The 30-Kilodalton Protein Present in Purified Fusicoccin Receptor Preparations Is a 14-3-3-Like Protein

Abstract

Pure preparations of FC receptors, obtained under nondenaturing conditions, showed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis two doublets of proteins with apparent molecular masses of 30 and 90 kD. In the present paper we describe the isolation and identification of the primary structure of the 30-kD doublet proteins. Sequencing studies of peptides resulting from the digestion of the 30-kD protein showed a full identity with a 14-3-3-like protein from corn, named CF14. The 14-3-3 family is a class of proteins that is widely distributed in eukaryotes and is known to play various regulatory roles. The 30-kD protein has been immunologically identified by specific antibodies prepared against a synthetic peptide based on the determined amino acid sequence. A similar protein is recognized in partially purified FC receptor preparations from bean and spinach leaves.Although in the last few years progress has been made in the elucidation of mode of action of FC (Aducci et al., 1988; Johansson et al., 1993; Rasi-Caldogno et al., 1993), the primary structure of the FC receptor is still unknown. Different groups have reported on the purification of FC-binding proteins from various plants (de Boer et al., 1989; Feyerabend and Weiler, 1989; Oecking and Weiler, 1991), showing that the putative FC receptor is a protein in the range of 30 to 35 kD.Recently we have described the purification of the FC receptor from com (Zea mays L.) shoots using HPLC (Aducci et al., 1993). SDS-PAGE of highly purified samples revealed the occurrence of two doublets corresponding to proteins of * ComesDondine author; fax 39-6-2023500. tritiated azido-FC, resulted in labeling of the 90-kD protein under conditions that did not affect FC binding. In contrast, labeling of the 30-kD protein occurred only under conditions that severely impaired the binding activity. From our data we inferred that the minimal functional FC receptor has an apparent molecular mass of 90 kD. However, the presence of the 30-kD protein in fractions retaining an FC-binding capacity after extensive purification (5000-fold increase of specific FC-binding activity) suggests a tight association of the two proteins during chromatographic separations, including gel filtration, when performed under nondissociating conditions. This suggests a possible regulatory role for the 30-kD protein in the FC signaling pathway. These results prompted us to further purify the FC receptor to obtain pure samples of the 30-and 90-kD proteins suitable for primary structure determination, cloning, and expression.In this paper we report on the separation of the 30-and 90-kD proteins from com by RP HPLC chromatography and partial elucidation of the primary structure of the 30-kD protein and its immunological identification by antibodies directed to a synthetic peptide based on the determined amino acid sequence. MATERIALS AND METHODS ChemicalsFC was prepared as described by Ballio et al. (1968).[3H]FC was obtained according to Ballio et al. (1980). Chemicals used for gel electrophor...

Summary

“…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?…”

“…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).…”

“…According to one model, fusicoccin interferes with a signal transduction chain ultimately leading to activation of the plant plasma membrane H ϩ -ATPase by a phosphorylation or dephosphorylation event (de Boer et al, 1994;van der Hoeven et al, 1996;Marra et al, 1994). The other model involves the fusicoccin binding protein directly regulating the H ϩ -ATPase (Marrè , 1979;De Michelis et al, 1996).…”

The H+‐ATPase purified from maize root plasma membranes retains fusicoccin in vivo activation

“…Upon binding to plasma membrane receptors [6][7][8], it rapidly and persistently stimulates the enzyme, thus triggering cell growth and membrane transport [5]. It has been established that activation of the H+-ATPase is more remarkable when FC is administered in vivo to plant tissues and that it is maintained after plasma membrane isolation [9].…”

“…One favours a regulation through a direct interaction between FC receptors and the H+-ATPase [13], whereas the other envisages the occurrence of a transduction chain in which phosphorylation/ dephosphorylation events may be involved [14]. Recently, clues about the FC signaling machinery have been obtained by the discovery that 14-3-3 proteins are present in purified FC receptor preparations [6][7][8]. Nevertheless, this finding does not help to favour one of the two above models, since these proteins are known to regulate protein kinase activities as well as to mediate protein-protein interactions [15].…”