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S Hohmann

Publications and source records attributed to S Hohmann.

At least 55 records · Page 3Linked to original sources
PubMed1994

The byp1-3 allele of the Saccharomyces cerevisiae GGS1/TPS1 gene and its multi-copy suppressor tRNA(GLN) (CAG): Ggs1/Tps1 protein levels restraining growth on fermentable sugars and trehalose accumulation.

Byp1-3 is an amber nonsense allele of the Saccharomyces cerevisiae GGS1/TPS1 gene which encodes the small subunit of the trehalose synthase complex. Mutations in this gene confer an inability to grow on glucose or fructose but the phenotype of byp1-3 mutants is leaky in a strain-dependent manner. Overexpression of the isolated byp1-3 allele suppressed the growth defect of a ggs1/tps1 delta mutant. Expression of an in-vitro-generated mutant allele of GGS1/TPS1 that lacks all the coding sequences downstream from the byp1-3 mutation led to the production of a shortened protein that did not complement the ggs1/tps1 delta mutant. We have isolated, as an allele-specific multi-copy suppressor of the growth defect of the byp1-3 mutant on fructose, the gene for tRNA(GLN) (CAG). Thus the leaky phenotype of byp1-3 mutants is due to a low level of read through of the internal nonsense codon by tRNA(GLN) (CAG). Using overexpression of the isolated byp1-3 allele, as well as of the tRNA(GLN) (CAG) gene, we were able to demonstrate that as little as about 10% of the normal Ggs1/Tps1 protein level is sufficient for slow growth on fructose. We also show a correlation between the level of Ggs1/Tps1, the ability to accumulate trehalose in stationary phase and the ability to grow on fermentable sugars. Sequence analysis of the cloned tRNA(GLN) (CAG) gene showed that it is located 700 bp upstream of URA10. However, we found considerable differences to the reported sequence of URA10, in particular in the non-coding region.

Alleles↗
PubMed1994

Characterization of the osmotic-stress response in Saccharomyces cerevisiae: osmotic stress and glucose repression regulate glycerol-3-phosphate dehydrogenase independently.

Micro-organisms have developed systems to adapt to sudden changes in the environment. Here we describe the response of the yeast Saccharomyces cerevisiae to osmotic stress. A drop in the water activity (aw) of the medium following the addition of NaCl led to an immediate shrinkage of the cells. During the 2 h following the osmotic shock the cells partially restored their cell volume. This process depended on active protein synthesis. During the recovery period the cells accumulated glycerol intracellularly as a compatible solute and very little glycerol was leaking out of the cell. We have investigated in more detail the enzymes of glycerol metabolism and found that only the cytoplasmic glycerol-3-phosphate dehydrogenase was strongly induced. The level of induction was dependent on the yeast strain used and the degree of osmotic stress. The synthesis of cytoplasmic glycerol-3-phosphate dehydrogenase is also regulated by glucose repression. Using mutants defective in glucose repression (hxk2 delta), or derepression (snf1 delta), and with invertase as a marker enzyme, we show that glucose repression and the osmotic-stress response system regulate glycerol-3-phosphate dehydrogenase synthesis independently. We infer that specific control mechanisms sense the osmotic situation of the cell and induce responses such as the production and retention of glycerol.

Enzyme Induction↗
PubMed1994

GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway.

The yeast Saccharomyces cerevisiae responds to osmotic stress, i.e., an increase in osmolarity of the growth medium, by enhanced production and intracellular accumulation of glycerol as a compatible solute. We have cloned a gene encoding the key enzyme of glycerol synthesis, the NADH-dependent cytosolic glycerol-3-phosphate dehydrogenase, and we named it GPD1. gpd1 delta mutants produced very little glycerol, and they were sensitive to osmotic stress. Thus, glycerol production is indeed essential for the growth of yeast cells during reduced water availability. hog1 delta mutants lacking a protein kinase involved in osmostress-induced signal transduction (the high-osmolarity glycerol response [HOG] pathway) failed to increase glycerol-3-phosphate dehydrogenase activity and mRNA levels when osmotic stress was imposed. Thus, expression of GPD1 is regulated through the HOG pathway. However, there may be Hog1-independent mechanisms mediating osmostress-induced glycerol accumulation, since a hog1 delta strain could still enhance its glycerol content, although less than the wild type. hog1 delta mutants are more sensitive to osmotic stress than isogenic gpd1 delta strains, and gpd1 delta hog1 delta double mutants are even more sensitive than either single mutant. Thus, the HOG pathway most probably has additional targets in the mechanism of adaptation to hypertonic medium.

