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.

Summary

“…In plants, pretreatment of intact tissue with fusicoccin results in an increased amount of 14-3-3 proteins associated with the plasma membrane after fractionation (Korthout and de Boer, 1994;Oecking et al, 1994Oecking et al, , 1997. The same phenomenon was observed in yeast, but only in yeast expressing full-length AHA2 (Figure 3).…”

“…Interestingly, expression of the deletion mutant aha2∆92 also failed to create fusicoccin binding sites (Figure 2). Apparently, the C-terminus of the pump is not only essential for autoinhibition (Regenberg et al, 1995) and for the interaction with 14-3-3 proteins (Jahn et al Oecking et al, 1997), but also for the formation of a functional fusicoccin receptor.…”

“…The same phenomenon was observed in yeast, but only in yeast expressing full-length AHA2 (Figure 3). This increase is most likely due to stabilization by fusicoccin of a complex between 14-3-3 protein and H ϩ -ATPase (Jahn et al, 1997;Oecking et al, 1997). …”

“…The low but significant fusicoccin binding activity in the absence of exogenously added recombinant 14-3-3 protein is probably due to the association of plant H ϩ -ATPase with endogenous 14-3-3 protein, which was indeed found to be associated with yeast plasma membranes. Interestingly, cytosolic 14-3-3 protein isolated from Commelina communis does not bind fusicoccin, whereas membrane-bound 14-3-3 protein copurifying with solubilized plasma membrane H ϩ -ATPase does bind fusicoccin (Oecking et al, 1997). Since membrane-bound 14-3-3 protein is recruited from the cytosol to the membrane, it was suggested that a fusicoccin receptor was generated after association of 14-3-3 protein with the H ϩ -ATPase (Oecking et al, 1997).…”

“…Single point mutations in the autoinhibitory domain can produce a constitutively active H ϩ -ATPase (Baunsgaard et al, 1996;Morsomme et al, 1996), and the C-terminal regulatory domain has recently been found to be associated with the 14-3-3 protein (Jahn et al, 1997;Oecking et al, 1997), of which there are several isoforms. 14-3-3 proteins have been implicated in the co-ordination of signal transduction pathways (Aitken, 1996) and in the regulation of an increasing number of enzymes (Sehnke and Ferl, 1996).…”