title: Activation of the plant plasma membrane H+-ATPase by phosphorylation and binding of 14-3-3 proteins converts a dimer into a hexamer.
abstract: 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.
authors.affiliation: Unité de Biochimie Physiologique, Institut des Sciences de la Vie, University of Louvain, Croix du Sud, 2-20, B-1348 Louvain-la-Neuve, Belgium.
authors.forename: Justyna
authors.initials: J
authors.lastname: Kanczewska
authors.forename: Sergio
authors.initials: S
authors.lastname: Marco
authors.forename: Caroline
authors.initials: C
authors.lastname: Vandermeeren
authors.forename: Olivier
authors.initials: O
authors.lastname: Maudoux
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Rigaud
authors.forename: Marc
authors.initials: M
authors.lastname: Boutry
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects; D006027:Glycosides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics
chemical_list: D048948:14-3-3 Proteins; D003470:Culture Media; D006027:Glycosides; D010769:Phosphothreonine; C007808:fusicoccin; D006180:Proton-Translocating ATPases