Fusicoccin effect on the in vitro interaction between plant 14-3-3 proteins and plasma membrane H+-ATPase.
A 17-amino acid peptide was selectively cleaved from the highly variant C terminus of the 33-kDa 14-3-3 isoform occurring in fusicoccin receptor preparations from maize and was sequenced. The determined C-terminal sequence was identical to that of the already known maize 14-3-3 homolog GF14-6, thus prompting the use of recombinant GF14-6 in an in vitro protein-protein interaction study. The cDNA of GF14-6 was expressed in Escherichia coli as a 32P-phosphorylatable glutathione S-transferase fusion protein and was used as a probe in overlay experiments with H+-ATPase partially purified from maize roots. The results demonstrated that the recombinant protein specifically bound to H+-ATPase. The binding was dependent on Mg2+ and was strongly increased by fusicoccin. Controlled trypsin digestion of H+-ATPase abolished the association with GF14-6, a finding that was suggestive of an interaction with the C terminus of the enzyme. To confirm this result, the C-terminal domain of H+-ATPase was expressed as a glutathione S-transferase fusion peptide and was used in overlay experiments. GF14-6 was also able to bind to the isolated C terminus, but only in the presence of fusicoccin.
Fullone
M R
MR
Dipartimento di Scienze Biochimiche "A. Rossi Fanelli," Università di Roma "La Sapienza," Piazzale Aldo Moro 5, I-00185 Roma, Italy.
Visconti
S
S
Marra
M
M
Fogliano
V
V
Aducci
P
P
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006027:Glycosides / Q000494:pharmacology*; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011961:Receptors, Fc / Q000737:chemistry; D011994:Recombinant Proteins; D014446:Tyrosine 3-Monooxygenase; D003313:Zea mays
D048948:14-3-3 Proteins; D004268:DNA-Binding Proteins; C091848:GRF1 protein, Zea mays; D006027:Glycosides; D010940:Plant Proteins; D011506:Proteins; D011961:Receptors, Fc; D011994:Recombinant Proteins; C007808:fusicoccin; D014446:Tyrosine 3-Monooxygenase; D006180:Proton-Translocating ATPases; D008274:Magnesium