Amino Acid Sequence↗
PubMed1993

Disruption of the Kluyveromyces lactis GGS1 gene causes inability to grow on glucose and fructose and is suppressed by mutations that reduce sugar uptake.

In the yeast Saccharomyces cerevisiae the GGS1 gene is essential for growth on glucose or other readily fermentable sugars. GGS1 is the same gene as TPS1 which was identified as encoding a subunit of the trehalose-6-phosphate synthase/phosphatase complex and it is allelic to the fdp1, byp1, glc6 and cif1 mutations. Its precise function in the regulation of sugar catabolism is unknown. We have cloned the GGS1 homologue from the distantly related yeast Kluyveromyces lactis. The KlGGS1 gene is 74% and 79% identical at the nucleotide and amino acid sequence level, respectively, to the S. cerevisiae counterpart. We also compared the sequence with the partly homologous products of the S. cerevisiae genes TPS2 and TSL1 which code for the larger subunits of the trehalose synthase complex and with a TSL1 homologue, TPS3, of unknown function. Multiple alignment of these sequences revealed several particularly well conserved elements. Disruption of GGS1 in K. lactis caused the same pleiotropic phenotype as in S. cerevisiae, i.e. inability to grow on glucose or fructose and strongly reduced trehalose content. We have also studied short-term glucose-induced regulatory effects related to cAMP and cAMP-dependent protein kinase, i.e. the cAMP signal, trehalase activation, trehalose mobilization and inactivation of fructose-1,6-bisphosphatase. These effects occur very rapidly in S. cerevisiae and are absent in the Scggs1 mutant. In K. lactis all these effects were much slower and largely unaffected by the Klggs1 mutation. On the other hand, glucose strongly induced pyruvate decarboxylase and activated the potassium transport system in K. lactis and both effects were absent in the Klggs1 mutant. Addition of glucose to galactose-grown cells of the Klggs1 mutant caused, as in S. cerevisiae, intracellular accumulation of free glucose and of sugar phosphates and a rapid drop of the ATP and inorganic phosphate levels. Glucose transport kinetics were the same for the wild type and the Klggs1 mutant in both derepressed cells and in cells incubated with glucose. We have isolated phenotypic revertants of the Klggs1 mutant for growth on fructose. The suppressors that we characterized had, to different extents, diminished glucose uptake in derepressed cells but cells incubated in glucose showed very different characteristics. The suppressor mutations prevented deregulation of glycolysis in the Klggs1 mutant but not the accumulation of free glucose. The mutants with higher residual uptake activity showed partially restored induction of pyruvate decarboxylase and activation of potassium transport.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Role of cysteines in the activation and inactivation of brewers' yeast pyruvate decarboxylase investigated with a PDC1-PDC6 fusion protein.

