Role of transmembrane segment M8 in the biogenesis and function of yeast plasma-membrane H+-ATPase

Guadalupe Guerra, Valery V. Petrov, Kenneth E. Allen, Manuel Miranda, Juan Pablo Pardo, Carolyn W. Slayman

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Abstract

Of the four transmembrane helices (M4, M5, M6, and M8) that pack together to form the ion-binding sites of P2-type ATPases, M8 has until now received the least attention. The present study has used alanine-scanning mutagenesis to map structure-function relationships throughout M8 of the yeast plasma-membrane H+-ATPase. Mutant forms of the ATPase were expressed in secretory vesicles and at the plasma membrane for measurements of ATP hydrolysis and ATP-dependent H+pumping. In secretory vesicles, Ala substitutions at a cluster of four positions near the extracytoplasmic end of M8 led to partial uncoupling of H+transport from ATP hydrolysis, while substitution of Ser-800 (close to the middle of M8) by Ala increased the apparent stoichiometry of H+transport. A similar increase has previously been reported following the substitution of Glu-803 by Gln (Petrov, V. et al., J. Biol. Chem. 275:15709-15718, 2000) at a position known to contribute directly to Ca2+binding in the Ca2+-ATPase of sarcoplasmic reticulum (Toyoshima, C., et al., Nature 405: 647-655, 2000). Four other mutations in M8 interfered with H+-ATPase folding and trafficking to the plasma membrane; based on homology modeling, they occupy positions that appear important for the proper bundling of M8 with M5, M6, M7, and M10. Taken together, these results point to a key role for M8 in the biogenesis, stability, and physiological functioning of the H+-ATPase. © 2007 Elsevier B.V. All rights reserved.
Original languageAmerican English
Pages (from-to)2383-2392
Number of pages10
JournalBiochimica et Biophysica Acta - Biomembranes
DOIs
StatePublished - 1 Oct 2007

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