Methylation of Elongation Factor La from the Fungus Mucor (Posttranslational Modification/Nt-Methyllysine/Protein Synthesis) WILLIAM R

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 _ .

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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. From the elution from CM-Sepharose yielded a preparation in which above this suggests at least sites of on polypeptide 1 was the only radioactive protein present as judged value, eight methylation by two-dimensional gel electrophoresis and fluorography. This the protein. radiochemically pure material was added to an unlabeled extract Methylated Peptides from EF-la. Totest further the pos- of Mucor that was then fractionated by the same procedure. sibility of multiple sites of methylation on EF-la, methylated Fractions were analyzed for the presence of 3H-labeled poly- tryptic peptides were analyzed. The preparation of EF-la peptide 1 and for EF-la activity. Polypeptide 1 and EF-la ac- shown in Fig. 3, which had been labeled with [methyl- tivity were copurified by chromatography of the material on 3H]methionine in the presence of cycloheximide, was exhaus- DEAE-Sephadex (Fig. 2A) and CM-Sepharose (Fig. 2B). Two- tively digested with chymotrypsin-free trypsin, and the pep- Aimensional gel analysis of the fraction that eluted from CM- tides were resolved by electrophoresis and chromatography Sepharose with the highest EF-la activity showed a nearly pure (Fig. 5). At least 16 methylated peptides were visible under preparation of radioactive polypeptide 1 (Fig. 3). these conditions. Downloaded by guest on September 24, 2021 Biochemistry: Hiatt et al. Proc. Natl Acad. Sci. USA 79 (1982) 3435

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33 36 39 42 300 2 E Retention time, min -200 c

I 100 Z FIG. 4. HPLC of a purified Mucor EF-la hydrolysate. Only the region of the gradient containing lysine and lysine derivatives is . O shown. Fluorescence was determined by a Laboratory Data Control model 1309 detector. Peaks: 1, Me2Lys; 2, Me3Lys; 3, lysine (off scale); Fraction 4, MeLys. FIG. 2. Cochromatography of polypeptide 1 and EF-la activity. A crude extract of Mucor to which H-labeled polypeptide 1 had been [3H]lysine and preparing total cell extracts. The proteins were added was fractionated on DEAE-Sephadex A-50 (A) and then by gra- separated by two-dimensional gel electrophoresis as in Fig. 1, dient elution from CM-Sepharose (B) with NH4Cl (A). Polypeptide 1 and the EF-la spot was removed and its radioactivity was de- was monitored by the amount of 3H radioactivity (o) in 100-,ul aliquots termined. The amount of EF-la was determined as a percent- of the fractions. EF-la activity (e) was determined as the amount of of ['4C]Phe-tRNA binding directed by 2-1.l aliquots of the fractions. age the total radioactivity applied to the gel. There was a >2.5-fold change in the amount of EF-la between yeast cells grown in a CO2 atmosphere and the germling stage (Table 2). Methylation as a Function of Cellular Morphology. The re- Mycelial cells showed an intermediate level of EF-la protein. sults presented above are from studies of EF-la from cells in the mycelial phase ofdevelopment. These are cells, germinated Table 1. Amino acid composition of EF-la from from spores, that have hyphal germ tubes with a length 5-8 Mucor racemosus times the diameter of the spore. We examined other forms of Amino acid Mol % the fungus to determine whether or not the amount of EF-la and the extent of its methylation might vary as a function of Lysine* 9.8 morphology. EF-la was quantitated by growing cells in Histidine 2.5 Arginine 4.1 Aspartic acid 9.5 .A B Threonine 8.1 Serine 5.7 Glutamic acid 7.9 Proline 4.9 Glycine 9.4 Alanine 8.9 Valine 9.1 Methionine 1.7 _~~~~~~~~~~~~------...... ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Isoleucine 7.1 Leucine 5.9

