Conservation and release of osmolytes by yeasts during hypo-osmotic stress.

In response to fluctuations in environmental osmolarity, yeast cells adjust their intracellular solute concentrations in order to maintain a constant turgor pressure and ensure continuation of cellular activity. In this study, the effect of hypo-osmotic stress on osmolyte content of osmotolerant yeasts Zygosaccharomyces rouxii and Pichia sorbitophila and the less tolerant Saccharomyes cerevisiae was investigated. All these yeasts released glycerol upon hypo-osmotic shock. However, Z. rouxii also released arabitol, whereas P. sorbitophila released erythritol in addition to arabitol and glycerol. Osmolyte release was very rapid and specific and was neither affected by reduced temperatures nor inhibited by the channel blocker gadolinium or the protonophore carbonyl cyanide m-chlorophenyl hydrazone. Extracellular osmolyte levels increased drastically suggesting that osmolytes were not metabolised but mainly released upon exposure to hypotonic conditions. The export process is well controlled, and the amount of osmolyte released was proportional to the shock intensity. Osmolyte release occurred with little cell lysis and thus the survival as well as the subsequent growth of yeast cells was largely unaffected after hypo-osmotic shock. The kinetics and patterns of osmolyte export suggest the involvement of channel proteins, but the molecular nature of this export pathway in yeasts, with exception of S. cerevisiae, remains to be established.

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

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 fps1delta 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 fps1delta 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.

Isolation and characterization of the TIM10 homologue from the yeast Pichia sorbitophila: a putative component of the mitochondrial protein import system.

The Saccharomyces cerevisiae TIM10 gene encodes one of the few essential mitochondrial proteins that are required for the import of nuclear-encoded precursor proteins from the cytosol and their subsequent sorting into the different mitochondrial compartments. We have isolated and characterized a putative homologue of TIM10 from the halotolerant yeast Pichia sorbitophila. The Pichia TIM10 gene encodes a protein of 90 amino acids with 66% identity to S. cerevisiae Tim10p. It was capable of suppressing the temperature sensitivity of tim10-1 mutant in S. cerevisiae, suggesting that Pichia TIM10 is both a functional and structural homologue of S. cerevisiae TIM10. The putative Pichia TIM10 gene product contains all the four conserved cysteine residues and the two CX(3)C motifs typical of the Tim family proteins in the mitochondrial intermembrane space. Using anti-Tim10p serum, Western blots detected a protein of about 10 kDa, suggesting that the Pichia Tim10p is a mitochondrial protein. The results suggest that mitochondrial import and sorting systems might be also strongly conserved in other fungi. The coding sequence of the P. sorbitophila TIM10 has been deposited in the EMBL Nucleotide Sequence Database under Accession No. AJ243940.