Journal Article

Analysis of the Phosphorylation Level in Guard-Cell Plasma Membrane H<sup>+</sup>-ATPase in Response to Fusicoccin

Toshinori Kinoshita and Ken-ichiro Shimazaki

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 42, issue 4, pages 424-432
Published in print April 2001 | ISSN: 0032-0781
Published online April 2001 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pce055
Analysis of the Phosphorylation Level in Guard-Cell Plasma
Membrane  H+-ATPase in Response to
Fusicoccin

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  • Biochemistry
  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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A fungal phytotoxin fusicoccin (FC) causes irreversible opening of stomata by activation of the plasma membrane H+-ATPase in guard cells. However, the mechanism by which FC activates the H+-ATPase is not fully understood with respect to the event of phosphorylation. In this study, we provide quantitative evidence that FC-dependent activation of H+-ATPase requires the phosphorylation of the C-terminus, and that FC maintains the activated state by preventing the dephosphorylation. The plasma membrane H+-ATPase in guard cells was phosphorylated on serine and threonine residues in the C-termini of both VHA1 and VHA2 by FC, and the phosphorylation level paralleled the rates of H+-pumping and ATP hydrolysis. An endogenous 14-3-3 protein was co-precipitated with the H+-ATPase, and the amount of 14-3-3 protein was proportional to the phosphorylation level of H+-ATPase. The recombinant 14-3-3 protein bound to the C-terminus only when it was phosphorylated, even in the presence of FC. The phosphorylated C-terminus was dephosphorylated by alkaline phosphatase, and the dephosphorylation was completely prevented when the C-terminus had been incubated with both FC and 14-3-3 protein. The results suggest that FC activates the H+-ATPase by accumulating the complex of phosphorylated H+-ATPase and 14-3-3 protein through inhibition of the dephosphorylation in guard cells.

Keywords: Key words: Fusicoccin — Guard cells — H+-ATPase (EC 3.6.1.35) — 14-3-3 protein — Protein phosphorylation — Vicia faba.; Abbreviations: AP, alkaline phosphatase; CNBr, cyanogen bromide; FC, fusicoccin; GCPs, guard cell protoplasts; GST, glutathione-S-transferase.

Journal Article.  6737 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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