Reference: Eraso P and Portillo F (1994) Molecular mechanism of regulation of yeast plasma membrane H(+)-ATPase by glucose. Interaction between domains and identification of new regulatory sites. J Biol Chem 269(14):10393-9

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Abstract


The carboxyl terminus of yeast plasma membrane H(+)-ATPase is an autoinhibitory 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 interacting 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 mutant ATPase gene resulted in 29 revertants. Six corresponded to full revertants of the initial double mutation. Fourteen suppressor (second-site) mutations are located within three functional domains of the enzyme. Four mutations (A165V, V169I/D170N, A350T, and A351T) are localized at the cytoplasmic ends of predicted transmembrane helices 2 and 4; six mutations (P536L, A565T, G587N, G648S, P669L, and G670S) map within the proposed ATP binding domain, and the other four substitutions (P890opa, S896F, R898K, and M907I) are located at the carboxyl terminus. These results demonstrate the interaction, direct or indirect, between these three domains far apart in the linear sequence of the ATPase. All the second-site mutations caused constitutive activation of the ATPase 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 introduction of a negative charge, in a S899D mutant constructed by site-directed mutagenesis, partially mimics the glucose effect on the ATPase.

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Journal Article | Research Support, Non-U.S. Gov't
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Eraso P, Portillo F
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