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Proc Natl Acad Sci U S A. 2005 Aug 16;102(33):11675-80. Epub 2005 Aug 4.

Activation of the plant plasma membrane H+-ATPase by phosphorylation and binding of 14-3-3 proteins converts a dimer into a hexamer.

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Unité de Biochimie Physiologique, Institut des Sciences de la Vie, University of Louvain, Croix du Sud, 2-20, B-1348 Louvain-la-Neuve, Belgium.


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.

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