Yeast Ribosomal Protein L12 Is a Substrate of Protein-arginine Methyltransferase 2*

Type III protein-arginine methyltransferase from the yeast Saccharomyces cerevisiae (RMT2) was expressed in Escherichia coli and purified to apparent homogene-ity. The cytosolic, ribosomal, and ribosome salt wash fractions from yeast cells lacking RMT2 were used as substrates for the recombinant RMT2. Using S- adenosyl-L -methionine as co-substrate, RMT2 methylated a pro- tein in the ribosome salt wash fraction. The same protein in the ribosomal fraction was also methylated by RMT2 after pretreating the sample with endonuclease. Amino acid analysis affirmed that the labeling products were (cid:1) - N -monomethylarginines. The methylated protein from the ribosomal or the ribosome salt wash fraction was isolated by two-dimensional gel electrophoresis and identified as ribosomal protein L12 by mass spectrometry. Using synthetic peptides, recombinant L12, and its mutant as substrates, we pinpointed Arg 67 on ribosomal protein L12 as the methyl acceptor. L12 was isolated from wild type yeast cells that have been grown in the presence of S -adenosyl- L -[ methyl - 3 H]methionine and subjected to amino acid analysis. The results indicate that L12 contains (cid:1) - N -monomethylarginines. Proteins can have a large variety of post-translational mod-ifications, including the N -methylation against a comprehensive nonredundant protein sequence data base (NCBInr) using the Mascot program (23) for protein identification. The postsource decay fragment ion spectra were acquired with adrenocorti- cotropic hormone fragment 18–39 as a standard.

the ␦-N-monomethylarginines. RMT2 is a protein of 412 amino acid residues, including the initiator methionine. It shares 23 and 22% sequence identity with yeast RMT1 and the rat PRMT1, respectively. Noticeably, the protein contains the sequence motif (GXGXG) conserved in S-adenosyl-L-methioninedependent methyltransferases (16). However, the substrate(s) for RMT2 has not been identified in either in vivo or in vitro labeling experiments.
We report here that ribosomal protein L12 from the YDR465c disruption mutant (⌬RMT2) can be specifically labeled by recombinant RMT2 heterologously expressed in Escherichia coli. We affirm that the reaction product is ␦-N-monomethylarginine. We demonstrate with synthetic peptides, recombinant L12, and its mutant that Arg 67 on ribosomal protein L12 is the methyl acceptor of RMT2. Furthermore, L12 isolated from wild type yeast strain contains ␦-N-monomethylarginine.
Expression and Purification of RMT1 and RMT2-Yeast ORFs encoding YBR034c and YDR465c were used as templates in PCR experiments. Primers employed in the cloning experiments are listed in Table I. YBR034c was amplified with PCR using primers 1 and 2. The product was restricted with NdeI and XhoI and then inserted into the pET-15b vector and transformed into E. coli BL21(DE3) for protein expression. The recombinant protein (His-RMT1) has a His tag at the N terminus and was purified on a Ni 2ϩ affinity column according to the manufacturer's instructions. The recombinant proteins in this study (His-RMT1, His-RMT2, and RMT2) have the tendency to form aggregates. All buffers used in the chromatographic steps contain 10% glycerol for protein stabilization. Soluble proteins (15 mg/ml for His-RMT1, 2-5 mg/ml for His-RMT2, 7-9 mg/ml for RMT2) were separated from precipitates by centrifugation and stored at Ϫ80°C in the elution buffer containing 1 M imidazole.
YDR465c contains an NdeI site near the C terminus of the coding sequence. PCR was carried out with primers 3 and 4 to eliminate this NdeI restriction site. The product was then combined with primer 5 in another PCR experiment to obtain the complete coding sequence of RMT2. The PCR product was restricted with NdeI and XhoI and then inserted into pET-15b vector. The resulting plasmid is designated as pET-RMT2. Transformation into bacterial cells, expression of the Histagged protein (His-RMT2), and purification with metal affinity column were carried out as outlined above. Samples from the affinity column were further separated from low molecular weight contaminants on a gel filtration column eluted with buffer I (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 6 mM ␤-mercaptoethanol, and 10% glycerol).
Alternatively, YDR465c was PCR-amplified from pET-RMT2 with primers 5 and 6. The product was inserted into the NdeI and SalI sites of the pBAce vector and transformed into Escherichia coli DH5␣ cells for protein expression at 30°C as described (17). The recombinant protein (RMT2) thus expressed does not have a His tag.
Cells containing RMT2 were lysed in buffer II (40 mM Tris-HCl, pH 8.0, 100 mM NaCl, 6 mM ␤-mercaptoethanol, and 10% glycerol), and the debris was removed by ultracentrifugation in a Beckman Type 70 Ti rotor at 50,000 rpm for 2.5 h. The supernatant was loaded directly onto a Fast Q anion exchanger equilibrated in buffer II. The column was eluted with an NaCl gradient, and RMT2 came off of the column at ϳ0.4 M NaCl. Fractions containing RMT2 were collected and diluted an with equal volume of buffer II. The sample was then loaded onto a prepacked Mono Q column (1.0 ϫ 10 cm) and eluted with an NaCl concentration gradient. The purity of the RMT2 preparation was examined by SDS-PAGE, and the molecular mass of the recombinant protein was confirmed by electrospray ionization-mass spectrometry (18).
