TOPOLOGY AND TARGET INTERACTION OF THE FUSICOCCIN-BINDING 14-3-3-HOMOLOG OF COMMELINA-COMMUNIS

Upon binding to a high affinity plasma membrane (PM) protein (a member of the 14-3-3 family of regulatory proteins), the fungal phytotoxin f usicoccin (FC) activates the H+- ATPase by hindering the inhibitory in teraction of the enzyme's C-terminus with its catalytic site. Protease protection experiments carried out with sealed PM vesicles of differe nt orientation proved that the FC-binding site faces the cytoplasmic s urface of the membrane. The in vivo induced activation of the H+-ATPas e by FC was retained during solubilization of PM proteins. Two-dimensi onal gel systems combining a native separation of membrane protein com plexes with a denaturing dimension as well as highperformance anion-ex change chromatography proved the existence of a labile ATPase:14-3-3 c omplex in plasma membranes. Stabilization of this complex could be ach ieved by FC treatment in vivo or in vitro. Mild proteolytic removal of the C-terminal auto-inhibitory domain of the H(+)ATPase liberated app arent hydrophobic 14-3-3 isoforms from the membrane in soluble form. D uring size exclusion chromatography of the proteolytically released pr oteins, co-elution of 14-3-3 dimers, protein-bound FC and the C-termin us of the H(+)ATPase was observed. Moreover, the data suggest that 14- 3-3 dimers themselves are not able to bind FC. Based on these results, it is proposed that the 'FC receptor' is represented by a labile comp lex between a 14-3-3 dimer and the H+-ATPase whose formation is part o f a mechanism regulating ATPase-activity under physiological condition s. In our working model, binding of FC stabilizes this labile complex, thus leading to a strong and persistent activation of the H+-ATPase i n vivo. The possibility that the C-terminus of the enzyme represents t he binding domain for 14-3-3 homologs is discussed.