.     

High-Copy Suppression of Glucose Transport Defects by Hxt4 and Regulatory Elements in the Promoters of the Hxt Genes in Saccharomyces Cerevisiae

HXT4, a new member of the hexose transporter (HXT) family in Saccharomyces cerevisiae was identified by its ability to suppress the snf3 mutation in multicopy. Multicopy HXT4 increases both high and low affinity glucose transport in snf3 strains and increases low and high affinity transport in wild-type strains. Characterization of HXT4 led to the discovery of a new class of multicopy suppressors of glucose transport defects: regulatory elements in the promoters of the HXT genes. We have designated these sequences DDSEs (DNA sequence dependent suppressing element). Multicopy HXT4 and DDSEs in the HXT1 HXT2, HXT3 and HXT4 promoters were found to restore growth to snf3 and grr1 strains on low glucose media. The DDSE in the HXT4 promoter was refined to a 340-bp sequence 450 bp upstream of the HXT4 translational start. This region was found to contain an 183-amino acid open reading frame. Extensive analysis indicates that the DNA sequence itself and not the encoded protein is responsible for suppression. The promoters of SNF3 and of other glycolytic genes examined did not suppress snf3 in multicopy. Suppression of snf3 by DDSE is dependent on the presence of either HXT2 or HXT3.     

A Suppressor of snf1 Mutations Causes Constitutive High-Level Invertase Synthesis in Yeast

The SNF1 gene product of Saccharomyces cerevisiae is required to derepress expression of many glucose-repressible genes, including the SUC2 structural gene for invertase. Strains carrying a recessive snf1 mutation are unable to ferment sucrose. We have isolated 30 partial phenotypic revertants of a snf1 mutant that were able to ferment sucrose. Genetic characterization of these revertants showed that the suppressor mutations were all recessive and defined eight complementation groups, designated ssn1 through ssn8 (suppressor of snf1 ). The revertants were assayed for secreted invertase activity, and although activity was detected in members of each complementation group, only the ssn6 strains contained wild-type levels. Synthesis of secreted invertase in ssn6 strains was found to be constitutive, that is, insensitive to glucose repression; moreover, the ssn6 mutations also conferred constitutivity in a wild-type ( SNF1 ) genetic background and are, therefore, not merely suppressors of snf1 . Pleiotropic defects were observed in ssn6 mutants. Genetic analysis suggested that the ssn6 mutations are allelic to the cyc8 mutation isolated by R. J. Rothstein and F. Sherman, which causes increased production of iso-2-cytochrome c. The data suggest a regulatory function for SSN6 .     

Altered Regulatory Responses to Glucose Are Associated with a Glucose Transport Defect in Grr1 Mutants of Saccharomyces Cerevisiae

The GRR1 gene of Saccharomyces cerevisiae affects glucose repression, cell morphology, divalent cation transport and other processes. We present a kinetic analysis showing that the grr1 mutant is also defective in high affinity glucose transport. In combination with a mutation in SNF3, a member of the glucose transporter gene family, grr1 strikingly impairs growth on glucose. These findings suggest that GRR1 and SNF3 affect glucose transport by distinct pathways. The mutation rgt1-1, a suppressor of snf3, restores both glucose transport and glucose repression to a grr1 mutant, but does not remedy the morphological defect. We suggest that GRR1 affects the glucose sensing process and that the association between transport and regulation may reflect the involvement of a transporter in glucose sensing.     

New Snf Genes, Gal11 and Grr1 Affect Suc2 Expression in Saccharomyces Cerevisiae

To identify new genes required for depression of the SUC2 (invertase) gene in Saccharomyces cerevisiae, we have isolated mutants with defects in raffinose utilization. In addition to mutations in SUC2 and previously identified SNF genes, we recovered recessive mutations that define four new complementation groups, designated snf7 through snf10. These mutations cause defects in the derepression of SUC2 in response to glucose limitation. We also recovered five alleles of gal11 and showed that a gal11 null mutation decreases SUC2 expression to 30% of the wild-type level. Finally, one of the mutants carries a grr1 allele that converts SUC2 from a glucose-inducible gene.     

High-affinity glucose transport in Saccharomyces cerevisiae is under general glucose repression control.

