Fusicoccin affects cytochrome c leakage and cytosolic 14-3-3 accumulation independent of H+-ATPase activation

Fusicoccin (FC) is a well known toxin acting as a 14-3-3 protein-mediated activator of the plasma membrane H+-ATPase and the biochemical and physiological changes induced in the cell by this toxin have, up to now, been ascribed to the increased rate of proton extrusion by this pump leading to external acidification and cell hyperpolarization. In a recent work (Malerba M et al. 2003, Physiologia Plantarum, 119: 480-488) it was shown that, besides the previously well studied changes, FC induces a large stimulation of H2O2 production, an activation of alternative respiration and a leakage of cytochrome c from mitochondria. In this article further studies on the relation between the H2O2 overproduction and medium acidification are reported. The increase in the rate of H2O2 accumulation is particularly evident when high concentrations of the toxin ensure a rapid acidification of the medium, but it is not obtained when the time-course of acidification is reproduced by external acid additions. The FC-dependent H2O2 overproduction is strongly inhibited by inhibitors of the H+-ATPase activity, such as vanadate and erythrosin B, and it does not occur when the activation of the H+-ATPase is prevented by phenylarsine oxide (PAO), an inhibitor of the activating interaction between the enzyme and its regulative 14-3-3 protein. Interestingly, all these inhibitors only partially prevent the leakage of cytochrome c from the mitochondria. A kinetic analysis of FC-dependent changes of 14-3-3s shows that the initial increase in the plasma membrane level of these proteins, presumably due to translocation of free cytosolic forms, is followed by a remarkable increase in the level of the 14-3-3 proteins located in the cytosol. This latter change is not prevented by inhibitors of the activity or activation of the H+-ATPase. These results suggest that, besides the H+-ATPase activation, FC can induce other cell changes possibly mediated by changes of the regulative 14-3-3 proteins.