Stalk segment 5 of the yeast plasma membrane H+-ATPase - Mutational evidence for a role in glucose regulation

In P-2-type ATPases, a stalk region connects the cytoplasmic part of the mo lecule, which binds and hydrolyzes ATP, to the membrane-embedded part throu gh which cations are pumped. The present study has used cysteine scanning m utagenesis to examine structure-function relationships within stalk segment 5 (S5) of the yeast plasma-membrane H+-ATPase. Of 29 Cys mutants that were made and examined, two (G670C and R682C) were blocked in biogenesis, presu mably due to protein misfolding, In addition, one mutant (S681C) had very l ow ATPase activity, and another (F685C) displayed a 40-fold decrease in sen sitivity to orthovanadate, reflecting a shift in equilibrium from the E-2 c onformational state toward E-1. By far the most striking group of mutants ( F666C, L671C, I674C, A677C, I684C, R687C, and Y689C) were constitutively ac tivated even in the absence of glucose, with rates of ATP hydrolysis and ki netic properties normally seen only in glucose-metabolizing cells. Previous work has suggested that activation of the wild-type H+-ATPase results from kinase-mediated phosphorylation in the auto-inhibitory C-terminal region o f the 100-kDa polypeptide, The seven residues identified in the present stu dy are located on one face of the S5 alpha -helix, consistent with the idea that mutations along this face serve to release the auto inhibition.