Responses of yeasts to hypo-osmotic stress

English: This study examined the responses of yeasts to hypo-osmotic stress with special emphasis to osmolyte export and facilitating proteins. All yeast strains studied (Z rouxii, P. sorbitophila, S. cerevisiae and S. pombe) rapidly release their intracellular osmolytes upon a decrease in external osmolarity (osmotic downshoek or hypo-osmotic shock). Osmolyte release is very rapid, specific and is not affected at reduced temperatures neither inhibited by the channel blocker gadolinium or the protonophore CCCP. The export process is well controlled and the amount of osmolyte released is proportional to the shock intensity. Osmolyte release occurs with minimal cell lysis and thus the survival as well as the subsequent growth of yeast cells is largely unaffected after hypo-osmotic shock. The patterns and export kinetics suggested the involvement of channel proteins similar to that of Fps1p previously reported in S. cerevisiae. However, search for FPS1 homologues from other yeasts using PCR and DNA probes resulted in weak hybridization signals suggesting that the putative glycerol channel encoding genes might have low sequence similarity to FPSl. It appears that although the mechanism of osmolyte release is conserved among yeasts, the proteins involved in this release might be divergent. This finding was in contrast to the general view that most of the genes involved in glycerol metabolism and stress responses such as GPDl (NAD+-dependent glycerol-3- phosphate dehydrogenase), DAKl (dihydroxyacetone kinase) and HOGl (MAP kinase of the HOG pathway) are well conserved in all yeasts. Isolation and cloning of the corresponding genees) involved in osmolyte export will shed more light on the molecular nature and physiological roles of these exporters. In yeast, osmolyte transport across the cell membrane occurs via active transport, mediated by channel proteins and by passive diffusion. The extent to which osmolytes permeates the cell membrane may then be influenced by the membrane lipid composition. In this study, the role of ergosterol (the most abundant sterol in yeast membranes) in osmolyte release and survival of yeast cells during hypo-osmotic stress was investigated. Cells lacking a glycerol facilitator (the fps1∆ strain) grow very poorly upon an osmotic downs hock, but apparently survived the shock better and recovered more rapidly if ergosterol was supplied. Furthermore, the rate and amount of glycerol release was markedly enhanced in the fps1∆ mutant when exogenous ergosterol was supplied. The erg-l disruption mutant which is unable to synthesise ergosterol, survived and recovered from the osmotic shock more successfully at the higher ergosterol concentration. Although the mechanism by which ergosterol improves glycerol release and survival of yeast cells is not well understood, it could presumably be related to the membrane stabilizing effects of ergosterol and the associated improvements in membrane fluidity. The polyene antibiotic nystatin, which affects membrane permeability in an ergosterol dependent way, caused S. cerevisiae cells to release a large amount of glycerol and equally inhibited the growth of wild-type and fsp1 deletion strains in medium containing 5% (w/v) Nael. This study demonstrated the role of ergosterol in glycerol efflux and survival in S. cerevisiae after an osmotic downshock and provided additional evidence for the significance of membrane permeability and glycerol con servation in yeast osmoregulation. The ability to regulate water and solute flux across cell membranes is critical in ensuring a constant turgor pressure as well as the proper functioning of biochemical processes. In most organisms, this process appears to be mediated by the MIP family transmembrane channel proteins, most of which have been characterized in higher animals. An in silicio phylogenetic analysis of microbial MIP channels revealed two major groups, the glycerol facilitators and the water channels (aquaporins), but further divided the glycerol facilitators into two subfamilies. Water channels seem to be important for growth after drastic changes in medium osmolarity, especially to lower osmolarity. Glycerol facilitators appear to exist in all microbial groups where they function in the uptake of glycerol and related compounds for their catabolism. The S. cerevisiae glycerol facilitator has been shown to be involved in osmoregulation by controlling the accumulation and release of glycerol. The occurrence of glycerol facilitators in other yeasts and their role in osmoregulation were investigated in this study. Blast searches in the S. pombe data bases revealed three putative glycerol transport proteins one of which shows considerably structural similarities to known MIP family glycerol facilitators. However, heterologous expression and subsequent functional analysis of this S. pombe mip l did not indicate its involvement in glycerol transport across the plasma membrane. It is still unknown whether the protein is involved in glycerol transport across other organelle membranes or whether it is involved in transport of a yet unidentified solute. The expression of S. pombe mip l is induced by osmotic stress suggesting a role in osmoregulation. However, deletion of S. pombe mip I does not cause any observable effects on growth of S. pombe cells during osmotic stress. Therefore, the physiological role of the S. pombe mipl as well as the actual transporter(s) controlling glycerol flux in S. pombe remains to be elucidated.