Differential stabilities of phosphorylated response regulator domains reflect functional roles of the yeast osmoregulatory SLN1 and SSK1 proteins

Osmoregulation in Saccharomyces cerevisiae involves a multistep phosphorela y system requiring three proteins, SLN1, YPD1, and SSK1, that are related t o bacterial two-component signaling proteins, in particular, those involved in regulating sporulation in Bacillus subtilis and anaerobic respiration i n Escherichia coli. The SLN1-YPD1-SSK1 phosphorelay regulates a downstream mitogen-activated protein kinase cascade which ultimately controls the conc entration of glycerol within the cell under hyperosmotic stress conditions. The C-terminal response regulator domains of SLN1 and SSK1 and full-length YPD1 have been overexpressed and purified from E. coli, A heterologous sys tem consisting of acetyl phosphate, the bacterial chemotaxis response regul ator CheY, and YPD1 has been developed as an efficient means of phosphoryla ting SLN1 and SSK1 in vitro. The homologous regulatory domains of SLN1 and SSK1 exhibit remarkably different phosphorylated half-lives, a finding that provides insight into the distinct roles that these phosphorylation-depend ent regulatory domains play in the yeast osmosensory signal transduction pa thway.