Cloning and characterization of genes required for osmoregulation in the yeast Saccharomyces cerevisiae

[摘要] Salt stress induces multiple physiological responses in unicellular organisms. Responses include both immediate effects upon membrane permeability to select cytoplasmic solutes and changes in gene expression. This study has sought to isolate genes required for osmoregulation in the yeast, Saccharomyces cerevisiae. Eighteen mutants, with an inability to grow under osmotically stressed conditions ($m Osmsp{s}$), and deficient in salt induced glycerol accumulation were isolated. Each of the mutants fell into one of four complementation groups hog1 through hog4 (High Osmolarity Glycerol response). Three genes, HOG1, HOG2, and HOG4, that rescued growth of representative mutants under osmotically stressed conditions were cloned and disruption alleles generated. Disruption mutants of each gene displayed $m Osmsp{S}$ growth and depressed glycerol accumulation following salt stress. Sequence analysis of HOG1 revealed striking sequence homology with mitogen-activated protein (MAP) kinases, including 50% identity with FUS3, a MAP kinase in the yeast mating response pathway. Immunoblots of normal and osmotically stressed cells detected an inducible tyrosine phosphorylation of HOG1, an event shown to activate homologous kinases. Site-directed mutagenesis of HOG1 on residues required for activation in related MAP kinases resulted in a loss of complementing activity on high salt plates and a lack of tyrosine phosphorylation of Hog1p when $m Tyrsp{176}$ was mutated to Phe. HOG4 was identified to be a previously sequenced gene, PBS2, with strong sequence homology to MAPK kinases, kinases upstream of MAP kinases and thought to be responsible for the activating phosphorylation events. Hog1p was not tyrosine phosphorylated in a pbs2 mutant consistent with PBS2 being the kinase responsible for Hog1p phosphorylation. Osmotic stress was shown to result in aberrant terminal morphology (elongate, multi-budded, multi-nucleate cells) in both hog1 and pbs2 null mutants. Further characterization of the mutants using time lapse microscopy revealed that salt stress induces bud abandonment and aberrant bud site selection. HOG1 and PBS2 therefore are two genes involved in carrying an osmostress induced signal to stimulate adaptive cellular responses and are required for normal bud site selection following salt stress.

[发布机构] Rice University

[效力级别] biology