MOLECULAR MECHANISM OF REGULATION OF YEAST PLASMA-MEMBRANE H-ATPASE BY GLUCOSE - INTERACTION BETWEEN DOMAINS AND IDENTIFICATION OF NEW REGULATORY SITES()

The carboxyl terminus of yeast plasma membrane H+-ATPase is an autoinh ibitory domain, and its effect is counteracted by modification of the enzyme triggered by glucose metabolism (Portillo, F., Larrinoa, I. F., and Serrano, R. (1989) FEBS Lett. 247, 381-385). To identify interact ing domains involved in this regulation, we have performed intragenic suppressor analysis. A double mutation at the carboxyl terminus (S911A /T912A) results in no activation of the ATPase by glucose and lack of yeast growth on this sugar (Portillo, F., Eraso, P., and Serrano, R. ( 1991) FEBS Lett. 287, 71-74). Random in vitro mutagenesis of this muta nt ATPase gene resulted in 29 revertants. Six corresponded to full rev ertants of the initial double mutation. Fourteen suppressor (second-si te) mutations are located within three functional domains of the enzym e. Four mutations (A165V, V169I/D170N, A350T, and A351T) are localized at the cytoplasmic ends of predicted transmembrane helices 2 and 4; s ix mutations (P536L, A565T, G587N, G648S, P669L,and G670S) map within the proposed ATP binding domain, and the other four substitutions (P89 0opa, S896F, R898K, and M907I) are located at the carboxyl terminus. T hese results demonstrate the interaction, direct or indirect, between these three domains far apart in the linear sequence of the ATPase. Al l the second-site mutations caused constitutive activation of the ATPa se in the absence of glucose metabolism. Second site mutations at the carboxyl terminus were close to Ser-899 and suggested phosphorylation of this amino acid during glucose activation. Accordingly, the introdu ction of a negative charge, in a S899D mutant constructed by site-dire cted mutagenesis, partially mimics the glucose effect on the ATPase.