Identification of a domain in the Vo subunit d that is critical for coupling of the yeast vacuolar proton-translocating ATPase

Vacuolar proton-translocating ATPase pumps consist of two domains, V-1 and V-o. 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%, whereas proton transport was not measurable (E328A) or was moderately reduced (E317A and C329A). Except for W325A, which had low levels of V1Vo, mutations allowed wild-type assembly regardless of the fact 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 Delta prevented V1Vo formation, although 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 Delta 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 3 for vacuolar proton-translocating ATPase function are discussed.