Vacuolar proton-translocating ATPase pumps consist of two domains, V(1) and V(o). Subunit d is a component of V(o) 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 V(1)V(o), 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 V(1)V(o) formation. Both N and C terminus were required for V(o) assembly. The N-terminal truncation 2-19Delta prevented V(1)V(o) 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 310-345Delta 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.