Methylation of Elongation Factor La from the Fungus Mucor (Posttranslational Modification/Nt-Methyllysine/Protein Synthesis) WILLIAM R
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Proc. Nati Acad. Sci. USA Vol. 79, pp. 3433-3437, June 1982 Biochemistry Methylation of elongation factor la from the fungus Mucor (posttranslational modification/Nt-methyllysine/protein synthesis) WILLIAM R. HIATT*4, ROBERTO GARCIA*§, WILLIAM C. MERRICKt, AND PAUL S. SYPHERD*¶ *Department of Microbiology, College of Medicine, University of California, Irvine, California 92717; and tDepartment of Biochemistry, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106 Communicated by Bernard D. Davis, February 22, 1982 ABSTRACT Abasic protein from the dimorphic fungus Mucor obtained from Sigma. Poly(U) was purchased from Miles, and racemosus, found to be highly methylated, is shown to be protein reagents for electrophoresis were as described (26). synthesis elongation factor la. This protein is the most abundant Culture Conditions. Mucor racemosus (M. lusitanicus) protein in hyphal cells but is less abundant in yeast cells. It is post- (ATCC 1216B) was grown as yeasts and induced to form aerobic translationally methylated with the formation of mono-, di-, and hyphae as described (26). Methyl-labeled EF-la was obtained trimethyllysine at as many as 16 sites. Nearly 20% of the 44 lysine by suspending mycelia from a 1-liter culture for 1 hr in 50 ml residues of elongation factor la from mycelia are modified while of a peptone-lacking medium containing [methyl-3H]methionine those from sporangiospores are virtually unmethylated. at 20 p.Ci/ml and cycloheximide at 400 Ag/ml. EF-la labeled with Mucor ['4C]lysine was prepared from cells grown in medium con- racemosus is a dimorphic fungus that exists in either taining peptone and [U-'4C]lysine at 10 ,uCi/ml. yeast or hyphal forms. These changes in morphology are ac- Purification of EF-la. Mycelia were ground in liquid N2, companied by changes in the translational system, evidenced suspended in 60 mM Tris acetate, pH 7.0/50 mM NH4CV5 mM by an acceleration in the rate of peptide bond formation (1, 2) 2-mercaptoethanol/10% glycerol, and then further disrupted and by a change in the level of phosphorylation of a ribosomal mechanical with protein (3). In the course of studying posttranslational modifi- by agitation glass beads (0.45-0.5 mm). The cations in suspension was centrifuged at 30,000 x g for 20 min, and the the fungus, we resolved several methylated peptides supernatant was adjusted to 0.5 M KC1 and centrifuged at by using two-dimensional polyacrylamide gel electrophoresis. for 2 hr in a We have now identified one of these peptides as the a subunit 50,000 rpm Spinco 60 Ti rotor. The resulting su- ofprotein synthesis elongation factor 1 (EF-la), the functional pernatant was fractionated as described by Skogerson (27). Rab- equivalent ofprokaryotic bit reticulocyte EF-la was prepared from the 100,000 X g su- elongation factor Tu (EF-Tu) (4). EF- pernatant of a reticulocyte lysate (28). The purification steps Tu from Salmonella typhimurium and Escherichia coli is meth- involved ylated (5, 6), and that in E. coli contains a single DEAE-cellulose chromatography (EF-la does not methyllysine bind at 50 mM KCl) and gradient elution from a phosphocel- at position 56. lulose column. The In certain proteins, acidic amino acids are methylated by a resulting preparation was at least 95% pure reversible carboxymethylation reaction, examples of which as judged by the Coomassie blue-staining material present in have been a NaDodSO4 slab gel (29, 30). The amount ofMucor EF-la was shown to play roles in bacterial chemotaxis (7-9), determined from the radioactivity in EF-la and total cellular leukocyte chemotaxis (10-12), and hormone secretion (13). The from cells in methylation of basic amino acids occurs at the E-amino group protein grown the presence of ['4C]lysine. of lysine, the guanidinium group of arginine, or the imidazole Assay for EF-la Activity. Phe-tRNA binding assays were group of histidine (14). Although these reactions show a high carried out essentially as described by Slobin and Moller (31). degree of specificity, there has been no clear functional role Mucor ribosomes were suspended in 20 mM Tris-HCl, pH 7.4/ assigned to the presence ofmethylated basic amino acids. Meth- 100 mM KC1/5 mM Mg(OAc)2/0.1 mM EDTA/1 mM dithio- ylated basic amino acids occur in histones (15, 16, 17), yeast threitoV0.25 M sucrose at a concentration of 243 A260 units/ cytochrome c (18), ribosomal proteins (19-21), Salmonella flag- ml, insoluble material was removed by centrifugation at 30,000 ella protein (22), actin (23), and myosin (24). X g for 20 min, and the supernatant was stored at -70°C. The high degree ofmethylation of EF-la from Mucor, shown Two-Dimensional Polyacrylamide Gel Electrophoresis. in this report, and the changes in the level of methylation oc- Sample processing, two-dimensional gel electrophoresis, gel curring