Phosphorylation of Thr-948 at the C terminus of the plasma membrane H+-ATPase creates a binding site for the regulatory 14-3-3 protein

The plant plasma membrane H+-ATPase is activated by the binding of 14-3-3 p rotein to the C-terminal region of the enzyme, thus forming an H+-ATPase-14 -3-3 complex that can be stabilized by the fungal toxin fusicoccin, A novel 14-3-3 binding motif, QQXYpT(948)V, at the C terminus of the H+-ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H+-ATP ase is identified. A synthetic peptide that corresponds to the C-terminal 1 6 amino acids of the H+-ATPase and that is phosphorylated on Thr-943 preven ts the in vitro activation of the H+-ATPase that is obtained in the presenc e of recombinant 14-3-3 and fusicoccin, Furthermore, binding of 14-3-3 to t he H+-ATPase in the absence of fusicoccin is absolutely dependent on the ph osphorylation of Thr-948, whereas binding of 14-3-3 in the presence of fusi coccin occurs independently of phosphorylation but still involves the C-ter minal motif YTV, Finally, by complementing yeast that lacks its endogenous H+-ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H+-ATPase isoform PMA2, we provide physiological evidence for the importanc e of the phosphothreonine motif in 14-3-3 binding and, hence, in the activa tion of the H+-ATPase in vivo. Indeed, replacing Thr-943 in the plant H+-AT Pase with alanine is lethal because this mutant fails to functionally repla ce the yeast H+-ATPase. Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H+-ATPase activity in the plant and thus for plant growth.