The fusicoccin receptor of plants is a member of the 14‐3‐3 superfamily of eukaryotic regulatory proteins

Abstract

The receptor for the wilt-inducing phytotoxin fusicoccin was purified to homogeneity from plasma membranes of Commelina communis as a complex with the radioligand [3H]9'-nor-8'-hydroxyfusicoccin. The preparation consisted of two polypeptides with apparent molecular masses of 30.5 kDa and 31.5 kDa and with isoelectric points of around pH 5.2 and 5.3, respectively. The proteins were N-terminally blocked. Internal amino acid sequences were obtained for both polypeptides of the fusicoccin-binding complex. Sequence information, as well as subsequent immunological analysis, proved that both polypeptides are members of the eukaryotic 143-3 family, which comprises structurally conserved regulatory proteins of widespread occurrence and a wide range of functions. 143-3 isoform(s) constituting the fusicoccin receptor are distinguishable from other cellular 143-3 proteins by their tight association with the plasma membrane. Applying temperature-induced Triton X-l 14 phase separation experiments, they, as well as the target enzyme of fusicoccin action, the H'-ATPase, partitioned into the phospholipid-rich fraction which contains the most hydrophobic proteins. The results discussed herein provide a basis for the elucidation of the molecular mechanism of fusicoccin action.

Summary

“…However, addition of fusicoccin resulted in an increased amount of 14-3-3 protein associated with the membranes (Figure 9b). This is in accordance with both the observation that in vivo treatment with fusicoccin results in increased amounts of 14-3-3 protein associated with the plasma membranes of yeast cells expressing AHA2 (Figure 3) and with results of in vivo treatment of plant material with fusicoccin prior to preparation (Jahn et al, 1997;Korthout and de Boer, 1994;Oecking et al, 1994). However, pretreatment with phosphoserine-Raf-259 peptide diminished the level of 14-3-3 protein in both the control and the fusicoccin incubated plasma membranes.…”

“…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).…”

“…Fusicoccin binding activity in plants copurifies with 14-3-3 proteins (Korthout and de Boer, 1994;Marra et al, 1994;Oecking et al, 1994). If the H ϩ -ATPase is an essential part of the fusicoccin receptor, as shown here, why do none of the aforementioned papers report the presence of the plasma membrane H ϩ -ATPase in their preparations?…”

“…(1) Activation of the H ϩ -ATPase by fusicoccin in vivo correlates with binding of the 14-3-3 protein to the C-terminus of the enzyme (Jahn et al, 1997;Oecking et al, 1997); (2) action by fusicoccin results in increased binding of the 14-3-3 protein to plasma membranes (Jahn et al, 1997;Korthout and de Boer, 1994;Oecking et al, 1994Oecking et al, , 1997; (3) there is a direct correlation between the amount of fusicoccin bound to plasma membranes and the degree of activation of the H ϩ -ATPase (De Michelis et al, 1996); and (4) provided fusicoccin is present, the plant H ϩ -ATPase is activated by protein fractions containing fusicoccin binding activity (Marra et al, 1992). These indirect pieces of evidence all point to a role for 14-3-3 proteins in a positive regulation of H ϩ -ATPase activity and thus support the observations presented in this study.…”

“…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). In plants, 14-3-3 proteins have been suggested to also play a role in the binding of fusicoccin (Korthout and de Boer, 1994;Marra et al, 1994;Oecking et al, 1994). Fusicoccin is known to mimic some of the physiological effects of auxin and has been used as a tool for studying the action of this phytohormone (de Boer, 1997;Marrè , 1979).…”