δ-N-Methylarginine Is a Novel Posttranslational Modification of Arginine Residues in Yeast Proteins

We have found a novel modification of protein arginine residues in the yeast Saccharomyces cerevisiae. Intact yeast cells lacking RMT1, the gene encoding the protein ω-N G-arginine methyltransferase, were labeled with the methyl donorS-adenosyl-l-methyl-3Hmethionine. The protein fraction was acid-hydrolyzed to free amino acids, which were then fractionated on a high resolution sulfonated polystyrene cation exchange column at pH 5.27 and 55 °C. In the absence of the ω-N G ,N G-3Hdimethylarginine product of the RMT1 methyltransferase, we were able to detect a previously obscured 3H-methylated species that migrated in the region of methylated arginine derivatives. The 3Hmethyl group(s) of this unknown species were not volatilized by treatment with 2 m NaOH at 55 °C for up to 48 h, suggesting that they were not modifications of the terminal ω-guanidino nitrogen atoms. However, this base treatment did result in the formation of a new 3H-methylated derivative that co-chromatographed with δ-N-methylornithine on high resolution cation exchange chromatography, on reverse phase high pressure liquid chromatography, and on thin layer chromatography. From these data, we suggest that the identity of the original unknown methylated residue is δ-N-monomethylarginine. The presence of this methylated residue in yeast cells defines a novel type of protein modification reaction in eukaryotes. We have found a novel modification of protein arginine residues in the yeast Saccharomyces cerevisiae. Intact yeast cells lacking RMT1, the gene encoding the protein ω-N G-arginine methyltransferase, were labeled with the methyl donorS-adenosyl-l-methyl-3Hmethionine. The protein fraction was acid-hydrolyzed to free amino acids, which were then fractionated on a high resolution sulfonated polystyrene cation exchange column at pH 5.27 and 55 °C. In the absence of the ω-N G ,N G-3Hdimethylarginine product of the RMT1 methyltransferase, we were able to detect a previously obscured 3H-methylated species that migrated in the region of methylated arginine derivatives. The 3Hmethyl group(s) of this unknown species were not volatilized by treatment with 2 m NaOH at 55 °C for up to 48 h, suggesting that they were not modifications of the terminal ω-guanidino nitrogen atoms. However, this base treatment did result in the formation of a new 3H-methylated derivative that co-chromatographed with δ-N-methylornithine on high resolution cation exchange chromatography, on reverse phase high pressure liquid chromatography, and on thin layer chromatography. From these data, we suggest that the identity of the original unknown methylated residue is δ-N-monomethylarginine. The presence of this methylated residue in yeast cells defines a novel type of protein modification reaction in eukaryotes. S-adenosyl-l-methionine S-adenosyl-l-methyl-3Hmethionine high pressure liquid chromatography. The activity of many proteins is modulated by the covalent posttranslational modification of specific amino acid residues. Some of these modification reactions, such as phosphorylation, are reversible whereas others appear to permanently modify residues effectively enlarging the repertoire of amino acids available to proteins. A major group of the latter reactions involves S-adenosylmethionine (AdoMet)1-dependent methylation of the side chain nitrogen atoms of histidine, lysine, and arginine residues (1Paik W.K. Kim S. Protein Methylation. John Wiley and Sons, Inc., New York1980Google Scholar, 2Paik W.K. Kim S. Protein Methylation. CRC Press, Inc., Boca Raton, FL1990Google Scholar, 3Clarke S. Curr Opin. Cell Biol. 1993; 5: 977-983Crossref PubMed Scopus (200) Google Scholar). Recent interest has focused on the formation of several methylated arginine derivatives in a variety of eukaryotic proteins involved in signal transduction, nuclear RNA processing, the structural integrity of myelin, and other functions (4Liu Q. Dreyfuss G. Mol. Cell. Biol. 1995; 15: 2800-2808Crossref PubMed Scopus (270) Google Scholar, 5Gary J.D. Lin W-J. Yang M.C. Herschmann H.R. Clarke S. J. Biol. Chem. 1996; 271: 12585-12594Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 6Tang J. Gary J.D. Clarke S. Herschman H.R. J. Biol. Chem. 1998; 273: 16935-16945Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 7Siebel C.W. Guthrie C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13641-13646Crossref PubMed