A Phosphopeptide Corresponding to the Cytosolic Stretch Connecting Transmembrane Segments 8 and 9 of the Plasma Membrane H~+-ATPase Binds 14-3-3 Proteins and Inhibits Fusicoccin-Induced Activation of the H~+-ATPase

摘要

A putative consensus domain for binding of 14-3-3 proteins to the plasma membrane (PM) H~+-ATPase was identified in the higher-conserved sequence RSR(p)SWSF [where (p)S is Ser776 of the maize isoform MHA2], localized in the cytosolic stretch connecting transmembrane segments 8 and 9. A 15 amino acid biotinylated phosphopeptide comprising this motif: i) bound a recombinant 14-3-3 protein, ii) inhibited fusicoccininduced stimulation of the PM H~+-ATPase activity both in PM isolated from germinating radish (Raphanus sativus L.) seedlings and in ER isolated from Saccharomyces cerevisiae expressing AHA1 (an isoform of Arabidopsis thaliana PM H~+-ATPase), and iii) inhibited fusicoccin binding to PM isolated from germinating radish seedlings. The corresponding non-phosphorylated peptide was inactive in all the performed assays. Together, these results suggest that the cytosolic strand connecting transmembrane segments 8 and 9 of the PM H~+-ATPase is a 14-3-3 binding site which might cooperate with the C-terminal domain of the enzyme in generating a stable association between the H~+-ATPase and 14-3-3 protein.