Published:
American Society of Plant Physiologists, 1999
Published in:
The Plant Cell, 11 (1999) 12, Seite 2379-2391
Language:
English
ISSN:
1040-4651
Origination:
Footnote:
Description:
The plant plasma membrane H<sup>+</sup>- ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an <tex-math>${\rm H}^{+}\text{-}{\rm ATPase}-14\text{-}3\text{-}3$</tex-math> complex that can be stabilized by the fungal toxin fusicoccin. A novel 14-3-3 binding motif, <tex-math>${\rm QQXYpT}_{948}{\rm V}$</tex-math>, at the C terminus of the H<sup>+</sup>- ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H<sup>+</sup>- ATPase is identified. A synthetic peptide that corresponds to the C-terminal 16 amino acids of the H<sup>+</sup>- ATPase and that is phosphorylated on Thr-948 prevents the in vitro activation of the H<sup>+</sup>- ATPase that is obtained in the presence of recombinant 14-3-3 and fusicoccin. Furthermore, binding of 14-3-3 to the H<sup>+</sup>- ATPase in the absence of fusicoccin is absolutely dependent on the phosphorylation of Thr-948, whereas binding of 14-3-3 in the presence of fusicoccin occurs independently of phosphorylation but still involves the C-terminal motif YTV. Finally, by complementing yeast that lacks its endogenous H<sup>+</sup>- ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H<sup>+</sup>- ATPase isoform PMA2, we provide physiological evidence for the importance of the phosphothreonine motif in 14-3-3 binding and, hence, in the activation of the H<sup>+</sup>- ATPase in vivo. Indeed, replacing Thr-948 in the plant H<sup>+</sup>- ATPase with alanine is lethal because this mutant fails to functionally replace the yeast H<sup>+</sup>- ATPase. Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H<sup>+</sup>- ATPase activity in the plant and thus for plant growth.