Molecular dissection of the C-terminal regulatory domain of the plant plasma membrane H+-ATPase AHA2: Mapping of residues that when altered give riseto an activated enzyme

The plasma membrane H+-ATPase is a proton pump belonging to the P-type ATPa se superfamily and is important for nutrient acquisition in plants, The H+- ATPase is controlled by an autoinhibitory C-terminal regulatory domain and is activated by 14-3-3 proteins which bind to this part of the enzyme. Alan ine-scanning mutagenesis through 87 consecutive amino acid residues was use d to evaluate the role of the C-terminus in autoinhibition of the plasma me mbrane H+-ATPase AHA2 from Arabidopsis thaliana. Mutant enzymes were expres sed in a strain of Saccharomyces cerevisiae with a defective endogenous H+- ATPase. The enzymes were characterized by their ability to promote growth i n acidic conditions and to promote H+ extrusion from intact cells, both of which are measures of plasma membrane H+-ATPase activity, and were also cha racterized with respect to kinetic properties such as affinity for H+ and A TP. Residues that when altered lead to increased pump activity group togeth er in two regions of the C-terminus, One region stretches from K863 to L885 and includes two residues (Q879 and R880) that are conserved between plant and fungal H+-ATPases. The other region, incorporating S904 to L919, is si tuated in an extension of the C-terminus unique to plant H+-ATPases, Altera tion of residues in both regions led to increased binding of yeast 14-3-3 p rotein to the plasma membrane of transformed cells. Taken together, our dat a suggest that modification of residues in two regions of the C-terminal re gulatory domain exposes a latent binding site for activatory 14-3-3 protein s.