Identification of binding proteins to the C-terminus of plasma membrane H⁺-ATPase in stomatal guard-cells

Abstract

Blue light, absorbed by phototropins, activates the plasma membrane H⁺-ATPase via phosphorylation of C-terminus and concomitant binding of 14-3-3 protein to the phosphorylated C-terminus in guard-cells. In this study, we identified the binding proteins to the C-terminus of H⁺-ATPase, by two-hybrid system using yeast-cells. The C-terminus of VHA1 (position 846-951) used as a bait, and cDNA library was constructed from guard-cells of Vicia faba. Eight clones were isolated as positive from 4.2 x 10⁷ clones. Sequence analysis showed that 5 clones were encoded unknown proteins, and that 3 clones were encoded vf14-3-3a and vf14-3-3d, which are the 14-3-3 proteins in Vicia faba. Binding affinity of vf14-3-3a to C-terminus is 4 times higher than that of vf14-3-3d. Analysis of amino acid substitution of C-terminal end showed that both vf14-3-3 required T₉₅₀ for binding. This result confirmed the previous results that 14-3-3 protein binds to Yphospho-T₉₅₀V in the C-terminal end of H⁺-ATPase.