Fusicoccin Effect on the in Vitro Interaction between Plant 14-3-3 Proteins and Plasma Membrane H+-ATPase

Abstract

A 17-amino acid peptide was selectively cleaved from the highly variant C terminus of the 33-kDa 14-3-3 isoform occurring in fusicoccin receptor preparations from maize and was sequenced. The determined C-terminal sequence was identical to that of the already known maize 14-3-3 homolog GF14-6, thus prompting the use of recombinant GF14-6 in an in vitro protein-protein interaction study. The cDNA of GF14-6 was expressed in Escherichia coli as a 32 P-phosphorylatable glutathione S-transferase fusion protein and was used as a probe in overlay experiments with H ؉ -ATPase partially purified from maize roots. The results demonstrated that the recombinant protein specifically bound to H ؉ -ATPase. The binding was dependent on Mg 2؉ and was strongly increased by fusicoccin. Controlled trypsin digestion of H ؉ -ATPase abolished the association with GF14-6, a finding that was suggestive of an interaction with the C terminus of the enzyme. To confirm this result, the Cterminal domain of H ؉ -ATPase was expressed as a glutathione S-transferase fusion peptide and was used in overlay experiments. GF14-6 was also able to bind to the isolated C terminus, but only in the presence of fusicoccin.

Blue light activates the plasma membrane H+-ATPase by phosphorylation of the C-terminus in stomatal guard cells

“…Recent investigations indicate that the H ϩ -ATPase-14-3-3 protein complex is stabilized by FC, and that the complex maintains the high-activity state of H ϩ -ATPase (Jahn et al, 1997;Baunsgaard et al, 1998;Fullone et al, 1998;Olsson et al, 1998;Piotrowski et al, 1998, Sze et al, 1999. Therefore, in guard cells, it is also likely that the H ϩ -ATPase-14-3-3 complex acts as a high-activity state of H ϩ -ATPase.…”

“…The increase in the amount of co-precipitated 32 kDa protein was suppressed through the inhibition of protein phosphorylation in the presence of K-252a ( Figure 8A). Recent reports using FC indicate that a 14-3-3 protein associates directly with the C-terminus of H ϩ -ATPase and activates the H ϩ -ATPase, and that the molecular mass of this protein is 30 kDa (Jahn et al, 1997;Oecking et al, 1997;Baunsgaard et al, 1998;Fullone et al, 1998;Sze et al, 1999). Thus it is possible that the 32 kDa protein is the 14-3-3 protein.…”

“…Protein-protein interaction was analysed by far Western blotting according to Fullone et al (1998) with modifications. Immunoprecipitated H ϩ -ATPase or GCP proteins were subjected to SDS-PAGE and electroblotted onto nitrocellulose membranes.…”

“…Recent studies have demonstrated that the 14-3-3 protein interacts directly with the C-terminal autoinhibitory domain of H ϩ -ATPase as a positive modulator and that the complex of H ϩ -ATPase and 14-3-3 protein is stabilized by FC (Jahn et al, 1997;Oecking et al, 1997;Fullone et al, 1998). The 14-3-3 protein may act as a natural ligand of H ϩ -ATPase, and interaction between the C-terminus of H ϩ -ATPase and 14-3-3 protein can be a physiological regulatory mechanism of H ϩ -ATPase activity (Baunsgaard et al, 1998).…”

14-3-3 Proteins Are Constituents of the Insoluble Glycoprotein Framework of the Chlamydomonas Cell Wall

“…The underlined residues are different from the previously cloned 30-kD 14-3-3 isoform. (Oecking et al, 1994;Baunsgaard et al, 1998;Fullone et al, 1998). Furthermore, 14-3-3 proteins occur in chloroplasts and plant mitochondria, where they regulate ATP synthase activities (Bunney et al, 2001;Aducci et al, 2002;Jarvis and Soll, 2002;Soll, 2002).…”