Showing papers by "Stefan Hohmann published in 2001"

The Saccharomyces cerevisiae Sko1p transcription factor mediates HOG pathway-dependent osmotic regulation of a set of genes encoding enzymes implicated in protection from oxidative damage.

Abstract: A major part of the transcriptional response of yeast cells to osmotic shock is controlled by the HOG pathway and several downstream transcription factors. Sko1p is a repressor that mediates HOG pathway-dependent regulation by binding to CRE sites in target promoters. Here, we report five target genes of Hog1p-Sko1p: GRE2, AHP1, SFA1, GLR1 and YML131w. The two CREs in the GRE2 promoter function as activating sequences and, hence, bind (an) activator protein(s). However, the two other yeast CRE-binding proteins, Aca1p and Aca2p, are not involved in regulation of the GRE2 promoter under osmotic stress. In the absence of the co-repressor complex Tup1p-Ssn6p/Cyc8p, which is recruited by Sko1p, stimulation by osmotic stress is still observed. These data indicate that Sko1p is not only required for repression, but also involved in induction upon osmotic shock. All five Sko1p targets encode oxidoreductases with demonstrated or predicted roles in repair of oxidative damage. Altered basal expression levels of these genes in hog1Delta and sko1Delta mutants may explain the oxidative stress phenotypes of these mutants. All five Sko1p target genes are induced by oxidative stress, and induction involves Yap1p. Although Sko1p and Yap1p appear to mediate osmotic and oxidative stress responses independently, Sko1p may affect Yap1p promoter access or activity. The five Sko1p target genes described here are suitable models for studying the interplay between osmotic and oxidative responses at the molecular and physiological levels.

Implications of FPS1 deletion and membrane ergosterol content for glycerol efflux from Saccharomyces cerevisiae.

Abstract: The deletion of the gene encoding the glycerol facilitator Fps1p was associated with an altered plasma membrane lipid composition in Saccharomyces cerevisiae. The S. cerevisiae fps1Δ strain respectively contained 18 and 26% less ergosterol than the wild-type strain, at the whole-cell level and at the plasma membrane level. Other mutants with deficiencies in glycerol metabolism were studied to investigate any possible link between membrane ergosterol content and intracellular glycerol accumulation. In these mutants a modification in intracellular glycerol concentration, or in intra- to extracellular glycerol ratio was accompanied by a reduction in plasma membrane ergosterol content. However, there was no direct correlation between ergosterol content and intracellular glycerol concentration. Lipid composition influences the membrane permeability for solutes during adaptation of yeast cells to osmotic stress. In this study, ergosterol supplementation was shown to partially suppress the hypo-osmotic sensitivity phenotype of the fps1Δ strain, leading to more efficient glycerol efflux, and improved survival. The erg-1 disruption mutant, which is unable to synthesise ergosterol, survived and recovered from the hypo-osmotic shock more successfully when the concentration of exogenously supplied ergosterol was increased. The results obtained suggest that a higher ergosterol content facilitates the flux of glycerol across the plasma membrane of S. cerevisiae cells.

Existence of a tightly regulated water channel in Saccharomyces cerevisiae.

Abstract: The Saccharomyces cerevisiae strain Σ1278b possesses two putative aquaporins, Aqy1-1p and Aqy2-1p. Previous work demonstrated that Aqy1-1p functions as a water channel in Xenopus oocyte. However, no function could be attributed to Aqy2-1p in this system. Specific antibodies were used to follow the expression of Aqy1-1p and Aqy2-1p in the yeast. Aqy1-1p was never detected whatever the growth phase and culture conditions tested. In contrast, Aqy2-1p was detected only during the exponential growth phase in rich medium containing glucose. Aqy2-1p expression was repressed by hyper-osmotic culture conditions. Both immunocytochemistry and biochemical subcellular fractionation demonstrated that Aqy2-1p is located on the endoplasmic reticulum (ER) as well as on the plasma membrane. In microsomal vesicles enriched in ER, a water channel activity due to Aqy2-1p was detected by stopped-flow analysis. Our results show that the expression of aquaporins is tightly controlled. The physiological relevance of aquaporin-mediated water transport in yeast is discussed.

Transposon mutagenesis reveals novel loci affecting tolerance to salt stress and growth at low temperature

Abstract: Using transposon mutagenesis in the haploid Saccharomyces cerevisiae strain W303-1A we have identified genes required for growth in high salt medium, survival of a hypo-osmotic shock and growth at 15 °C. Screening 25,000 transposon insertions revealed a total of 61 insertions that caused salt-sensitivity; and those insertions affected 31 genes. Only 12 of those genes were previously known to be required for salt-tolerance. Among the 61 insertions, three caused general osmo-sensitivity. We identified one single insertion mutant in the already-known gene, FPS1, required for survival of hypo-osmotic shock. A total of 31 insertions caused failure to grow at low temperature. Those identified ten different genes, three of which had previously been reported to affect cold-tolerance. Four genes were identified in both the salt and the cold-sensitivity screen. We found several unusual insertion mutations: (1) insertions in or close to essential genes, (2) insertion in an intergenic region and (3) insertions causing stress-sensitivity in W303-1A, while the deletion mutant in BY4741 did not show such a phenotype. Surprisingly, our mutant set and that reported in the large-scale transposon insertion project (TRIPLES, http://ygac.med.yale.edu/triples/triples.htm) only marginally overlap. We discuss some of the features of transposon mutagenesis in light of the availability of the complete set of yeast deletion mutants and we discuss the possible roles of the genes we identified.

Recombinant saccharomyces cerevisiae expressing chimeric glucose transporters

Abstract: The present invention provides a modified Saccharomyces yeast which produces significantly lower levels of ethanol than wild-type yeast under aerobic conditions and saccharide concentrations of 2% glucose, and which exhibits a growth rate of at least 30% of the wild-type yeast, preferably containing a chimeric construct of at least 2 saccharide transporters, nucleic acid molecules encoding the chimeras and polypeptides encoded by such sequences, and methods of using the modified yeast for preparing products in the yeast.