Methylation of elongation factor la from the fungus Mucor ( posttranslational modification / Nt - methyllysine / protein synthesis )

A basic protein from the dimorphic fungus Mucor racemosus, found to be highly methylated, is shown to be protein synthesis elongation factor la. This protein is the most abundant protein in hyphal cells but is less abundant in yeast cells. It is posttranslationally methylated with the formation of mono-, di-, and trimethyllysine at as many as 16 sites. Nearly 20% of the 44 lysine residues of elongation factor la from mycelia are modified while those from sporangiospores are virtually unmethylated. Mucor racemosus is a dimorphic fungus that exists in either yeast or hyphal forms. These changes in morphology are accompanied by changes in the translational system, evidenced by an acceleration in the rate of peptide bond formation (1, 2) and by a change in the level of phosphorylation of a ribosomal protein (3). In the course of studying posttranslational modifications in the fungus, we resolved several methylated peptides by using two-dimensional polyacrylamide gel electrophoresis. We have now identified one of these peptides as the a subunit of protein synthesis elongation factor 1 (EF-la), the functional equivalent of prokaryotic elongation factor Tu (EF-Tu) (4). EFTu from Salmonella typhimurium and Escherichia coli is methylated (5, 6), and that in E. coli contains a single methyllysine at position 56. In certain proteins, acidic amino acids are methylated by a reversible carboxymethylation reaction, examples of which have been shown to play roles in bacterial chemotaxis (7-9), leukocyte chemotaxis (10-12), and hormone secretion (13). The methylation of basic amino acids occurs at the E-amino group of lysine, the guanidinium group of arginine, or the imidazole group of histidine (14). Although these reactions show a high degree of specificity, there has been no clear functional role assigned to the presence ofmethylated basic amino acids. Methylated basic amino acids occur in histones (15, 16, 17), yeast cytochrome c (18), ribosomal proteins (19-21), Salmonella flagella protein (22), actin (23), and myosin (24). The high degree ofmethylation of EF-la from Mucor, shown in this report, and the changes in the level of methylation occurring during morphogenesis make this a useful model for studying the functional significance of this posttranslational modification. MATERIALS AND METHODS Materials. L-[phenylalanyl-(U)-14C]Phe-tRNA (10.5 mCi/ mmol; 1 Ci = 3.7 x 10"° becquerels) and carrier-free H235SO4 were purchased from New England Nuclear. [35S]Methionine (450 Ci/mmol) was prepared by the method of Crawford and Gesteland (25). L-[methyl-3H]Methionine (27 Ci/mmol) was purchased from Amersham. CM-Sepharose, DEAE-Sephadex A-50, and diphenylcarbamoyl chloride-treated trypsin were obtained from Sigma. Poly(U) was purchased from Miles, and reagents for electrophoresis were as described (26). Culture Conditions. Mucor racemosus (M. lusitanicus) (ATCC 1216B) was grown as yeasts and induced to form aerobic hyphae as described (26). Methyl-labeled EF-la was obtained by suspending mycelia from a 1-liter culture for 1 hr in 50 ml of a peptone-lacking medium containing [methyl-3H]methionine at 20 p.Ci/ml and cycloheximide at 400 Ag/ml. EF-la labeled with ['4C]lysine was prepared from cells grown in medium containing peptone and [U-'4C]lysine at 10 ,uCi/ml. Purification of EF-la. Mycelia were ground in liquid N2, suspended in 60mM Tris acetate, pH 7.0/50 mM NH4CV5mM 2-mercaptoethanol/10% glycerol, and then further disrupted by mechanical agitation with glass beads (0.45-0.5 mm). The suspension was centrifuged at 30,000 x g for 20 min, and the supernatant was adjusted to 0.5 M KC1 and centrifuged at 50,000 rpm for 2 hr in a Spinco 60 Ti rotor. The resulting supernatant was fractionated as described by Skogerson (27). Rabbit reticulocyte EF-la was prepared from the 100,000 X g supernatant of a reticulocyte lysate (28). The purification steps involved DEAE-cellulose chromatography (EF-la does not bind at 50 mM KCl) and gradient elution from a phosphocellulose column. The resulting preparation was at least 95% pure as judged by the Coomassie blue-staining material present in a NaDodSO4 slab gel (29, 30). The amount ofMucor EF-la was determined from the radioactivity in EF-la and total cellular protein from cells grown in the presence of ['4C]lysine. Assay for EF-la Activity. Phe-tRNA binding assays were carried out essentially as described by Slobin and Moller (31). Mucor ribosomes were suspended in 20 mM Tris-HCl, pH 7.4/ 100 mM KC1/5 mM Mg(OAc)2/0.1 mM EDTA/1 mM dithiothreitoV0.25 M sucrose at a concentration of 243 A260 units/ ml, insoluble material was removed by centrifugation at 30,000 X g for 20 min, and the supernatant was stored at -70°C. Two-Dimensional Polyacrylamide Gel Electrophoresis. Sample processing, two-dimensional gel electrophoresis, gel processing, and fluorographywere carried out as described (32). Identification of Methylated Amino Acids. Aliquots of EFla purified by either the column method or by gel electrophoresis were dialyzed 24 hr in 1 M propionic acid, lyophilized, and hydrolyzed with 6 M HC1 at reduced pressure for 24 hr at 110°C. Then, basic amino acids were partially purified by ionexchange chromatography (33), and the resulting fractions were analyzed by two-dimensional TLC on cellulose plates (34) and by dansylation followed by HPLC (35). Abbreviations: EF-la, elongation factor la; EF-Tu, elongation factor Tu; MeLys, Me2Lys, and Me3Lys, Ne-methyl-, Ne,Ne-dimethyl-, and N',N8,Nt-trimethyllysine, respectively. t Present address: Calgene, Inc., 1410 Marina Circle, Davis, CA 95616. § Present address: Department of Biology, Ball State University, Muncie, IN 47306. ¶To whom reprint requests should be addressed. 3433 The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Proc. Nati. Acad. Sci. USA 79 (1982)