Plant and Cell Physiology, 2001, Vol. 42, No. 4 424-432
© 2001 Oxford University Press
Analysis of the Phosphorylation Level in Guard-Cell Plasma Membrane H+-ATPase in Response to Fusicoccin
Department of Biology, Faculty of Sciences, Kyushu University, Ropponmatsu, Fukuoka, 810-8560 Japan
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.
1 Corresponding author: E-mail, kenrcb@mbox.nc.kyushu-u.ac.jp; Fax, +81-92-726-4758.
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