title: Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin, fusicoccin.
abstract: The high-affinity fusicoccin-binding protein (FCBP) was solubilized from plasma-membrane vesicles prepared from leaves of Vicia faba L. by aqueous two-phase partitioning. Conditions for the solubilization of intact FCBP-radioligand complexes were worked out. About 60-70% of the complexes can be solubilized with 50-60 mM nonanoyl-N-methylglucamide in the presence of 1 mg· ml(-1) soybean phosphatidylcholine, type IV S, and 20% (v/v) glycerol at pH 5.5. The slow dissociation of the radioligand, 9'-nor-fusicoccin-8'-alcohol-[(3)H] from the FCBP at low temperatures permits the purification of FCBP-radioligand complexes at 4-10° C by fast protein liquid chromatography on anion-exchange and gel permeation columns. The FCBP, extracted from plasma membranes with cholate and chromatographed in the presence of this detergent, gave an apparent molecular mass (Mr) of 80±20 kDa on gel permeation columns under the conditions used. By comparison of the elution profiles of the fraction most enriched in FCBP-radioligand complexes with polypeptide patterns obtained on sodium dodecyl sulfate-polyacrylamide gels, a polypeptide with an Mr of approx. 34kDa co-separated with the radioactivity profile. A second, faint band of approx. 31 kDa was sometimes also observed co-electrophoresing. Photoaffinity labeling of plasma-membrane vesicles with the new compound 9'-nor-8'[(3,5-[(3)H]-4-azidobenzoy)ethylenediamine]-fusicoccin ([(3)H]ABE-FC) and subsequent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis labeled a single band with an Mr of 35±1 kDa. Labeling in this band was strongly reduced when the membranes were incubated with [(3)H]ABE-FC in the presence of 0.1-1 μM fusicoccin. From our data, we conclude (i) that the 34-35-kDa polypeptide represents the FCBP and (ii) that in detergent extracts of plasma membranes this polypeptide is probably present as a di- or trimeric structure.
authors.affiliation: Arbeitsgruppe Pflanzenphysiologie, Fachbereich Biologie/Chemie, Universität Osnabrück, Postfach 4469, D-4500, Osnabrück, Federal Republic of Germany.
authors.forename: M
authors.initials: M
authors.lastname: Feyerabend
authors.forename: E W
authors.initials: EW
authors.lastname: Weiler