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Topic 19.7

The Mechanism of Fusicoccin Activation of the Plasma Membrane H⁺-ATPase

Our examination of the auxin response would be incomplete without a discussion of the mechanism of fusicoccin (FC) . The structure of fusicoccin A, a phytotoxin produced by the fungus Fusicoccum amygdali. This phytotoxin is produced by the fungus Fusicoccum amygdali, a parasite of peach and almond trees. Once released into the leaves, the toxin stimulates the H⁺-ATPases of the guard cells. At the biochemical level it induces a rapid (10 to 30 seconds) hyperpolarization of the plasma membrane, accompanied by an acidification of the cell wall. This triggers an irreversible opening of the stomata (see textbook Chapter 18), resulting in wilting of the leaves and, eventually, the death of the tree.

FC causes membrane hyperpolarization and proton extrusion in nearly all plant tissues. Treatment of coleoptiles and stem sections with FC leads to a transient growth response, a result that provides support for the acid growth hypothesis for auxin-induced growth. But whereas auxin-induced proton extrusion has a lag time of about 10 minutes and is inhibited by cycloheximide, FC-induced proton extrusion begins after only 1 to 2 minutes, and the response is insensitive to cycloheximide. Moreover, FC-treated cells can acidify down to a much lower extracellular pH than auxin-treated cells can (pH 3 versus pH 4).

Because of these effects, FC has sometimes been referred to as a "super auxin." However, FC acts by a mechanism entirely different from that of auxin. For example, FC does not stimulate the expression of any of the auxin-induced genes, nor can it mimic the effects of IAA on other developmental processes, such as cell division. Thus the effects of FC on plants are much more limited than those of IAA, which is not surprising since FC is a toxin, not an endogenous plant hormone. In the case of proton extrusion, FC is known to activate preexisting H⁺-ATPases on the plasma membrane, probably by displacing the autoinhibitory C-terminal domain of the enzyme from the catalytic site (see textbook Chapter 6) (Jahn et al. 1996). In contrast, auxin may promote proton extrusion in part by activation of preexisting H⁺-ATPases (perhaps via acidification of the cytosol) and in part by causing the de novo synthesis of the H⁺-ATPase.

Fusicoccin was first identified as a phytotoxin in the 1960s, but it was not until 1977 that a radioactively labeled version of this toxin was available that could be used to characterize its ligand. Studies with radioactively labeled FC have shown that it binds tightly to a protein on the plasma membrane. Ultimately, these studies led to the purification and identification of the fusicoccin-binding protein (FCBP).

Initial attempts to purify FCBP after photoaffinity labeling led to conflicting results. Some laboratories identified a homodimer, composed of two 30 kDa subunits. The ability of the purified 30 kDa polypeptides to stimulate the plasma membrane H⁺-ATPase in the presence of FC was tested in in vitro proton-pumping experiments. The 30 kDa polypeptide was reconstituted along with purified plasma membrane ATPase into artificial membrane vesicles called proteoliposomes. The addition of FC to the reconstituted proteoliposomes caused an increase in their ATP-driven H⁺-pumping activity.

Cloning and sequencing of the gene for the 30 kDa polypeptide revealed it to be a member of the so-called 14-3-3 family of regulatory proteins (Korthout and de Boer 1994; Oecking et al. 1994; Marra et al. 1995). Regulatory proteins of the 14-3-3 family are widespread among plants and animals. Originally identified in mammalian brain tissue (and named after the positions they occupy in chromatographs), 14-3-3 proteins make up a heterogeneous family of soluble proteins ranging from 25 to 32 kDa, which associate to form dimers (Ferl 1996). The precise function of 14-3-3 proteins has not yet been elucidated. However, the numerous functions that have been attributed to them indicate that they play multiple roles in signal transduction pathways.

Although it was initially believed that the 30 kDa 14-3-3 protein represented the complete FCBP, another laboratory reported that a 90 kDa polypeptide consistently copurified with the two 30 kDa polypeptides (Aducci et al. 1993). More recently it has been demonstrated that the 14-3-3 protein is unable to bind FC by itself. To bind FC, the 14-3-3 protein requires the presence of H⁺-ATPase (Oecking et al. 1997). This finding confirms the earlier report that a 90 kDa polypeptide (the H⁺-ATPase) copurifies with the 14-3-3 protein. According to the current model, FC activates the H⁺-ATPase by stabilizing a transient complex that forms between the autoinhibitory C-terminal domain of the H⁺-ATPase and the 14-3-3 dimer (Web Figure 19.6.A) locking the enzyme into its most active state.

Web Figure 19.6.A   Model for the mechanism of fusicoccin activation of H+-ATPases. (A) A pair of plasma membrane H+-ATPases associate to form an active dimer. The activity of the pump is limited by the autoinhibitory effect of the C terminus of each monomer. (B) Dimers of 14-3-3 proteins form a transient complex with the C terminus of the H+-ATPase and possibly another protein. When the autoinhibitory C terminus is bound to the 14-3-3 protein, the activity of the H+-ATPase increases, but the effect is transient because of the instability of the complex. (C) The binding of fusicoccin to the complex stabilizes the complex, locking the H+-ATPase into an active state. (After Oecking et al. 1997.) (Click image to enlarge.)