GLUCOSE-INDEPENDENT INHIBITION OF YEAST PLASMA-MEMBRANE H-ATPASE BY CALMODULIN ANTAGONISTS()

Glucose metabolism causes activation of the yeast plasmamembrane H+-AT Pase. The molecular mechanism of this, regulation is not known, but it is probably mediated by phosphorylation of the enzyme. The involvemen t in this process of several kinases has been suggested but their actu al role has not been proved. The physiological role of a calmodulin-de pendent protein kinase in glucose-induced activation was investigated by studying the effect of specific calmodulin antagonists on the gluco se-induced ATPase kinetic changes in wild-type and two mutant strains affected in the glucose regulation of the enzyme. Preincubation of the cells with calmidazolium or compound 48/80 impeded the increase in AT Pase activity by reducing the V-max of the enzyme without modifying th e apparent affinity for ATP in the three strains. In one mutant, pma1- T912A, the putative calmodulin-dependent protein kinase-phosphorylatab le Thr-912 was eliminated, and in the other, pma1-P536L, H+-ATPase was constitutively activated, suggesting that the antagonistic effect was not mediated by a calmodulin-dependent protein kinase and not related to glucose regulation. This was corroborated when the in vitro effect of the calmodulin antagonists on H+-ATPase activity was tested. Purif ied plasma membranes from glucose-starved or glucose-fermenting cells from both pma1-P890X, another constitutively activated ATPase mutant, and wild-type strains were preincubated with calmidazolium or melittin , In all cases, ATP hydrolysis was inhibited with an IC50 of approxima te to 1 mu M. This inhibition was reversed by calmodulin. Analysis of the calmodulin-binding protein pattern in the plasma-membrane fraction eliminates ATPase as the calmodulin target protein, We conclude that H+-ATPase inhibition by calmodulin antagonists is mediated by an as ye t unidentified calmodulin-dependent membrane protein.