Mutational analysis of the interaction between 14-3-3 proteins and plant plasma membrane H+-ATPase

In this study, we report on mutational studies performed to investigate the mechanism of binding of 14-3-3 proteins to the plasma membrane H+-ATPase of plant cells. In fact, although the molecular basis of the interaction between 14-3-3 and the known mode-1 and mode-2 consensus sequences are well characterized, no information is available regarding the association with the H+-ATPase, which contains the novel binding site YTV totally unrelated to the 14-3-3 canonical motifs. To this purpose, different mutants of the maize 14-3-3 GF14-6 isoform were produced and used in interaction studies with the plasma membrane H+-ATPase and with a peptide reproducing the 14-3-3 binding site of the enzyme. The ability of 14-3-3 mutants to stimulate H+-ATPase activity was also tested. To investigate the mechanism of fusicoccin-dependent interaction, binding experiments between 14-3-3 proteins and mutants of the extreme portion of the H+-ATPase C terminus were also carried out. The results demonstrate that mutations of Lys56 and Val185 within the amphipathic groove disrupt the ability of GF14-6 to interact with H+-ATPase and to stimulate its activity. Moreover, substitution of Asp938 and Asp940 in the MHA2 H+-ATPase C terminus greatly decreased association with GF14-6, thereby demonstrating a crucial role of negatively charged residues in the fusicoccin-dependent interaction.