Reference: Owegi MA, et al. (2006) Identification of a Domain in the Vo Subunit d That Is Critical for Coupling of the Yeast Vacuolar Proton-translocating ATPase. J Biol Chem 281(40):30001-14

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Abstract

V-ATPases proton pumps consist of two domains, V1 and Vo. Subunit d is a component of Vo located in a central stalk that rotates during catalysis. By generating mutations, we showed that subunit d couples ATP hydrolysis and proton transport. The mutation F94A strongly uncoupled the enzyme preventing proton transport but not ATPase activity. C-terminal mutations changed coupling as well; ATPase activity was decreased by 59-72% while proton transport was not measurable (E328A) or moderately reduced (E317A, C329A). Except for W325A which had low levels of V1Vo, mutations allowed wild-type assembly regardless that subunits E and d were reduced at the membrane. N- and C-terminal deletions of various lengths were inhibitory and gradually destabilized subunit d, limiting V1Vo formation. Both N- and C-terminus were required for Vo assembly. The N-terminal truncation 2-19 prevented V1Vo formation even though subunit d was available. The C-terminus was required for retention of subunits E and d at the membrane. In addition, the C-terminus of its bacterial homolog (subunit C from T. thermophilus) stabilized the yeast subunit d mutant 310-345 deletion and allowed assembly of the rotor structure with subunits A and B. Structural features conserved between bacterial and eukaryotic subunit d and the significance of domain three for V-ATPase function are discussed.

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Journal Article
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Owegi MA, Pappas DL Jr, Finch MW, Bilbo SA, Resendiz CA, Jacquemin LJ, Warrier A, Trombley JD, McCulloch KM, Margalef KL, ... Show all
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