The Plant Plasma Memrbane H+-ATPase: regulation by phosphorylation and 14-3-3 proteins

Abstract: The plant plasma membrane H+-ATPase is a predominant membrane enzyme that provides the energy for secondary active transport across the plasma membrane. Consequently, the H+-ATPase is thought to play a major role in many cell processes, and it is implicated to be regulated by a number of factors, including hormones, blue light, and fungal toxins The plant plasma membrane H+-ATPase is regulated via an autoinhibitory domain located within the C-terminal region of the enzyme. Removal or displacement of this regulatory domain results in an activated enzyme with a lower Km, a higher Vmax, a more alkaline pH optimum, and an improved coupling between ATP hydrolysis and proton pumping. Characterization of the H+-ATPase activities of leaf and root plasma membranes from tobacco (Nicotiana tabacum) revealed a difference in the activation state of the enzymes in the two organs. This discovery led to the suggestion that there are at least two regulatory sites within the C-terminal autoinhibitory domain, one regulating Vmax, and another regulating Km and pH optimum. The fungal toxin fusicoccin activates the H+-ATPase by a mechanism involving a displacement of the C-terminal autoinhibitory domain. We have shown that the fusicoccin ¨receptor¨, a 14-3-3 protein, binds directly to the C-terminal region of the H+-ATPase and that fusicoccin stabilizes a 14-3-3/H+-ATPase complex, which represents the activated state of the enzyme. 14-3-3 proteins bind to phosphorylated motifs in their target proteins. In vivo phosphorylation of the plasma membrane H+-ATPase from spinach leaves in the presence of fusicoccin made it possible to identify a phosphorylated amino acid in the C terminus. The phosphorylation of this amino acid residue, Thr-948, the penultimate amino acid in the C terminus, was protected by the fusicoccin-dependent binding of 14-3-3 to the C terminus. Characterization of this novel 14-3-3 binding motif, QQXYpT948V, revealed that phosphorylation of Thr-948 is a prerequisite for binding of 14-3-3. Moreover, we could show that the fusicoccin-dependent 14-3-3 binding occurs independently of phosphorylation but still involves the three ultimate amino acids, YT948V. Finally, we demonstrate that the phosphothreonine motif is important for the binding of 14-3-3, and hence in the activation the H+-ATPase, also in vivo. Taken together our data show that 14-3-3 is a natural ligand of the H+-ATPase regulating H+-pumping, that phosphorylation of Thr-948 is a prerequisite for 14-3-3 binding, and that fusicoccin replaces the need for phosphorylation of Thr-948.