Possible roles of the Cys side chains in the activation and inactivation mechanisms of brewers' yeast pyruvate decarboxylase were investigated by comparing the behavior of the tetrameric enzyme pdc1 containing four cysteines/subunit (positions 69, 152, 221, and 222) with that of a fusion enzyme (pdc1-6, a result of spontaneous gene fusion between PDC1 and PDC6 genes) that is 84% identical in sequence with pdc1 and has only Cys221 (the other three Cys being replaced by aliphatic side chains). The two forms of the enzyme are rather similar so far as steady-state kinetic parameters and substrate activation are considered, as tested for activation by the substrate surrogate pyruvamide. Therefore, if a cysteine is responsible for substrate activation, it must be Cys221. The inactivation of the two enzymes was tested with several inhibitors. Methylmethanethiol sulfonate, a broad spectrum sulfhydryl reagent, could substantially inactivate both enzymes, but was slightly less effective toward the fusion enzyme. (p-Nitrobenzoyl)formic acid is an excellent alternate substrate, whose decarboxylation product p-nitrobenzaldehyde inhibited both enzymes possibly at a Cys221, the only one still present in the fusion enzyme. Exposure of the fusion enzyme, just as of pdc1, to (E)-2-oxo-4-phenyl-3-butenoic acid type inhibitors/alternate substrates enabled detection of the enzyme-bound enamine intermediate at 440 nm. However, unlike pdc1, the fusion enzyme was not irreversibly inactivated by these substrates. These substrates are now known to cause inactivation of pdc1 with concomitant modification of one Cys of the four [Zeng, X.; Chung, A.; Haran, M.; Jordan, F. (1991) J. Am. Chem. Soc. 113, 5842-49].(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning, Molecular↗
PubMed1993

Characterisation of PDC2, a gene necessary for high level expression of pyruvate decarboxylase structural genes in Saccharomyces cerevisiae.

The regulatory gene PDC2 was identified in a screen for mutations affecting pyruvate decarboxylase activity in yeast. I have cloned and sequenced this gene. The predicted protein of 925 amino acids has no homology to any sequence in the databases. However, the protein sequence is rich in asparagine and serine residues, as is often found for transcriptional regulators. The PDC2 deletion mutant exhibits a phenotype very similar to, but more severe than that of the point mutant: a strongly reduced pyruvate decarboxylase specific activity, slow, respiration-dependent growth on glucose, and accumulation of pyruvate. The activity of other glycolytic enzymes seems to be unaffected by the pdc2 delta mutation. Synthesis of pyruvate decarboxylase is regulated by PDC2 at the transcriptional level. Expression of the major structural gene for pyruvate decarboxylase, PDC1, is strongly reduced in pdc2 delta mutants. Transcription of the generally more weakly expressed PDC5 gene appears to be entirely abolished. However, glucose induction of pyruvate decarboxylase synthesis is unaffected. Thus, PDC2 is either important for a high basal level of PDC gene expression or it plays a positive role in the autoregulation that controls expression of PDC1 and PDC5.

Amino Acid Sequence↗
PubMed1993

The growth and signalling defects of the ggs1 (fdp1/byp1) deletion mutant on glucose are suppressed by a deletion of the gene encoding hexokinase PII.

Yeast cells defective in the GGS1 (FDP1/BYP1) gene are unable to adapt to fermentative metabolism. When glucose is added to derepressed ggs1 cells, growth is arrested due to an overloading of glycolysis with sugar phosphates which eventually leads to a depletion of phosphate in the cytosol. Ggs1 mutants lack all glucose-induced regulatory effects investigated so far. We reduced hexokinase activity in ggs1 strains by deleting the gene HXK2 encoding hexokinase PII. The double mutant ggs1 delta, hxk2 delta grew on glucose. This is in agreement with the idea that an inability of the ggs1 mutants to regulate the initiation of glycolysis causes the growth deficiency. However, the ggs1 delta, hxk2 delta double mutant still displayed a high level of glucose-6-phosphate as well as the rapid appearance of free intracellular glucose. This is consistent with our previous model suggesting an involvement of GGS1 in transport-associated sugar phosphorylation. Glucose induction of pyruvate decarboxylase, glucose-induced cAMP-signalling, glucose-induced inactivation of fructose-1,6-bisphosphatase, and glucose-induced activation of the potassium transport system, all deficient in ggs1 mutants, were restored by the deletion of HXK2. However, both the ggs1 delta and the ggs1 delta, hk2 delta mutant lack detectable trehalose and trehalose-6-phosphate synthase activity. Trehalose is undetectable even in ggs1 delta strains with strongly reduced activity of protein kinase A which normally causes a very high trehalose content. These data fit with the recent cloning of GGS1 as a subunit of the trehalose-6-phosphate synthase/phosphatase complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene Deletion↗
PubMed1993

Molecular cloning of a gene involved in glucose sensing in the yeast Saccharomyces cerevisiae.

