One of the most abundant proteins in the yeast plasma membrane is the P-type H(+)-ATPase that pumps protons out of the cell, supplying the driving force for a wide array of H(+)-dependent cotransporters. The ATPase is a 100 kDa polypeptide, anchored in the lipid bilayer by 10 transmembrane alpha-helices. It is structurally and functionally related to the P-type Na(+),K(+)-, H(+),K(+)- and Ca(2+)-ATPases of animal cells and the H(+)-ATPases of plant cells, and it shares with them a characteristic reaction mechanism in which ATP is split to ADP and inorganic phosphate (P(i)) via a covalent beta-aspartyl phosphate intermediate. Cryoelectron microscopic images of the H(+)-ATPase of Neurospora crassa and the sarcoplasmic reticulum Ca(2+)-ATPase of animal cells have recently been obtained at 8 nm resolution. The membrane-embedded portion of the molecule, which presumably houses the cation translocation pathway, is seen to be connected via a narrow stalk to a large, multidomained cytoplasmic portion, known to contain the ATP-binding and phosphorylation sites. In parallel with the structural studies, efforts are being made to dissect structure/function relationships in several P-type ATPases by means of site-directed mutagenesis. This paper reviews three phenotypically distinct classes of mutant that have resulted from work on the yeast PMA1 H(+)-ATPase: (1) mutant ATPases that are poorly folded and retained in the endoplasmic reticulum; (2) mutants in which the conformational equilibrium has been shifted from the E(2) state, characterized by high affinity for vanadate, to the E(1) state, characterized by high affinity for ATP; and (3) mutants with altered coupling between ATP hydrolysis and proton pumping. Although much remains to be learned before the transport mechanism can be fully understood, these mutants serve to identify critical parts of the polypeptide that are required for protein folding, conformational change and H(+):ATP coupling.

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