Saccharomyces cerevisiae mutants defective in growth on low glucose concentration (lgn mutants) were isolated and screened for abnormal glucose transport. Nine complementation groups were identified, falling into two broad groups: those unable to significantly derepress high-affinity (low-Km) glucose uptake (lgn1, lgn4, lgn5, lgn7, and lgn8), and those with elevated repressed levels of high-affinity uptake that either derepress to normal or near normal levels of high-affinity uptake with loss of low-affinity transport (lgn2 and lgn3) or derepress only slightly, appearing to have an intermediate yet constitutive level of high-affinity transport (lgn6 and lgn9). Further analysis of the lgn mutations revealed pleiotropic phenotypes most consistent with the true defect being in regulation or expression of glucose repression and derepression. The kinetics of glucose uptake in strains carrying known mutations preventing derepression of glucose-repressible functions (snf1, snf2, snf4, and snf6) demonstrated that three of these mutations (snf1, snf4, and snf6) were similarly defective in derepression of high-affinity glucose uptake. The snf2 and snf5 mutations had no apparent effect on glucose uptake. Two mutations resulting in constitutive expression of glucose-repressible functions, cid1 and reg1, resulted in constitutive expression of high-affinity glucose uptake. These data support the conclusion that high-affinity glucose uptake in Saccharomyces cerevisiae is under general glucose repression control. The implications of other properties of these mutants are discussed.     

The HXT1 gene product of Saccharomyces cerevisiae is a new member of the family of hexose transporters.

Two novel genes affecting hexose transport in the yeast Saccharomyces cerevisiae have been identified. The gene HXT1 (hexose transport), isolated from plasmid pSC7, was sequenced and found to encode a hydrophobic protein which is highly homologous to the large family of sugar transporter proteins from eucaryotes and procaryotes. Multicopy expression of the HXT1 gene restored high-affinity glucose transport to the snf3 mutant, which is deficient in a significant proportion of high-affinity glucose transport. HXT1 was unable to complement the snf3 growth defect in low copy number. The HXT1 protein was found to contain 12 putative membrane-spanning domains with a central hydrophilic domain and hydrophilic N- and C-terminal domains. The HXT1 protein is 69% identical to GAL2 and 66% identical to HXT2, and all three proteins were found to have a putative leucine zipper motif at a consensus location in membrane-spanning domain 2. Disruption of the HXT1 gene resulted in loss of a portion of high-affinity glucose and mannose transport, and wild-type levels of transport required both the HXT1 and SNF3 genes. Unexpectedly, expression of beta-galactosidase activity by using a fusion of the lacZ gene to the HXT1 promoter in a multicopy plasmid was maximal during lag and early exponential phases of growth, decreasing approximately 100-fold upon further entry into exponential growth. Deletion analysis of pSC7 revealed the presence of another gene (called ORF2) capable of suppressing the snf3 null mutant phenotype by restoring high-affinity glucose transport and increased low-affinity transport.     

Genes Affecting the Regulation of SUC2 Gene Expression by Glucose Repression in SACCHAROMYCES CEREVISIAE

Mutants of Saccharomyces cerevisiae with defects in sucrose or raffinose fermentation were isolated. In addition to mutations in the SUC2 structural gene for invertase, we recovered 18 recessive mutations that affected the regulation of invertase synthesis by glucose repression. These mutations included five new snf1 (sucrose nonfermenting) alleles and also defined five new complementation groups, designated snf2, snf3, snf4, snf5, and snf6. The snf2, snf4, and snf5 mutants produced little or no secreted invertase under derepressing conditions and were pleiotropically defective in galactose and glycerol utilization, which are both regulated by glucose repression. The snf6 mutant produced low levels of secreted invertase under derepressing conditions, and no pleiotropy was detected. The snf3 mutants derepressed secreted invertase to 10-35% the wild-type level but grew less well on sucrose than expected from their invertase activity; in addition, snf3 mutants synthesized some invertase under glucose-repressing conditions.--We examined the interactions between the different snf mutations and ssn6, a mutation causing constitutive (glucose-insensitive) high-level invertase synthesis that was previously isolated as a suppressor of snf1. The ssn6 mutation completely suppressed the defects in derepression of invertase conferred by snf1, snf3, snf4 and snf6, and each double mutant showed the constitutivity for invertase typical of ssn6 single mutants. In contrast, snf2 ssn6 and snf5 ssn6 strains produced only moderate levels of invertase under derepressing conditions and very low levels under repressing conditions. These findings suggest roles for the SNF1 through SNF6 and SSN6 genes in the regulation of SUC2 gene expression by glucose repression.     

Gss1 protein of the methylotrophic yeast Pichia pastoris is involved in glucose sensing, pexophagy and catabolite repression

In the yeast Saccharomyces cerevisiae, the one-at-a-time deletions of either the high-affinity glucose sensor gene SNF3 or the low-affinity glucose sensor gene RGT2 only slightly reduced pexophagy; however, deleting both genes greatly reduced pexophagy, evincing interaction beyond the sum of the additive effects, as recently shown. The present study identifies the only ScSNF3/RGT2 ortholog in the methylotrophic yeast Pichia pastoris (designated as PpGSS1, from GlucoSe Sensor) and describes its roles in autophagic pathways (non-selective and selective). GSS1 knock-out strain has been constructed. The experiments support the hypothesis that Gss1 plays an important role in autophagic degradation of peroxisomes and glucose catabolite repression in P. pastoris.     

