PHOSPHORYLATION OF YEAST PLASMA-MEMBRANE H-ATPASE BY CASEIN KINASE-I()

The plasma membrane H+-ATPase of Saccharomyces cerevisiae is subject t o phosphorylation by a casein kinase I activity in vitro. We show this casein kinase I activity to result from the combined function of YCK1 and YCK2, two highly similar and plasma membrane-associated casein ki nase I homologues. First, H+-ATPase phosphorylation is severely impair ed in the plasma membrane of YCK-deficient yeast strains. Furthermore, the wild-type level of the phosphoprotein is restored by the addition of purified mammalian casein kinase I to the mutant membranes. We use d the H+-ATPase as web as a synthetic peptide substrate that contains a phosphorylation site for casein kinase I to compare kinase activity in membranes prepared from yeast cells grown in the presence or absenc e of glucose, The addition of glucose results in increased H+-ATPase a ctivity which is associated with a decline in the phosphorylation leve l of the enzyme. Mutations in both YCK1 and YCK2 affect this regulatio n, suggesting that H+; ATPase activity is modulated by glucose via a c ombination of a ''down-regulating'' casein kinase I activity and anoth er, yet uncharacterize, ''up-regulating'' kinase activity. Biochemical mapping of phosphorylated H+- ATPase identifies a major phosphopeptid e that contains a consensus phosphorylation site (Ser-507) for casein kinase I. Site-directed mutagenesis of this consensus sequence indicat es that Glu-504 is important for glucose-induced decrease in the appar ent K-m for ATP.