Cells of the yeast Saccharomyces cerevisiae display a wide range of glucose-induced regulatory phenomena, including glucose-induced activation of the RAS-adenylate cyclase pathway and phosphatidylinositol turnover, rapid post-translational effects on the activity of different enzymes as well as long-term effects at the transcriptional level. A gene called GGS1 (for General Glucose Sensor) that is apparently required for the glucose-induced regulatory effects and several ggs1 alleles (fdp1, byp1 and cif1) has been cloned and characterized. A GGS1 homologue is present in Methanobacterium thermoautotrophicum. Yeast ggs1 mutants are unable to grow on glucose or related readily fermentable sugars, apparently owing to unrestricted influx of sugar into glycolysis, resulting in its rapid deregulation. Levels of intracellular free glucose and metabolites measured over a period of a few minutes after addition of glucose to cells of a ggs1 delta strain are consistent with our previous suggestion of a functional interaction between a sugar transporter, a sugar kinase and the GGS1 gene product. Such a glucose-sensing system might both restrict the influx of glucose and activate several signal transduction pathways, leading to the wide range of glucose-induced regulatory phenomena. Deregulation of these pathways in ggs1 mutants might explain phenotypic defects observed in the absence of glucose, e.g. the inability of ggs1 diploids to sporulate.

Alleles↗
PubMed1992

The cell division cycle gene CDC60 encodes cytosolic leucyl-tRNA synthetase in Saccharomyces cerevisiae.

The cdc60 mutation (for cell division cycle) of the yeast, Saccharomyces cerevisiae, confers arrest at the START point of the cell cycle upon shift to the restrictive temperature [Bedard et al., Curr. Genet. 4 (1981) 205-214]. We have cloned the CDC60 gene by complementation of the temperature-sensitive phenotype. Sequence analysis revealed a single open reading frame of 3270 bp and the deduced amino acid sequence showed 50.5% sequence identity to the cytosolic leucyl-tRNA synthetase (LeuRS) from Neurospora crassa, implying that CDC60 encodes the corresponding yeast protein. Thus, CDC60 does not appear to be involved directly in the regulation of the cell cycle. Rather, the cdc60 mutation leads to cell-cycle arrest at the nutrient control point START due to a deficiency of charged leucyl-tRNA. The CDC60 gene product also shows homology to LeuRSs from other organisms and to aminoacyl-RS for isoleucine, valine and methionine.

Amino Acid Sequence↗
PubMed1992

Glucose-induced regulatory defects in the Saccharomyces cerevisiae byp1 growth initiation mutant and identification of MIG1 as a partial suppressor.

Saccharomyces cerevisiae byp1-3 mutants displayed a long lag phase when shifted from a nonfermentable carbon source to a medium containing glucose. The byp1-3 mutation also caused several defects in regulatory phenomena which occur during the transition from the derepressed state to the repressed state. As opposed to wild-type cells, the addition of glucose to cells of the byp1-3 mutant grown on nonfermentable carbon sources did not induce a cyclic AMP signal. Fructose-2,6-bisphosphate formation and inactivation of fructose-1,6-bisphosphatase were severely delayed, but trehalase activation was not affected. In addition, the induction of pyruvate decarboxylase both at the level of activity and that of transcription was very slow compared with that in wild-type cells. These pleotropic defects in glucose-induced regulatory phenomena might be responsible for the very long lag phase of byp1-3 cells and the inability of ascospores to initiate growth after germination on glucose media. Screening of a yeast gene library for clones complementing the byp1-3 phenotype resulted in the isolation of a truncated form of the previously described zinc finger transcription repressor MIG1. The entire MIG1 gene and the truncated form suppressed even on a single-copy vector the growth initiation defect but not the regulatory abnormalities of the byp1-3 mutant. MIG1 is not allelic to byp1-3.

