Topology and target interaction of the fusicoccin-binding 14-3-3 homologs 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 fusicoccin (FC) activates the H+- ATPase by hindering the inhibitory interaction of the enzyme's C-terminus with its catalytic site. Protease protection experiments carried out with sealed PM vesicles of different orientation proved that the FC-binding site faces the cytoplasmic surface of the membrane. The in vivo induced activation of the H+-ATPase by FC was retained during solubilization of PM proteins. Two-dimensional gel systems combining a native separation of membrane protein complexes with a denaturing dimension as well as highperformance anion-exchange chromatography proved the existence of a labile ATPase:14-3-3 complex in plasma membranes. Stabilization of this complex could be achieved by FC treatment in vivo or in vitro. Mild proteolytic removal of the C-terminal auto-inhibitory domain of the H(+)ATPase liberated apparent hydrophobic 14-3-3 isoforms from the membrane in soluble form. During size exclusion chromatography of the proteolytically released proteins, co-elution of 14-3-3 dimers, protein-bound FC and the C-terminus 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 complex between a 14-3-3 dimer and the H+-ATPase whose formation is part of a mechanism regulating ATPase-activity under physiological conditions. In our working model, binding of FC stabilizes this labile complex, thus leading to a strong and persistent activation of the H+-ATPase in vivo. The possibility that the C-terminus of the enzyme represents the binding domain for 14-3-3 homologs is discussed.