pH H- Tyrosine 1.6 Phenylalanine 3.8 FIG. 3. Two-dimensional polyacrylamide gel analysis of fraction- Mucor EF-la was hydrolyzed at reduced pressure for 24 hr, and du- ated EF-1. The CM-Sepharose fraction (Fig. 2B) having the highest plicate samples of the hydrolysate were analyzed for amino acid com- EF-1a activity was dialyzed against H20, lyophilized, and analyzed by position by using a Durrum D-500 analyzer. Values for cysteine and two-dimensional gel electrophoresis using a first-dimension ampho- tryptophan were not determined. lyte of pH 3-10. (A) Polypeptides were stained with Coomassie blue. * Includes the methylated forms, which account for 19% of the total (B) 3H-Labeled polypeptides were visualized by fluorography. lysine content. Downloaded by guest on September 24, 2021 ,1-36IIA'l Biochemistry: Hiatt et al. Proc. Natl. Acad. Sci. USA 79 (1982) Table 3. Extent of EF-la methylation Cell type % lysine methylated Spore 1.2 ± 1.0 Germling 12.5 ± 2.1 Mycelium 16.6 ± 2.8 Methylated residues were detected by TLC of acid hydrolysates of 0~ EF-la. The protein was prepared from cells grown on [14C]lysine. Re- a) sults are mean ± SEM and include all methylated species. 0