Antimycin A↗
PubMed1991

A yeast homologue of the bovine lens fibre MIP gene family complements the growth defect of a Saccharomyces cerevisiae mutant on fermentable sugars but not its defect in glucose-induced RAS-mediated cAMP signalling.

Recently a new family of membrane proteins comprising the bovine lens fibre major intrinsic protein, soybean nodulin-26 protein and the Escherichia coli glycerol facilitator has been described [M.E. Baker and M.H. Saier, Jr (1990) Cell, 60, 185-186]. These proteins have six putative membrane spanning domains and one (probably intracellular) intermembrane fragment is particularly well conserved. We have identified a new member of this family in the yeast Saccharomyces cerevisiae. It also possesses the six transmembrane domains and the highly conserved intermembrane sequence. In contrast to the other three proteins which are all approximately 280 amino acids long, the yeast protein has an N-terminal extension of approximately 250 amino acids, which contains a string of 17 asparagine residues and a C-terminal extension of approximately 150 amino acids. The gene, which we called FPS1 (for fdp1 suppressor), suppresses in single copy the growth defect on fermentable sugars of the yeast fdp1 mutant but it is not allelic to FDP1. The deficiency of the fdp1 mutant in glucose-induced RAS-mediated cAMP signalling and in rapid glucose-induced changes in the activity of certain enzymes was not restored. Deletion of FPS1 does not cause any of the phenotypic deficiencies of the fdp1 mutant.

Amino Acid Sequence↗
PubMed1991

PDC6, a weakly expressed pyruvate decarboxylase gene from yeast, is activated when fused spontaneously under the control of the PDC1 promoter.

Three structural genes encode the pyruvate decarboxylase isoenzymes in the yeast Saccharomyces cerevisiae. PDC1 and PDC5 are active during glucose fermentation where PDC1 is expressed about six times more strongly than PDC5. Expression of PDC6 is weak and seems to be induced in ethanol medium. Consequently, pdc1 delta pdc5 delta double mutants do not ferment glucose and do not grow on glucose medium. Spontaneous mutants, derived from such a pdc1 pdc5 strain, were isolated which could again ferment glucose. They showed pyruvate decarboxylase activity due to a duplication of PDC6. The second copy of PDC6 was expressed under the control of the PDC1 promoter, which was still present in the pdc1 strain. However, the resulting PDC1-PDC6 fusion gene could only partially substitute for PDC1: to achieve normal growth and high pyruvate decarboxylase activity strains carrying PDC1-PDC6 required a functional PDC5 gene which is dispensable in a PDC1 wild-type background. Thus, expression of PDC5 depends on the state of the PDC1 locus: low in the PDC1 wild-type background and high in PDC1-PDC6 fusion strains and, as shown previously, in pdc1 mutants. The activation of PDC5 expression in PDC1-PDC6 strains may be due to particular properties of the PDC1-PDC6 fusion protein or simply to the weaker expression of PDC1-PDC6 in comparison to the wild-type PDC1 gene.

Alleles↗
PubMed1991

Characterization of PDC6, a third structural gene for pyruvate decarboxylase in Saccharomyces cerevisiae.

Pyruvate decarboxylase is the key enzyme in alcoholic fermentation in yeast. Two structural genes, PDC1 and PDC5 have been characterized. Deletion of either of these genes has little or no effect on the specific pyruvate decarboxylase activity, but enzyme activity is undetectable in mutants lacking both PDC1 and PDC5 (S. Hohmann and H. Cederberg, Eur. J. Biochem. 188:615-621, 1990). Here I describe PDC6, a gene structurally closely related to PDC1 and PDC5. The product of PDC6 does not seem to be required for wild-type pyruvate decarboxylase activity in glucose medium; delta pdc6 mutants have no reduced specific enzyme activity, and the PDC6 deletion did not change the phenotype or the specific enzyme activity of mutants lacking either or both of the other two structural genes. However, in cells grown in ethanol medium the PDC6 deletion caused a reduction of pyruvate decarboxylase activity. Northern (RNA) blot analysis showed that PDC6 is weakly expressed, and expression seemed to be higher during growth in ethanol medium. This behavior remained obscure since pyruvate decarboxylase catalyzes an irreversible reaction. Characterization of all combinations of PDC structural gene deletion mutants, which produce different amounts of pyruvate decarboxylase activity, showed that the enzyme is also needed for normal growth in galactose and ethanol medium and in particular for proper growth initiation of spores germinating on ethanol medium.

