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 Lys(56) and Val(185) within the amphipathic groove disrupt the ability of GF14-6 to interact with H(+)-ATPase and to stimulate its activity. Moreover, substitution of Asp(938) and Asp(940) 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.
Visconti
Sabina
S
Department of Biology, University of Rome Tor Vergata, via della Ricerca Scientifica, Italy.
Camoni
Lorenzo
L
Fullone
Maria Rosaria
MR
Lalle
Marco
M
Marra
Mauro
M
Aducci
Patrizia
P
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D008239:Lysine / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014633:Valine / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology*
D048948:14-3-3 Proteins; D017931:DNA Primers; D008565:Membrane Proteins; D014446:Tyrosine 3-Monooxygenase; D006180:Proton-Translocating ATPases; D014633:Valine; D008239:Lysine