Scopus (73) Google Scholar, 8Kim S. Lim I.K. Park G-H. Paik W.K. Int. J. Biochem. Cell Biol. 1997; 29: 743-751Crossref PubMed Scopus (71) Google Scholar, 9Abramovich C. Yakobson B. Chebath J. Revel M. EMBO J. 1997; 16: 260-266Crossref PubMed Scopus (153) Google Scholar, 10Shen E.C. Henry M.F. Weiss V.H. Valentini S.R. Silver P.A. Lee M.E. Genes Dev. 1998; 12: 679-691Crossref PubMed Scopus (251) Google Scholar, 11Gary J.D. Clarke S. Prog. Nucleic Acid Res. Mol. Biol. 1998; 61: 65-131Crossref PubMed Google Scholar). There are at least three distinct types of protein arginineN-methyltransferases that have been classified by their reaction products. The type I enzymes catalyze the formation of ω-N G-monomethylarginine and asymmetric ω-N G,N G-dimethylarginine residues, the type II enzymes catalyze the formation of ω-N G-monomethylarginine and symmetric ω-N G,N′ G-dimethylarginine residues, whereas the type III enzyme forms only the ω-N G-monomethylarginine derivative (for a review, see Ref. 11Gary J.D. Clarke S. Prog. Nucleic Acid Res. Mol. Biol. 1998; 61: 65-131Crossref PubMed Google Scholar). The precise functional role of these methylated arginine residues has not been established. We have been interested in studying the modification of yeast proteins by methylation. The complete genome of Saccharomyces cerevisiae has been sequenced (12Goffeau A. et al.Nature. 1997; 387 suppl.: 1-105Google Scholar), and the presence of methyltransferase sequence motifs in many of these enzymes aids in the identification of new modification enzymes (13Kagan R.M. Clarke S. Arch. Biochem. Biophys. 1994; 310: 417-427Crossref PubMed Scopus (423) Google Scholar). Yeast cells, in contrast to almost all other cell types, actively import the biological methyl donor S-adenosylmethionine (14Murphy J.T. Spence K.D. J. Bacteriol. 1972; 109: 499-504Crossref PubMed Google Scholar), and this allows radiolabeling of proteins in intact cells under in vivoconditions to determine the relevant substrates. Finally, yeast knockout mutants can be readily constructed and can be useful in the pairing of specific methyltransferase genes with their substrates. We recently identified the gene responsible for the majority of protein arginine methylation in yeast (5Gary J.D. Lin W-J. Yang M.C. Herschmann H.R. Clarke S. J. Biol. Chem. 1996; 271: 12585-12594Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). This gene, RMT1, encodes a type I protein arginine methyltransferase capable of sequentially methylating arginine residues to ω-N G-monomethylarginine and ω-N G ,N G-dimethylarginine in a variety of proteins (5Gary J.D. Lin W-J. Yang M.C. Herschmann H.R. Clarke S. J. Biol. Chem. 1996; 271: 12585-12594Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 11Gary J.D. Clarke S. Prog. Nucleic Acid Res. Mol. Biol. 1998; 61: 65-131Crossref PubMed Google Scholar). Mutants lacking this enzyme appear to be completely deficient in the formation of ω-N G ,N G-dimethylarginine residues, suggesting that this enzyme is wholly responsible for this modification. However, these mutants contain residual ω-N G-monomethylarginine residues as well as an additional unknown methylated residue that chromatographs between the positions of the dimethyl- and monomethylarginine derivatives (5Gary J.D. Lin W-J. Yang M.C. Herschmann H.R. Clarke S. J. Biol. Chem. 1996; 271: 12585-12594Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). In this paper, we provide evidence that this latter methylated residue represents a novel modification of arginine residues giving rise to δ-N-monomethylarginine through the methylation of the internal (or δ) guanidino nitrogen atom and suggest that a new type of protein arginine methyltransferase exists in nature. S. cerevisiae strain JDG9100–2 (MATa, prc1–407, prb1–1122, pep4–3, leu2, trp1, ura3–52, ycl57wΔ::URA3, rmt1::LEU2) (5Gary J.D. Lin W-J. Yang M.C. Herschmann H.R. Clarke S. J. Biol. Chem. 1996; 271: 12585-12594Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) was grown at 30 °C to early log phase (approximately 5 × 107 cells/ml;A 600 nm = 0.8) in YPD medium (1% (w/v) yeast extract (Difco Laboratories), 2% (w/v) bacto-peptone (Difco), and 2% (w/v) d-glucose). Cells from a 6-ml culture were harvested at 1000 × g for 3 min in a 15-ml polystyrene centrifuge tube at room temperature. Cells were washed 3 times with 1 ml of YPD medium and resuspended in 0.82 ml of YPD medium. The suspension was transferred to a 1.5-ml polypropylene microcentrifuge