Fusicoccin binding to its plasma membrane receptor and the activation of the plasma membrane H(+)-ATPase. IV. Fusicoccin induces the association between the plasma membrane H(+)-ATPase and the fusicoccin receptor.
Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H(+)-ATPase in plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H(+)-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H(+)-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H(+)-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H(+)-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H(+)-ATPase antiserum and with an anti-14-3-3 antiserum indicated an FC-induced association of FCBP with the PM H(+)-ATPase. Analysis of the activation state of PM H(+)-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme.
Olivari
C
C
Dipartimento di Biologia, Università di Milano, Italy. fimca@imiucca.csi.unimi.it
Meanti
C
C
De Michelis
M I
MI
Rasi-Caldogno
F
F
D001937:Brassica / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D006027:Glycosides / Q000378:metabolism*; D010940:Plant Proteins; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
D006027:Glycosides; D010940:Plant Proteins; D011956:Receptors, Cell Surface; C080425:fusicoccin receptor protein, plant; C007808:fusicoccin; D006180:Proton-Translocating ATPases