Amino Acid Sequence↗
PubMed1990

Molecular analysis of the structural gene for yeast transaldolase.

We have cloned the structural gene for yeast transaldolase. Transformants carrying the TAL1 gene on a multicopy plasmid over-produced transaldolase. A deletion mutant which was constructed using the cloned gene did not show any detectable transaldolase activity in vitro. Furthermore, both transaldolase isoenzymes which were detected in wild-type crude extracts by immunoblotting were missing in the deletion mutants. Thus, TAL1 is the only transaldolase structural gene in yeast. TAL1 is not an essential gene. Deletion of the transaldolase gene did not affect growth on complete media with different carbon sources or on synthetic media. However, the transaldolase-deficient strains accumulated sedoheptulose 7-phosphate, an intermediate of the pentose-phosphate pathway. Mutants lacking both transaldolase and phosphoglucose isomerase grew more slowly than the single mutants. They accumulated more sedoheptulose 7-phosphate on medium containing fructose than on glucose medium. This shows that fructose 6-phosphate and glyceraldehyde 3-phosphate, metabolites of glycolysis, can enter the nonoxidative part of the pentose-phosphate pathway.

Amino Acid Sequence↗
PubMed1990

Autoregulation may control the expression of yeast pyruvate decarboxylase structural genes PDC1 and PDC5.

Recently we deleted the pyruvate decarboxylase structural gene PDC1 from the genome of the yeast Saccharomyces cerevisiae. The pdc1 deletion mutants had pyruvate decarboxylase activity due to the presence of a second structural gene [Schaaff, I., Green, J. B. A., Gozalbo, D. & Hohmann, S. (1989) Curr. Genet. 15, 75-81]. We cloned and sequenced this gene which we call PDC5. The predicted amino acid sequences of PDC1 and PDC5 are 88% identical. Deletion of PDC5 did not cause any decrease in the specific pyruvate decarboxylase activity while pdc1 deletion mutants had 80% of the wild-type activity. Deletion mutants lacking both PDC1 and PDC5 did not show any detectable pyruvate decarboxylase activity in vitro and were unable to ferment glucose. This indicates that PDC1 and PDC5 are the only structural genes for pyruvate decarboxylase in yeast. The PDC5 isoenzyme showed a slightly higher Km value for its substrate pyruvate than the PDC1 product (PDC5: Km = 8 mM; PDC1: Km = 5 mM), as measured in crude extract of pdc1 and pdc5 deletion mutants, respectively. PDC5 is only expressed in pdc1 deletion mutants. No mRNA transcribed from PDC5 could be detected in wild-type cells. Thus, in addition to the control by glucose induction, pyruvate decarboxylase activity seems to be subject to autoregulation. Similar phenomena have been described previously for tubulin, histones and a ribosomal protein but not for metabolic enzymes.

Alleles↗
PubMed1989

The naturally occurring silent invertase structural gene suc2 zero contains an amber stop codon that is occasionally read through.

The yeast invertase structural gene SUC2 has two naturally occurring alleles, the active one and a silent allele called suc2 zero. Strains carrying suc2 zero are unable to ferment sucrose and do not show detectable invertase activity. We have isolated suc2 zero and found an amber codon at position 232 of 532 amino acids. However, transformants carrying suc2 zero on a multicopy plasmid were able to ferment sucrose and showed detectable invertase activity. Full-length invertase was found in gels stained for active invertase and in immunoblots. Therefore we concluded that the amber codon is occasionally read as an amino acid. The calculated frequency of read-through is about 4% of all translation events.

Alleles↗