tube and mixed with 0.18 ml of 3HAdoMet (Amersham Life Science, 79 Ci/mmol, 1 mCi/ml in dilute HCl (pH 2.0–2.5):ethanol (9:1, v/v) (final concentration of 1.4 μm) and incubated while shaking at 30 °C for 30 min. Cells were then pelleted at 13,600 × g at room temperature for 1 min, washed twice with 1 ml of water, and used immediately or stored at −80 °C. Isotopically labeled cells were resuspended in 50 μl of 1% sodium dodecyl sulfate (w/v) and 0.67 mm phenylmethylsulfonyl fluoride. Acid-washed glass beads (0.5 mm diameter, Biospec Products) were then added (0.2 g) to the cell suspension, and the tube was vortexed for 1 min followed by an incubation on ice for 1 min. This cycle was repeated 7 times. Approximately 40 μl of this extract was removed with a micropipette tip and placed into a new tube. The beads were washed with an additional 50 μl of the lysis solution and combined with the cell lysate. Aliquots (30 μl) of the cell lysate were mixed with an equal volume of 25% (w/v) trichloroacetic acid in a 6 × 50-mm glass vial and incubated at room temperature for 20 min. The precipitated material was pelleted at 4000 × g for 40 min at 25 °C. The pellets were washed once with acetone (100 μl) at −20 °C, dried, and then acid-hydrolyzed in the presence of 200 μl of 6 n HCl at 110 °C for 20 h in a Waters Pico-Tag vapor-phase apparatus. The hydrolyzed samples were resuspended in 200 μl of water for analysis by cation exchange chromatography. δ-N-Methylornithine was synthesized after the general reductive methylation strategy described by Benoiton (15Benoiton L. Can. J. Chem. 1964; 42: 2043-2047Crossref Google Scholar). α-N-Acetyl-l-ornithine (29 μmol, Sigma) was dissolved in 280 μl of 1 m NaOH in a 12 × 75-mm borosilicate test tube. A molar equivalent of formaldehyde (2.5 μl of 37% (w/w), Fisher) was then added. The reaction proceeded at room temperature, and the solution was mixed periodically over a 20-min period. N-Acetylation of the α-nitrogen atom of the substrate ensured that only the δ-nitrogen atom of ornithine would be modified. Four molar equivalents (116 μmol; 116 μl) of freshly prepared NaBH4 dissolved in 1 m NaOH were added to the mixture, vortexed, and incubated at 15 °C for 1 h with periodic mixing. Approximately 150 μl of 6 n HCl was added dropwise to the mixture until the pH of the solution was less than 2. Aliquots (100 μl) were placed into 6 × 50-mm glass vials and dried under vacuum at room temperature using a Speed Vac (Savant). The sample was then acid-hydrolyzed to remove the α-N-acetyl protection group, as described above. The material in each tube was dissolved in 50 μl of water and then combined and stored at −20 °C. The reaction products were analyzed by thin layer chromatography at room temperature on a 20-cm sheet coated with a 0.2-mm layer of silica 60 (EM Separations, Gibbstown, NJ, no. 5748) with a mobile phase consisting of CH3OH:∼14.8 n NH4OH (3:1, v/v). After heating the sheet at 105 °C in a vacuum oven for 5 min to evaporate the solvents, it was sprayed with ninhydrin (10 mg/ml in acetone) and incubated at 55 °C. Three ninhydrin-positive spots were detected at RF values of 0.37, 0.42, and 0.63. The areas corresponding to these spots on an adjacent lane were scraped into a microcentrifuge tube and submitted to the UCLA Center for Molecular and Medical Sciences Mass Spectroscopy for electrospray analysis. The samples in each tube were dissolved in 400 μl of H2O:CH3CN (50:50, v/v) and centrifuged momentarily to pellet silica debris. An aliquot (20 μl) was mixed with 10 μl of H2O:CH3CN:HCOOH (50:50:0.1, v/v) and scanned in positive ion mode from 120 to 220m/z. We found that the compound migrating atRF = 0.42 had a mass of 133 corresponding to ornithine, the RF = 0.37 compound with a mass of 147 corresponds to δ-N-monomethylornithine, and theRF = 0.63 compound with a mass of 161 corresponds to δ-N,N-dimethylornithine. The reaction products were purified on a preparative basis using the cation exchange column described above. Two ninhydrin-positive peaks were detected at 43–47 min and 50–55 min that were identified by thin layer chromatography as ornithine and as a mixture of δ-N-monomethylornithine and δ-N,N-dimethylornithine, respectively. To separate the mono- from the dimethylornithine species, the 50–55-min peak was pooled, and 1 ml was injected onto a reverse phase HPLC column (Econosphere C18 5 μm column from Alltech, 4.6 mm by 250 mm) equilibrated in 0.1% trifluoroacetic acid/H2O