Roles of multiple glucose transporters in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, TRK1 and TRK2 are required for high- and low-affinity K+ transport. Among suppressors of the K+ transport defect in trk1 delta trk2 delta cells, we have identified members of the sugar transporter gene superfamily. One suppressor encodes the previously identified glucose transporter HXT1, and another encodes a new member of this family, HXT3. The inferred amino acid sequence of HXT3 is 87% identical to that of HXT1, 64% identical to that of HXT2, and 32% identical to that of SNF3. Like HXT1 and HXT2, overexpression of HXT3 in snf3 delta cells confers growth on low-glucose or raffinose media. The function of another new member of the HXT superfamily, HXT4 (previously identified by its ability to suppress the snf3 delta phenotype; L. Bisson, personal communication), was revealed in experiments that deleted all possible combinations of the five members of the glucose transporter gene family. Neither SNF3, HXT1, HXT2, HXT3, nor HXT4 is essential for viability. snf3 delta hxt1 delta hxt2 delta hxt3 delta hxt4 delta cells are unable to grow on media containing high concentrations of glucose (5%) but can grow on low-glucose (0.5%) media, revealing the presence of a sixth transporter that is itself glucose repressible. This transporter may be negatively regulated by SNF3 since expression of SNF3 abolishes growth of hxt1 delta hxt2 delta hxt3 delta hxt4 delta cells on low-glucose medium. HXT1, HXT2, HXT3, and HXT4 can function independently: expression of any one of these genes is sufficient to confer growth on medium containing at least 1% glucose. A synergistic relationship between SNF3 and each of the HXT genes is suggested by the observation that SNF2 hxt1 delta hxt2 delta hxt3 delta hxt4 delta cells and snf3 delta HXT1 HXT2 HXT3 HXT4 cells are unable to grow on raffinose (low fructose) yet SNF3 in combination with any single HXT gene is sufficient for growth on raffinose. HXT1 and HXT3 are differentially regulated. HXT1::lacZ is maximally expressed during exponential growth whereas HXT3::lacZ is maximally expressed after entry into stationary phase.     

Molecular and Genetic Analysis of the Snf7 Gene in Saccharomyces Cerevisiae

Mutations in the SNF7 gene of Saccharomyces cerevisiae prevent full derepression of the SUC2 (invertase) gene in response to glucose limitation. We report the molecular cloning of the SNF7 gene by complementation. Sequence analysis predicts that the gene product is a 27-kDa acidic protein. Disruption of the chromosomal locus causes a fewfold decrease in invertase derepression, a growth defect on raffinose, temperature-sensitive growth on glucose, and a sporulation defect in homozygous diploids. Genetic analysis of the interactions of the snf7 null mutation with ssn6 and spt6/ssn20 suppressor mutations distinguished SNF7 from the SNF2, SNF5 and SNF6 genes. The snf7 mutation also behaved differently from mutations in SNF1 and SNF4 in that snf7 ssn6 double mutants displayed a synthetic phenotype of severe temperature sensitivity for growth. We also mapped SNF7 to the right arm of chromosome XII near the centromere.     

Relationship of the Camp-Dependent Protein Kinase Pathway to the Snf1 Protein Kinase and Invertase Expression in Saccharomyces Cerevisiae

The SNF1 protein kinase and the associated SNF4 protein are required for release of glucose repression in Saccharomyces cerevisiae. To identify functionally related proteins, we selected genes that in multicopy suppress the raffinose growth defect of snf4 mutants. Among the nine genes recovered were two genes from the cAMP-dependent protein kinase (cAPK) pathway, MSI1 and PDE2. Increased dosage of these genes partially compensates for defects in nutrient utilization and sporulation in snf1 and snf4 null mutants, but does not restore invertase expression. These results suggest that SNF1 and cAPK affect some of the same cellular responses to nutrients. To examine the role of the cAPK pathway in regulation of invertase, we assayed mutants in which the cAPK is not modulated by cAMP. Expression of invertase was regulated in response to glucose and was dependent on SNF1 function. Thus, a cAMP-responsive cAPK is dispensable for regulation of invertase.     

Mutational analysis of the SNF3 glucose transporter of Saccharomyces cerevisiae.