during morphogenesis make this a useful model for processing, and fluorographywere carried out as described (32). studying the functional significance of this posttranslational Identification of Methylated Amino Acids. Aliquots of EF- modification. la purified by either the column method or by gel electropho- resis were dialyzed 24 hr in 1 M propionic acid, lyophilized, and MATERIALS AND METHODS hydrolyzed with 6 M HC1 at reduced pressure for 24 hr at Materials. L-[phenylalanyl-(U)-14C]Phe-tRNA (10.5 mCi/ 110°C. Then, basic amino acids were partially purified by ion- mmol; 1 Ci = 3.7 x 10"° becquerels) and carrier-free H235SO4 exchange chromatography (33), and the resulting fractions were were purchased from New England Nuclear. [35S]Methionine analyzed by two-dimensional TLC on cellulose plates (34) and (450 Ci/mmol) was prepared by the method ofCrawford and by dansylation followed by HPLC (35). Gesteland (25). L-[methyl-3H]Methionine (27 Ci/mmol) was purchased from Amersham. CM-Sepharose, DEAE-Sephadex Abbreviations: EF-la, elongation factor la; EF-Tu, elongation factor A-50, and Tu; MeLys, Me2Lys, and Me3Lys, Ne-methyl-, Ne,Ne-dimethyl-, and diphenylcarbamoyl chloride-treated trypsin were N',N8,Nt-trimethyllysine, respectively. t Present address: Calgene, Inc., 1410 Marina Circle, Davis, CA 95616. The publication costs ofthis article were defrayed in part by page charge § Present address: Department ofBiology, Ball State University, Mun- payment. This article must therefore be hereby marked "advertise- cie, IN 47306. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. ¶To whom reprint requests should be addressed. 3433 Downloaded by guest on September 24, 2021 3434 Biochemistry: Hiatt et al. Proc. Nati. Acad. Sci. USA 79 (1982) Mr x10-3 - 155 -56 1 _b 1 _ . 4 _ 31 A B - pH FIG. 1. Comparison of 35S-labeled and methylated Mucor basic polypeptides. Polypeptides were resolved by two-dimensional polyacrylamide gel electrophoresis as described (32). (A) Autoradiograph of mycelial polypeptides labeled with [35S]methionine at 80 ACi/ml for 1 hr. (B) Fluo- rograph of mycelial polypeptides labeled with [methyl-3Hlmethionine at 20 pACi/ml for 1 hr in the presence of trichodermin at 180 Ag/ml. Molecular weights were determined as described (32). RESULTS Methylated Amino Acids from EF-la. The presence of Identification of EF-la. Resolution of the basic 'S-labeled methylated amino acids in EF-la was suggested by the fact that Mucor polypeptides by two-dimensional polyacrylamide gel the polypeptide was labeled in vivo with [methyl-3H]methionine electrophoresis (Fig. 1A) showed an abundant polypeptide in the presence of trichodermin (Fig. 1B). Nonradioactive EF- (numbered 1) that had a nominal molecular weight of 53,000 la preparations were hydrolyzed, and the amino acids were and a pI of -9.5. When cells were labeled with [methyl- dansylated and then analyzed by HPLC. As shown in Fig. 4, 3H]methionine in the presence oftrichodermin, an inhibitor of in addition to unmodified lysine, Ne-methyllysine (MeLys), protein synthesis, polypeptide 1 was the predominantly labeled NeN8-dimethyllysine (Me2Lys), and N',N',N8-trimethyllysine species, presumdbly'by methyl transfer via S-adenosylmethio- (Me3Lys) were also found in the preparations. TLC of an acid nine(Fig. 1B). Similar results were obtained when protein syn- hydrolysate of EF-la labeled with [methyl-3H]methionine in thesis was inhibited with cycloheximide (32), which, like tri- the presence of cycloheximide showed that MeLys, Me2Lys, chodermin, completely inhibited incorporation of [3S]methio- and Me3Lys were the only methylated amino acids present. nine into protein under these conditions. To determine the number of methylated lysine residues, The pI, the molecular weight, and the abundance of poly- Mucor was grown in the presence of ['4C]lysine and EF-la peptide 1 were characteristic of the a subunit of EF-la (4). was purified from extracts by two-dimensional gel electropho- Coelectrophoresis of rabbit reticulocyte EF-la with a Mucor resis. Ofthe total [14C]lysine incorporated into EF-la, 81% was extract showed that rabbit EF-la and polypeptide 1 had nearly present as lysine and 5.4%, 4.6%, and 9.4% were present as identical electrophoretic mobilities on two-dimensional poly- MeLys, Me2Lys, and Me3Lys, respectively. By this determi- acrylamide gels. Mucor polypeptide 1 appeared slightly larger nation, 19% ofthe lysine residues in Mucor EF-la were meth- and more basic. ylated. Amino acid analysis of purified Mucor EF-la showed To confirm the identity ofpolypeptide 1, an extract ofMucor that lysine and lysine derivatives account for 9.8 mol % of the labeled with [methyl-3H]methionine in the presence of cyclo- amino acids found in an acid hydrolysate (Table 1). With a mo- heximide was fractionated as described by Skogerson (27). lecular weight of 53,000, EF-la should have =442 residues, Chromatography on DEAE-Sephadex followed by gradient 43 of which appear to be lysine and its derivatives.