0 E 0 peptide. Calculation of the number of methylated sites by de- -C termining the percent total lysine residues methylated indi- u cated approximately eight sites on EF-la. However, the accuracy of this value depends on the homogeneity of the EF- la preparation and the specificity of the methyltransferase. Since it is possible that only a portion of the lysine residues at a given site are methylated (5, 6), this value represents only the origin average number of methylated residues per molecule of EF-la. I More direct evidence on the extent of methylation was pro- vided by tryptic maps ofmethylated peptides from EF-la. Be- cause of the ability of trypsin to cleave polypeptides at lysine + Electrophoresis residues, including methylated ones (38, 39), each radioactive peptide likely contained only one lysine residue representing FIG. 5. Methylated peptides from Mucor EF-la. The preparation a site of methylation. While 16 labeled peptides from EF-la of 3H-labeled EF-1 shown in Fig. 3 was digested with trypsin and the were found, 9 were more prominent and represented lysine resulting peptides were resolved by electrophoresis at 400 V for 1 hr residues preferentially methylated. followed by chromatography on thin-layer cellulose (36). Labeled (and Differences in the amount ofradioactivity in the tryptic pep- therefore methylated) peptides were visualized by fluorography (37). tides may have arisen from several sources. MeLys, Me2Lys, Approximately 40,000 cpm of radioactivity was applied to the plate or Me3Lys could occur at the same site (6) in each peptide, with and exposure was for 45 days. variations existing among protein molecules. More intensely labeled peptides may contain lysine residues at sites prefer- The activity of EF-la did not parallel the changes in EF-1 pro- entially labeled, while faintly labeled ones may contain sites tein (unpublished data). recognized infrequently. In that regard, charge heterogeneity The extent of methylation of EF-la was determined at dif- is a common property of EF-la preparations from a number of ferent stages of hyphal development, from spores to mycelia eukaryotic sources (40). This heterogeneity may be a contrib- (Table 3). EF-la from spores had quite low levels of methyla- uting factor to the large differences in the extent oflabeling of tion, in some cases below detection levels. The spore data were tryptic peptides of EF-la. derived from spores recovered from sporulating hyphae that Protein methylation in eukaryotes is usually restricted to one had been grown on radioactive solid medium. The EF-la from or two sites (4), with Mucor EF-la by far the most heavily germlings had considerably higher levels of methylation, and methylated eukaryotic protein reported. However, the func- EF-la from older mycelia had the highest quantity of meth- tional significance of methylation of basic amino acids in any ylated lysine residues. The relatively large standard errors protein is the subject of considerable speculation. It is of in- found probably reflect poor and variable synchrony in the mor- terest that many methylated proteins, including ribosomal pro- phogenetic events. The data show, however, that EF-la from teins, nuclear and cytoplasmic RNA-associated proteins, his- the dormant sporangiospore has very low levels of methylated tones, EF-Tu and EF-la interact with nucleic acids. Baxter and lysine and that this value increases during the morphogenesis Byovet (41) have shown that increasing methyl substitution on to mycelia. Furthermore, cells of different morphology have lysine increased the positive charge in the region ofthe 6-amino various amounts of the elongation factor. group, and the affinity for the phosphates ofnucleic acids. Sim- ilarly, dimethylarginine, which is found in substantial amounts DISCUSSION in ribonucleoproteins, has been postulated to play a role in The results presented here show that Mucor EF-la contains RNA-protein interaction (42). The possibility exists, therefore, methylated derivatives of lysine at multiple sites in the poly- that methylation may provide a subtle means for regulating the interactions of proteins with nucleic acids. Table 2. Proportion of total cellular protein present as EF-la EF-la from Mucor should prove valuable in studies of the Growth functional role ofprotein methylation. We have found that the Morphology atmosphere EF-la, % total rate ofpolypeptide chain elongation in M. racemosus increases sharply during the appearance of hyphal germ tubes from Yeast C02/N2 0.60 spores (1) and during the conversion of yeasts to hyphal cells Germling Air 1.63 (2). In addition, we have isolated a conditional morphology Mycelium Air 1.04 mutant of M. racemosus that requires high exogenous concen- The amount of EF-la is expressed as % total proteinapplied to a two- trations ofmethionine to carry out the normal yeast-to-hyphae dimensional gel. Radioactivity in the EF-la spot was 1.9-3.5 x 104 transition (43). Analysis of EF-la isolated from this mutant has cpm. Germlings are defined as cells having short germ tubes emerging revealed an altered pattern ofmethylated tryptic peptides un- from the spore body. The germ tubes were 1- to 2-spore-diameters long. Mycelia had well-developed hyphal processes averaging 5- to 8-spore- der conditions in which morphogenesis does not occur. More- diameters long. over, spores of M. racemosus appear to contain an essentially Downloaded by guest on September 24, 2021 Biochemistry: Hiatt et al. Proc. NatL Acad. Sci. USA 79 (1982) 3437 unmethylated form of EF-la. EF-la, like EF-Tu, interacts 17. Borun, T. W., Pearson, D. & Paik, W. K. (1972)J. Biot Chem. with a variety of substrates including GTP, GDP, aminoacyl- 247, 4288-4298. tRNAs, and ribosomes. These interactions are all readily as- 18. Paik, W. K., Polastro, E. & Kim, S. (1980) in Current Topics in sayed, and the role of methylation, if any, can be determined. Cellular Regulation, eds. Horecker, B. L. & Stadtman, E. R. Finally, as in other (Academic, New York), pp. 87-111. rapidly growing cells, EF-la is an extremely 19. Alix, J. H., Hayes, D. H. & Nierhaus, K. H. (1979)J. Mot Biot abundant protein in Mucor. The high affinity ofthis protein for 127, 375-388. nucleic acids (44, 45) and the observation that significant 20. Colson, C. & Smith, H. 0. (1977) Mot Gen. Genet. 154, 167-173. amounts of EF-la are found in the nucleus of some cells (44) 21. Chang, F. N., Navickas, I. J., Au, C. & Budzilowicz, C. (1978) suggests potential regulatory roles other than polypeptide elon- Biochim. Biophys. Acta 518, 89-94. gation. 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Byvoet, P. & Baxter, C. S. (1975) in Chromosomal Proteins and (1978) Biochim. Biophys. Acta 519, 398-405. Their Role in the Regulation of Gene Expression, eds. Stein, G. 45. Ovchinnikov, L. P., Spirin, A. S., Erni, B. & Staehelin, T. (1978) S. & Kleinsmith, L. J. (Academic, New York), pp. 120-133. FEBS Lett. 88, 21-26. Downloaded by guest on September 24, 2021