SummaryUsing the two-hybrid technique we identi®ed a novel protein whose N-terminal 88 amino acids (aa) interact with the C-terminal regulatory domain of the plasma membrane (PM) H + -ATPase from Arabidopsis thaliana (aa 847±949 of isoform AHA1). The corresponding gene has been named Ppi1 for Proton pump interactor 1. The encoded protein is 612 aa long and rich in charged and polar residues, except for the extreme C-terminus, where it presents a hydrophobic stretch of 24 aa. Several genes in the A. thaliana genome and many ESTs from different plant species share signi®cant similarity (50±70% at the aa level over stretches of 200±600 aa) to Ppi1. The PPI1 N-terminus, expressed in bacteria as a fusion protein with either GST or a His-tag, binds the PM H + -ATPase in overlay experiments. The same fusion proteins and the entire coding region fused to GST stimulate H + -ATPase activity. The effect of the His-tagged peptide is synergistic with that of fusicoccin (FC) and of tryptic removal of a C-terminal 10 kDa fragment. The His-tagged peptide binds also the trypsinised H + -ATPase. Altogether these results indicate that PPI1 N-terminus is able to modulate the PM H + -ATPase activity by binding to a site different from the 14-3-3 binding site and is located upstream of the trypsin cleavage site.

No abstract

Interaction of 14-3-3 proteins with their targets depends not only on the phosphorylation status of the target but also on that of 14-3-3 (Fu et al., 2000). In this work we demonstrated that the maize 14-3-3 isoform GF14-6 is a substrate of the tyrosine kinase insulin growth factor receptor 1. By means of site-directed mutants of GF14-6, we identified Tyr-137 as the specific tyrosine residue phosphorylated by the insulin growth factor receptor 1. Phosphorylation of GF14-6 on Tyr-137 lowered its affinity for a peptide mimicking the 14-3-3 binding site of the plant plasma membrane H+-ATPase. Moreover, phosphorylation in planta of 14-3-3 tyrosine residues, resulting from incubation with the tyrosine phosphatase inhibitor, phenylarsine oxide, decreased their association to the H+-ATPase.

A putative consensus domain for binding of 14‐3‐3 proteins to the plasma membrane (PM) H+‐ATPase was identified in the highly‐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 fusicoccin‐induced 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.