Topology and target interaction of the fusicoccin‐binding 14‐3‐3 homologs of Commelina communis

Abstract

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 high‐performance 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.