at room temperature. One-min fractions were dried under vacuum, and pellets were resuspended in 10 μl of water and analyzed by thin layer chromatography as described above. δ-N-Monomethylornithine was found to elute within the first 8 min whereas the bulk of δ-N,N-dimethylornithine elutes between 8 and 11 min. We also performed this synthesis with 3HNaBH4 to obtain radiolabeled methyl derivatives. The same procedure was used except 4.4 μmol of sodium methyl-3Hborohydride (NEN Life Science Products, 222 mCi/mmol dissolved in 1 m NaOH, pH 9) was used with 4.4 μmol of NaBH4. Thin layer chromatography showed radioactivity in species at RF = 0.33 and RF = 0.60 corresponding to δ-N-monomethylornithine and δ-N,N-dimethylornithine, respectively. When protein arginine methylation was characterized in a yeastrmt1 mutant deficient in ω-N G ,N G-dimethylarginine formation, a peak of residual 3Hmethyl radioactivity was found to elute just after the expected position of ω-N G ,N G-dimethylarginine (5Gary J.D. Lin W-J. Yang M.C. Herschmann H.R. Clarke S. J. Biol. Chem. 1996; 271: 12585-12594Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). We have now investigated whether this material might represent a new type of methylated arginine derivative. Intact yeast cells were incubated with the isotopically labeled biological methyl donorS-adenosyl-l-methyl-3Hmethionine. Following disruption of the cells, acid hydrolysates of the protein fraction were applied to a high resolution amino acid analysis cation exchange column along with standards of ω-N G-monomethylarginine and ω-N G ,N G-dimethylarginine (Fig. 1). Although the bulk of radioactivity was found in species eluting prior to the methylated arginine derivatives, a well defined peak of 3H radioactivity consistently eluted between the two standards in a position similar to that of the symmetric ω-N G ,N′ G-dimethylarginine derivative. A diagnostic test for arginine derivatives is their lability to base treatment. Breakdown products of arginine in base include ornithine, citrulline, urea, carbon dioxide, and ammonia (16Greenstein J.P. Winitz M. Chemistry of the Amino Acids. II. John Wiley and Sons, Inc., New York1961: 1612Google Scholar). Arginine residues methylated at the terminal (ω) nitrogen atoms have been shown to yield methylamine and methylurea (17Paik W.K. Kim S. J. Biol. Chem. 1968; 243: 2108-2114Abstract Full Text PDF PubMed Google Scholar, 18Paik W.K. Kim S. J. Biol. Chem. 1970; 245: 88-92Abstract Full Text PDF PubMed Google Scholar, 19Nakajima T. Matsuoka Y. Kakimoto Y. Biochim. Biophys. Acta. 1971; 230: 212-222Crossref PubMed Scopus Google Scholar, W.K. Kim S. Biochem. Biophys. Res. PubMed Scopus Google Scholar) base treatment. with ω-N G ,N G-3Hdimethylarginine from similar to that shown in 1 with type Ref. 5Gary J.D. Lin W-J. Yang M.C. Herschmann H.R. Clarke S. J. Biol. Chem. 1996; 271: 12585-12594Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) or with ω-N that a fraction of the radioactivity is to a as methylamine or by treatment with m NaOH for h When the unknown derivative eluting at min (Fig. was to base we radioactivity suggesting that it did not represent of the ω-N forms of of methyl arginine amino m NaOH NaOH m NaOH G,N fractions from the cation exchange column ω-N G,N ω-N and the novel 3H-methylated peak were analyzed for radiolabeled products after base treatment. Aliquots μl) were mixed with NaOH (final concentration of 1 or 2 in microcentrifuge and placed into vials 5 ml of The vials were and placed into a 55 °C oven for or 48 h to of products from the vial to the The vials were then removed and for values are as of in a new fractions from the cation exchange column ω-N G,N ω-N and the novel 3H-methylated peak were analyzed for radiolabeled products after base treatment. Aliquots μl) were mixed with NaOH (final concentration of 1 or 2 in microcentrifuge and placed into vials 5 ml of The vials were and placed into a 55 °C oven for or 48 h to of products from the vial to the The vials were then removed and for values are as of We then the that the derivative an arginine species methylated on the internal nitrogen atom of the guanidino group (Fig. with the from base treatment of such a species would the methylated product δ-N-methylornithine (17Paik W.K. Kim S. J. Biol. Chem. 1968; 243: 2108-2114Abstract Full Text PDF PubMed Google Scholar, 18Paik W.K. Kim S. J. Biol. Chem. 1970; 245: 88-92Abstract Full Text PDF PubMed Google Scholar, 19Nakajima T. Matsuoka Y. Kakimoto Y. Biochim. Biophys. Acta. 1971; 230: 212-222Crossref PubMed Scopus Google Scholar, Y. A. T. Kakimoto Y. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). We synthesized a of δ-N-methylornithine for with the product of the A fraction of the peak from the cation exchange column in 1 was mixed with NaOH to a concentration of 2 m NaOH and incubated at 55 °C for h, as described in than the radioactivity as in the sample was on the cation exchange column and with standards to the We found that the radioactivity in the original unknown 3H-methylated peak at min and was transferred to a new peak of radioactivity in the region (Fig. 3 This peak with the δ-N-methylornithine added as an internal (Fig. 3 the unknown 3H-methylated peak was on the cation exchange column or base treatment (Fig. 3 or incubated at 55 °C for h base (Fig. 3 that the formation of the new 3H-methylated product with δ-N-methylornithine is base treatment of the novel methylated amino acid derivative a species that with the unknown peak eluting just after the ω-N G ,N G-dimethylarginine peak on the cation exchange column from 1 were an aliquot of the μl) was as shown in an aliquot of the μl) was incubated for h at 55 °C and as an aliquot μl) of the was mixed with NaOH to a concentration of 2 The mixture was incubated at 55 °C for h and mixed with 200 μl of 12 n and 40 μl of δ-N-methylornithine was added. The reaction mixture was then with ml of water and as To the identification of fractions the 3H-methylated peak at min in 3 were pooled, and 1 ml was injected onto a reverse phase HPLC column as described under The radioactivity the unknown peak eluted from the HPLC column in a peak with the and in a distinct peak at 8 min not This latter material was dried and to thin layer chromatography as described under We found that radioactivity from the species with the δ-N-monomethylornithine (Fig. provide evidence that the product of the base treatment is and that is in the of the yeast The of methylated arginine derivatives in proteins focused on the of mono- and arginine residues in and (17Paik W.K. Kim S. J. Biol. Chem. 1968; 243: 2108-2114Abstract Full Text PDF PubMed Google Scholar, 18Paik W.K. Kim S. J. Biol. Chem. 1970; 245: 88-92Abstract Full Text PDF PubMed Google Scholar, 19Nakajima T. Matsuoka Y. Kakimoto Y. Biochim. Biophys. Acta. 1971; 230: 212-222Crossref PubMed Scopus Google Scholar, W.K. Kim S. Biochem. Biophys. Res. 29: PubMed Scopus Google Scholar, W.K. Kim S. Biochem. Biophys. Res. 1970; PubMed Scopus Google Scholar). In Paik and Kim W.K. Kim S. J. Biol. Chem. 1970; 245: 88-92Abstract Full Text PDF PubMed Google Scholar) the of in hydrolysates of they found that treatment of derivatives radiolabeled methylurea and product co-chromatographed with a δ-N-methylornithine et T. Matsuoka Y. Kakimoto Y. Biochim. Biophys. Acta. 1971; 230: 212-222Crossref PubMed Scopus Google Scholar) with this result in similar using hydrolysates of proteins from and There appear to be of the of residues in proteins these were In of of these residues in yeast it might now be of interest to whether residues are in cell proteins at they are obscured by the derivatives. The by the methylation of arginine residues at the δ-nitrogen atom is However, it is that such methylation would effectively the of arginine to at this atom P.A. Lee Protein Sci. 1994; PubMed Scopus Google Scholar, L. J.P. Protein Sci. 1995; PubMed Scopus Google Scholar) and for specific the atoms of the same The of the in yeast cells that contain residues, as well as the identification of the that catalyze be in the role of this modification. Although have been of the presence of residues in is a in the on the of the free amino acid in the of Y. Y. M. of the of of Scholar). free the product of has been to in by analysis of Y. A. T. Kakimoto Y. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). δ-N-methylornithine to be a of in J. Chem. PubMed Scopus Google Scholar). The the that these free amino acids from the and of proteins In of the of derivatives on A. M. M. T. Int. 1997; Full Text PDF PubMed Scopus Google Scholar), it would be to has similar that be of in Finally, we that is for the methylation of the δ-nitrogen atom in a arginine derivative than in protein as A well characterized methyltransferase to in the to in and cells 1998; PubMed Scopus Google Scholar). We for mass analysis at the UCLA Center for Molecular and Medical Sciences Mass We also in the for on this