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 binding of the 14-3-3 protein to the C terminus in guard cells. Because several isoforms of the 14-3-3 protein are expressed in plants, we determined which 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 isolated from broad bean (Vicia faba) guard cells. Northern analysis revealed that mRNAs encoding vf14-3-3a and vf14-3-3b proteins were expressed predominantly 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. The H(+)-ATPase immunoprecipitated from mesophyll cell protoplasts, which had been stimulated by fusicoccin, coprecipitated with the 32.5-kD 14-3-3 protein, although three 14-3-3 isoproteins were found in mesophyll cell protoplasts. Digestions of the bound 14-3-3 protein and recombinant vf14-3-3a with cyanogen bromide gave the identical migration profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but that of vf14-3-3b gave a different profile. Mass profiling of trypsin-digested 14-3-3 protein bound to the H(+)-ATPase gave the predicted peptide masses of vf14-3-3a. Far western analysis 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 key role in the activation of H(+)-ATPase in guard cells.
Emi
T
T
Department of Biology, Faculty of Sciences, Kyushu University, Ropponmatsu, Fukuoka 810-8560, Japan.
Kinoshita
T
T
Shimazaki
K
K
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology*; D017931:DNA Primers; D006027:Glycosides / Q000494:pharmacology*; D008027:Light; D008969:Molecular Sequence Data; D009183:Mycotoxins / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D020033:Protein Isoforms / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D006180:Proton-Translocating ATPases / Q000378:metabolism* / Q000528:radiation effects; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014446:Tyrosine 3-Monooxygenase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D031307:Vicia faba / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects
D048948:14-3-3 Proteins; D017931:DNA Primers; D006027:Glycosides; D009183:Mycotoxins; D020033:Protein Isoforms; C007808:fusicoccin; D014446:Tyrosine 3-Monooxygenase; D006180:Proton-Translocating ATPases