Functional complementation of yeast vma1 Delta cells by a plant subunit A homolog rescues the mutant phenotype and partially restores vacuolar H+-ATPase activity

The ability of a vacuolar H+-ATPase (V-ATPase) subunit homolog (subunit A) from plants to rescue the vma mutant phenotype of yeast was investigated as a first step towards investigating the structure and function of plant sub units in molecular detail. Heterologous expression of cotton cDNAs encoding near-identical isoforms of subunit A in mutant vma id yeast cells successf ully rescued the mutant vma phenotype, indicating that subunit A of plants and yeast have retained elements essential to V-ATPases during the course o f evolution. Although vacuoles become acidified, the plant-yeast hybrid hol oenzyme only partially restored V-ATPase activity (approximately 60%) in mu tant yeast cells. Domain substitution of divergent N- or C-termini only sli ghtly enhanced V-ATPase activity, whereas swapping both domains acted syner gistically, increasing coupled ATP hydrolysis and proton translocation by a pproximately 22% relative to the native plant subunit. Immunoblot analysis indicated that similar amounts of yeast, plant or plant-yeast chimeric subu nits are membrane-bound. These results suggest that subunit A terminal doma ins contain structural information that impact V-ATPase structure and funct ion.