The SNF3 gene of Saccharomyces cerevisiae encodes a high-affinity glucose transporter that is homologous to mammalian glucose transporters. Point mutations affecting the function of the transporter were recovered from the genomes of four snf3 mutants and characterized. Two of the mutations introduced a charged amino acid into the first and second predicted membrane-spanning regions, respectively. The analogs of a bifunctional SNF3-lacZ fusion containing these two mutations were constructed, and the mutant fusion proteins were not localized to the plasma membrane, as judged by immunofluorescence microscopy. The third mutation produced a valine-to-isoleucine substitution in hydrophobic region 8, and the corresponding mutant fusion protein was correctly localized. The finding that this conservative change causes a transport defect is consistent with the possibility that this transmembrane region, which could exist as an amphipathic alpha-helix, forms part of the glucose channel through the membrane. The fourth snf3 allele harbored an ochre mutation midway through the coding sequence. We have also constructed mutations in the cloned SNF3 gene. A major difference between the yeast SNF3 protein and mammalian glucose transporters is the presence in the SNF3 protein of an additional 303 amino acids at the C terminus. Analysis of a series of C-terminal deletions and fusions to lacZ showed that this C-terminal region is important, but not essential, for transport function. We also report the genetic mapping of the SNF3 locus on the left arm of chromosome IV.     

Suppressors Reveal Two Classes of Glucose Repression Genes in the Yeast Saccharomyces Cerevisiae

We selected and analyzed extragenic suppressors of mutations in four genes-GRR1, REG1, GAL82 and GAL83-required for glucose repression of the GAL genes in the yeast Saccharomyces cerevisiae. The suppressors restore normal or nearly normal glucose repression of GAL1 expression in these glucose repression mutants. Tests of the ability of each suppressor to cross-suppress mutations in the other glucose repression genes revealed two groups of mutually cross-suppressed genes: (1) REG1, GAL82 and GAL83 and (2) GRR1. Mutations of a single gene, SRG1, were found as suppressors of reg1, GAL83-2000 and GAL82-1, suggesting that these three gene products act at a similar point in the glucose repression pathway. Mutations in SRG1 do not cross-suppress grr1 or hxk2 mutations. Conversely, suppressors of grr1 (rgt1) do not cross-suppress any other glucose repression mutation tested. These results, together with what was previously known about these genes, lead us to propose a model for glucose repression in which Grr1p acts early in the glucose repression pathway, perhaps affecting the generation of the signal for glucose repression. We suggest that Reg1p, Gal82p and Gal83p act after the step(s) executed by Grr1p, possibly transmitting the signal for repression to the Snf1p protein kinase.     

Molecular analysis of SNF2 and SNF5, genes required for expression of glucose-repressible genes in Saccharomyces cerevisiae.

The SNF2 and SNF5 genes are required for derepression of SUC2 and other glucose-repressible genes of Saccharomyces cerevisiae in response to glucose deprivation. Previous genetic evidence suggested that SNF2 and SNF5 have functionally related roles. We cloned both genes by complementation and showed that the cloned DNA was tightly linked to the corresponding chromosomal locus. Both genes in multiple copy complemented only the cognate snf mutation. The SNF2 gene encodes a 5.7-kilobase RNA, and the SNF5 gene encodes a 3-kilobase RNA. Both RNAs contained poly(A) and were present in low abundance. Neither was regulated by glucose repression, and the level of SNF2 RNA was not dependent on SNF5 function or vice versa. Disruption of either gene at its chromosomal locus still allowed low-level derepression of secreted invertase activity, suggesting that these genes are required for high-level expression but are not directly involved in regulation. Further evidence was the finding that snf2 and snf5 mutants failed to derepress acid phosphatase, which is not regulated by glucose repression. The SNF2 and SNF5 functions were required for derepression of SUC2 mRNA.     

Mutations Causing Constitutive Invertase Synthesis in Yeast: Genetic Interactions with snf Mutations

We have selected 210 mutants able to grow on sucrose in the presence of 2-deoxyglucose. We identified recessive mutations in three major complementation groups that cause constitutive (glucose-insensitive) secreted invertase synthesis. Two groups comprise alleles of the previously identified HXK2 and REG1 genes, and the third group was designated cid1 (constitutive invertase derepression). The effect of cid1 on SUC2 expression is mediated by the SUC2 upstream regulatory region, as judged by the constitutive expression of a SUC2-LEU2-lacZ fusion in which the LEU2 promoter is under control of SUC2 upstream sequences. A cid1 mutation also causes glucose-insensitive expression of maltase. The previously isolated constitutive mutation ssn6 is epistatic to cid1, reg1 and hxk2 for very high level constitutive invertase expression. Mutations in SNF genes that prevent derepression of invertase are epistatic to cid1, reg1 and hxk2; we have previously shown that ssn6 has different epistasis relationships with snf mutations. The constitutive mutation tup1 was found to resemble ssn6 in its genetic interactions with snf mutations. These findings suggest that CID1, REG1 and HXK2 are functionally distinct from SSN6 and TUP1.