Effect of Mutations in the Extracytosolic Domain of the Saccharomyces cerevisiae H⁺-ATPase on Its Activity and Regulation

Abstract—

Plasma membrane H⁺-ATPase (PMA1), the key enzyme of yeast metabolism, undergoes multiple phosphorylation during biogenesis and functioning. In the course of this process, the ATPase is getting activated (glucose effect). We have employed site-directed mutagenesis to determine the functional role of potentially phosphorylable amino acid residues located in the extracytosolic L9-10 loop (846-SENWTD). The mutant enzyme forms were expressed at the plasma membrane to examine the effect of substitutions on biogenesis and ATPase activity. Immunobloting revealed that the mutant ATPase expression was not significantly impaired. In the absence of glucose, basal activity of the mutant enzymes differed insignificantly from that of the wild type. At the same time, regulation of the mutant E847A, T850A, and D851A enzymes has been impaired; the level of enzyme activation by glucose was lower by 2.0−2.5-fold. On the contrary, the S846A mutant displayed elevated basal activity, maintaining the ability to undergo further activation. These data indicate that these residues (especially, Ser-846, Thr-850, and Asp-851) are essential for the normal functioning of the PMA1 and its regulation by glucose.