scholarly journals Opposite roles of trehalase activity in heat-shock recovery and heat-shock survival in Saccharomyces cerevisiae

1999 ◽  
Vol 343 (3) ◽  
pp. 621-626 ◽  
Author(s):  
Stefaan WERA ◽  
Ellen DE SCHRIJVER ◽  
Ilse GEYSKENS ◽  
Solomon NWAKA ◽  
Johan M. THEVELEIN

A variety of results has been obtained consistent with activation of neutral trehalase in Saccharomyces cerevisiae through direct phosphorylation by cAMP-dependent protein kinase (PKA). A series of neutral trehalase mutant alleles, in which all evolutionarily conserved putative phosphorylation sites were changed into alanine, was tested for activation in vitro (by PKA) and in vivo (by glucose addition). None of the mutations alone affected the activation ratio, whereas all mutations combined resulted in an inactive enzyme. All mutant alleles were expressed to similar levels, as shown by Western blotting. Several of the point mutations significantly lowered the specific activity. Using this series of mutants with different activity levels we show an inverse relationship between trehalase activity and heat-shock survival during glucose-induced trehalose mobilization. This is consistent with a stress-protective function of trehalose. On the other hand, reduction of trehalase activity below a certain threshold level impaired recovery from a sublethal heat shock. This suggests that trehalose breakdown is required for efficient recovery from heat shock, and that the presence of trehalase protein alone is not sufficient for efficient heat-stress recovery.

1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875 ◽  
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.


1990 ◽  
Vol 10 (11) ◽  
pp. 5679-5687
Author(s):  
C K Barlowe ◽  
D R Appling

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.


1990 ◽  
Vol 10 (11) ◽  
pp. 5679-5687 ◽  
Author(s):  
C K Barlowe ◽  
D R Appling

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.


1989 ◽  
Vol 9 (1) ◽  
pp. 34-42 ◽  
Author(s):  
J Yu ◽  
M S Donoviel ◽  
E T Young

A 22-base-pair (bp) inverted repeat present in the ADH2 promoter is an upstream activation sequence (UAS1) which confers ADR1-dependent activation upon a heterologous Saccharomyces cerevisiae promoter. UAS1 was nonfunctional when placed within an intron 3' to the transcription start site. The 11-bp sequence which constitutes one-half of the UAS1 palindrome did not activate transcription in a single copy, as direct repeats, or in an inverted orientation opposite to that of ADH2 UAS1. Furthermore, two pairs of symmetrical point mutations within UAS1 significantly reduced activation. This result suggests that a specific orientation of sequences within UAS1 is necessary for ADR1-dependent activation. We determined that an ADR1-dependent complex was formed with UAS1 and, to a lesser extent, with the nonfunctional 11-bp half palindrome. However, the 11 bp did not confer UAS activity, suggesting that ADR1 binding is not sufficient for activation in vivo. ADR1 did not bind to mutant UAS1 sequences in vitro, indicating that their decreased activation is attributable to a reduced affinity of ADR1 for these sequences. We also identified an additional 20-bp ADH2 element (UAS2) that increased the expression of CYC1-lacZ 20-fold when combined with UAS1. UAS2 permitted ADR1-independent, glucose-regulated expression of the hybrid gene. Consistent with this observation, ADR1 did not form a detectable complex with UAS2. Deletion of UAS2 at the chromosomal ADH2 locus virtually abolished ADH2 derepression and had no effect on glucose repression.


Genetics ◽  
1995 ◽  
Vol 141 (4) ◽  
pp. 1263-1274
Author(s):  
S J Deminoff ◽  
J Tornow ◽  
G M Santangelo

Abstract The GCR1 gene of Saccharomyces cerevisiae encodes a transcriptional activator that complexes with Rap1p and, through UASRPG elements (Rap1p DNA binding sites), stimulates efficient expression of glycolytic and translational component genes. To map the functionally important domains in Gcr1p, we combined multiple rounds of random mutagenesis in vitro with in vivo selection of functional genes to locate conserved, or hypomutable, regions. We name this method unigenic evolution, a statistical analysis of mutations in evolutionary variants of a single gene in an otherwise isogenic background. Examination of the distribution of 315 mutations in 24 variant alleles allowed the localization of four hypomutable regions in GCR1 (A, B, C, and D). Dispensable N-terminal (intronic) and C-terminal portions of the evolved region of GCR1 were included in the analysis as controls and were, as expected, not hypomutable. The analysis of several insertion, deletion, and point mutations, combined with a comparison of the hypomutability and hydrophobicity plots of Gcr1p, suggested that some of the hypomutable regions may individually or in combination correspond to functionally important surface domains. In particular, we determined that region D contains a putative leucine zipper and is necessary and sufficient for Gcr1p homodimerization.


1996 ◽  
Vol 16 (2) ◽  
pp. 475-480 ◽  
Author(s):  
X Mao ◽  
B Schwer ◽  
S Shuman

RNA (guanine-7-)-methyltransferase is the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA. The Saccharomyces cerevisiae enzyme is a 436-amino-acid protein encoded by the essential ABD1 gene. In this study, deletion and point mutations in ABD1 were tested for the ability to support growth of an abd1 null strain. Elimination of 109 amino acids from the N terminus had no effect on cell viability, whereas a more extensive N-terminal deletion of 155 residues was lethal, as was a C-terminal deletion of 55 amino acids. Alanine substitution mutations were introduced at eight conserved residues within a 206-amino-acid region of similarity between ABD1 and the methyltransferase domain of the vaccinia virus capping enzyme. ABD1 alleles H253A (encoding a substitution of alanine for histidine at position 253), T282A, E287A, E361A, and Y362A were viable, whereas G174A, D178A, and Y254A were either lethal or severely defective for growth. Alanine-substituted and amino-truncated ABD1 proteins were expressed in bacteria, purified, and tested for cap methyltransferase activity in vitro. Mutations that were viable in yeast cells had either no effect or only a moderate effect on the specific methyltransferase activity of the mutated ABD1 protein, whereas mutations that were deleterious in vivo yielded proteins that were catalytically defective in vitro. These findings substantiate for the first time the long-held presumption that cap methylation is an essential function in eukaryotic cells.


