phosphothreonine and fusicoccin

Plant plasma membrane H+-ATPases (PMAs) can be activated by phosphorylation of their penultimate residue (a Thr) and the subsequent binding of regulatory 14-3-3 proteins. Although 14-3-3 proteins usually exist as dimers and can bind two targets, the in vivo effects of their binding on the quaternary structure of H+-ATPases have never been examined. To address this question, we used a Nicotiana tabacum cell line expressing the Nicotiana plumbaginifolia PMA2 isoform with a 6-His tag. The purified PMA2 was mainly nonphosphorylated and 14-3-3-free, and it was shown by blue native gel electrophoresis and chemical cross-linking to exist as a dimer. Fusicoccin treatment of the cells resulted in a dramatic increase in Thr phosphorylation, 14-3-3 binding, and in vivo and in vitro ATPase activity, as well as in the conversion of the dimer into a larger, possibly hexameric, complex. PMA2 phosphorylation and 14-3-3 binding were observed also when cells in stationary growth phase were metabolically activated by transfer to fresh medium. When expressed in yeast, PMA2 was also phosphorylated and formed a complex with 14-3-3 proteins without requiring fusicoccin; no complex was observed when phosphorylation was prevented by mutagenesis. Single-particle analysis by cryoelectron microscopy showed that the PMA2-14-3-3 complex is a wheel-like structure with a 6-fold symmetry, suggesting that the activated complex consists of six H+-ATPase molecules and six 14-3-3 molecules.

Topics: 14-3-3 Proteins; Cell Line; Cell Membrane; Culture Media; Dimerization; Enzyme Activation; Glycosides; Hydrogen-Ion Concentration; Microscopy, Electron; Nicotiana; Phosphorylation; Phosphothreonine; Protein Binding; Protein Structure, Quaternary; Proton-Translocating ATPases; Saccharomyces cerevisiae

Several authors previously showed that the interaction between 14-3-3 proteins and plasma membrane H(+)-ATPase leads to an activated complex in which the enzyme is endowed with more favorable kinetic parameters and a more physiological pH optimum. In this paper we report immunological studies with antibodies covering a different specific region of the protein, including the N- and the C-terminal ends. The results showed that, beside a free and a complexed form, a third form of H(+)-ATPase in the cell must exist with low activity and no more activation due to the loss of a part of the C-terminal regulatory domain. A model in which 14-3-3 proteins activate H(+)-ATPase by protecting it from a specific proteolytic attack is presented and its generalization is discussed.

Topics: 14-3-3 Proteins; Amino Acid Sequence; Arsenicals; Blotting, Western; Cell Membrane; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Inhibitors; Ethylmaleimide; Glycosides; Glycosylation; Hydrogen-Ion Concentration; Immunoblotting; Models, Biological; Molecular Sequence Data; Phosphorylation; Phosphothreonine; Phosphotyrosine; Plant Proteins; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; Proton-Translocating ATPases; Sequence Homology, Amino Acid; Tyrosine 3-Monooxygenase

We have isolated the plasma membrane H+-ATPase in a phosphorylated form from spinach (Spinacia oleracea L.) leaf tissue incubated with fusicoccin, a fungal toxin that induces irreversible binding of 14-3-3 protein to the C terminus of the H+-ATPase, thus activating H+ pumping. We have identified threonine-948, the second residue from the C-terminal end of the H+-ATPase, as the phosphorylated amino acid. Turnover of the phosphate group of phosphothreonine-948 was inhibited by 14-3-3 binding, suggesting that this residue may form part of a binding motif for 14-3-3. This is the first identification to our knowledge of an in vivo phosphorylation site in the plant plasma membrane H+-ATPase.

Topics: 14-3-3 Proteins; Amino Acid Sequence; Cell Membrane; Glycosides; Phosphorus Radioisotopes; Phosphorylation; Phosphothreonine; Protein Binding; Proteins; Proton-Translocating ATPases; Sequence Homology, Amino Acid; Spinacia oleracea; Tyrosine 3-Monooxygenase