Specific binding of vf14-3-3a isoform to the plasma membrane H+-ATPase in response to blue light and fusicoccin in guard cells of broad bean

The plasma membrane H+-ATPase is activated by blue light with concomitant b inding of the 14-3-3 protein to the C terminus in guard cells. Because seve ral isoforms of the 14-3-3 protein are expressed in plants, we determined w hich isoform(s) bound to the H+-ATPase in vivo. Four cDNA clones (vf14-3-3a , vf14-3-3b, vf14-3-3c, and vf14-3-3d) encoding 14-3-3 proteins were isolat ed from broad bean (Vicia faba) guard cells. Northern analysis revealed tha t mRNAs encoding vf14-3-3a and vf14-3-3b proteins were expressed predominan tly in guard cells. The 14-3-3 protein that bound to the H+-ATPase in guard cells had the same molecular mass as the recombinant vf14-3-3a protein. Th e H+-ATPase immunoprecipitated from mesophyll cell protoplasts, which had b een stimulated by fusicoccin, coprecipitated with the 32.5-kD 14-3-3 protei n, although three 14-3-3 isoproteins were found in mesophyll cell protoplas ts. Digestions of the bound 14-3-3 protein and recombinant vf14-3-3a with c yanogen bromide gave the identical migration profiles on sodium dodecyl sul fate-polyacrylamide gel electrophoresis, but that of vf14-3-3b gave a diffe rent profile. Mass profiling of trypsin-digested 14-3-3 protein bound to th e H+-ATPase gave the predicted peptide masses of vf14-3-3a. Far western ana lysis revealed that the H+-ATPase had a higher affinity for vf14-3-3a than for vf14-3-3b. These results suggest that the 14-3-3 protein that bound to the plasma membrane H+-ATPase in vivo is vf14-3-3a and that it may play a k ey role in the activation of H+-ATPase in guard cells.