2000 ◽  
Vol 150 (1) ◽  
pp. 65-76 ◽  
Author(s):  
C. Randell Brown ◽  
Jameson A. McCann ◽  
Hui-Ling Chiang

Fructose-1,6-bisphosphatase (FBPase) is targeted to the vacuole for degradation when Saccharomyces cerevisiae are shifted from low to high glucose. Before vacuolar import, however, FBPase is sequestered inside a novel type of vesicle, the vacuole import and degradation (Vid) vesicles. Here, we reconstitute import of FBPase into isolated Vid vesicles. FBPase sequestration into Vid vesicles required ATP and cytosol, but was inhibited if ATP binding proteins were depleted from the cytosol. The heat shock protein Ssa2p was identified as one of the ATP binding proteins involved in FBPase import. A Δssa2 strain exhibited a significant decrease in the rate of FBPase degradation in vivo as compared with Δssa1, Δssa3, or Δssa4 strains. Likewise, in vitro import was impaired for the Δssa2 strain, but not for the other Δssa strains. The cytosol was identified as the site of the Δssa2 defect; Δssa2 cytosol did not stimulate FBPase import into import competent Vid vesicles, but wild-type cytosol supported FBPase import into competent Δssa2 vesicles. The addition of purified recombinant Ssa2p stimulated FBPase import into Δssa2 Vid vesicles, providing Δssa2 cytosol was present. Thus, Ssa2p, as well as other undefined cytosolic proteins are required for the import of FBPase into vesicles.


1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.


1989 ◽  
Vol 9 (1) ◽  
pp. 34-42
Author(s):  
J Yu ◽  
M S Donoviel ◽  
E T Young

A 22-base-pair (bp) inverted repeat present in the ADH2 promoter is an upstream activation sequence (UAS1) which confers ADR1-dependent activation upon a heterologous Saccharomyces cerevisiae promoter. UAS1 was nonfunctional when placed within an intron 3' to the transcription start site. The 11-bp sequence which constitutes one-half of the UAS1 palindrome did not activate transcription in a single copy, as direct repeats, or in an inverted orientation opposite to that of ADH2 UAS1. Furthermore, two pairs of symmetrical point mutations within UAS1 significantly reduced activation. This result suggests that a specific orientation of sequences within UAS1 is necessary for ADR1-dependent activation. We determined that an ADR1-dependent complex was formed with UAS1 and, to a lesser extent, with the nonfunctional 11-bp half palindrome. However, the 11 bp did not confer UAS activity, suggesting that ADR1 binding is not sufficient for activation in vivo. ADR1 did not bind to mutant UAS1 sequences in vitro, indicating that their decreased activation is attributable to a reduced affinity of ADR1 for these sequences. We also identified an additional 20-bp ADH2 element (UAS2) that increased the expression of CYC1-lacZ 20-fold when combined with UAS1. UAS2 permitted ADR1-independent, glucose-regulated expression of the hybrid gene. Consistent with this observation, ADR1 did not form a detectable complex with UAS2. Deletion of UAS2 at the chromosomal ADH2 locus virtually abolished ADH2 derepression and had no effect on glucose repression.


2005 ◽  
Vol 4 (8) ◽  
pp. 1364-1374 ◽  
Author(s):  
Damian J. Krysan ◽  
Elizabeth L. Ting ◽  
Claudia Abeijon ◽  
Lee Kroos ◽  
Robert S. Fuller

ABSTRACT The yeast cell wall is a crucial extracellular organelle that protects the cell from lysis during environmental stress and morphogenesis. Here, we demonstrate that the yapsin family of five glycosylphosphatidylinositol-linked aspartyl proteases is required for cell wall integrity in Saccharomyces cerevisiae. Yapsin null mutants show hypersensitivity to cell wall perturbation, and both the yps1Δ2Δ mutant and the quintuple yapsin mutant (5ypsΔ) undergo osmoremedial cell lysis at 37°C. The cell walls of both 5ypsΔ and yps1Δ2Δ mutants have decreased amounts of 1,3- and 1,6-β-glucan. Although there is decreased incorporation of both 1,3- and 1,6-β-glucan in the 5ypsΔ mutant in vivo, in vitro specific activity of both 1,3- and 1,6-β-glucan synthesis is similar to wild type, indicating that the yapsins affect processes downstream of glucan synthesis and that the yapsins may be involved in the incorporation or retention of cell wall glucan. Presumably as a response to the significant alterations in cell wall composition, the cell wall integrity mitogen-activated kinase signaling cascade (PKC1-MPK pathway) is basally active in 5ypsΔ. YPS1 expression is induced during cell wall stress and remodeling in a PKC1-MPK1-dependent manner, indicating that Yps1p is a direct, and important, output of the cell wall integrity response. The Candida albicans (SAP9) and Candida glabrata (CgYPS1) homologues of YPS1 complement the phenotypes of the yps1Δ mutant. Taken together, these data indicate that the yapsins play an important role in glucan homeostasis in S. cerevisiae and that yapsin homologues may play a similar role in the pathogenic yeasts C. albicans and C. glabrata.


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