Creation of Yeast Strains Lacking RMT1 (⌬RMT1) and RMT2 (⌬RMT2)-S. cerevisiae strain SEY6210 was used in this study. A DNA fragment in which TRP1 is flanked by the 5Ј and 3Ј sequences of RMT1 was obtained by PCR amplification of a plasmid encoding the TRP1 gene (pRS404). The primers used for amplification contain the flanking sequences of TRP1 (underlined) and either the 5Ј (primer 7) or 3Ј (primer 8) sequence of RMT1 (Table I). The amplified DNA fragment was transformed into the haploid strain SEY6210 to disrupt the genomic RMT1 by homologous recombination. Transformants with disrupted RMT1 gene (⌬RMT1) were screened and verified by PCR.
The yeast strain lacking RMT2 (⌬RMT2) was obtained similarly. A DNA fragment in which HIS3 is flanked by the 5Ј and 3Ј sequences of RMT2 was PCR-amplified from pRS403 with hybrid primers containing the flanking sequences of HIS3 (underlined) and either the 5Ј (primer 9) or 3Ј (primer 10) sequence of RMT2 (Table I). The amplified DNA fragment was transformed into the haploid strain SEY6210 for homologous recombination. Transformants with disrupted RMT2 were screened and verified by PCR.
Expression and Purification of Yeast Ribosomal Protein L12 and Its Mutant-YDR418W, the ORF encoding yeast ribosomal protein L12B, was obtained by amplifying genomic DNA from S. cerevisiae strain S288C with primers 11 and 12 (Table I). The PCR product was restricted with NdeI and SalI and then inserted into the pBAce vector (pBA-L12) for protein expression as outlined above.
Yeast ribosomal protein L12 with an Arg 67 to lysine substitution was generated with the QuikChange site-directed mutagenesis kit according to the manufacturer's instructions. The pBA-L12 vector was used as template, and primer 13 (Table I) was used as the mutation primer. Cell transformation and protein expression were carried out as outlined above.
Ribosomal protein L12 and its mutant were expressed as inclusion bodies. DH5␣ cells containing recombinant proteins were lysed in buffer III (50 mM sodium phosphate, pH 7.2, 6 mM ␤-mercaptoethanol, and 0.25 M sucrose) at a concentration of 75 A 600 units/ml. After cell lysis, the debris and the recombinant proteins were pelleted in a Sorvall SS34 rotor at 10,000 rpm for 20 min. The recombinant proteins were then dissolved in buffer IV (0.25 M Tris, pH 8.5, 6 M guanidinium chloride, and 1 mM EDTA) and separated from cell debris and organelles by centrifugation in a Beckman Type 70.1 Ti rotor at 50,000 rpm for 3 h. The recombinant proteins were then aliquoted and stored at Ϫ80°C until use.
Fractionation of Yeast Extracts-Yeast strains were grown with constant shaking at 30°C in YPD medium (1% (w/v) yeast extract, 2% (w/v) bactopeptone, and 2% D-glucose) until A 600 ϭ 2. Cells were collected by centrifugation and washed three times with buffer V (50 mM sodium phosphate, pH 7.0, 1 mM EDTA and 1 mM EGTA. The wet pastes were used immediately or stored at Ϫ80°C. Cells from 4 liters of culture were suspended in 50 ml of buffer V in the presence of protease inhibitors (1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 2 g/ml of aprotinin, leupeptin, and pepstatin A). Cell breakage was induced by passing the suspension through a Microfluidizer (Avestin, Ottawa, Canada) at 25,000 p.s.i. 7-9 times. Cell debris was removed by centrifugation in a Beckman Type 70 Ti rotor at 20,000 rpm for 30 min. The supernatant was collected and layered on top of a 10% sucrose cushion, and centrifugation was carried out in a Type 70 Ti rotor at 50,000 rpm for 4 h. The supernatant was designated as the cytosolic fraction (C) and stored at Ϫ80°C after dialysis against buffer V. The crude ribosomal pellet was resuspended overnight at 4°C in buffer VI (50 mM sodium phosphate, pH 7.0, 20 mM MgCl 2 , and 0.5 M NH 4 Cl). Insoluble particulate was removed by spinning in a Type 70 Ti rotor at 20,000 rpm for 30 min. The supernatant was then spun down in a 10% sucrose cushion as mentioned above and designated as the ribosome salt wash (S) fraction. The pelleted ribosome (R) was redissolved in buffer V. Both fractions were dialyzed extensively against buffer V before storage at Ϫ80°C.
Protein-arginine Methyltransferase Assay-The cytosolic (C), R, and S fractions from SEY6210, ⌬RMT1, and ⌬RMT2 strains were used as methyl acceptors in the assays. The reactions were carried out in buffer V at a final volume of 40 l. The reaction mixture contained 340 g of protein, 5 g of methyltransferase, and 0.  . Incubation was carried out at 30°C for 2 h. Proteins were precipitated with trichloroacetic acid and collected on glass fiber filters for scintillation counting or redissolved in sample buffer for SDS-PAGE analysis.
Electrophoretic Analysis of Proteins-The methylated proteins were boiled with an equal volume of SDS-PAGE sample buffer (19) for 5 min. Typically, one-twentieth of the assay mixture was separated on a precast Novex 4 -20% polyacrylamide gel at 125 V for ϳ120 min. Proteins on the gels were visualized with Coomassie Blue dye staining. After destaining, the gels were soaked in Enlightening Enhancer, dried, and exposed to x-ray films (X-Omat AR; Eastman Kodak Co.) at Ϫ80°C.
Isoelectric focusing was carried out on 13-cm Immobiline DryStrips (pH 3-10 and 6 -11) with the IPGphor system. The DryStrips were rehydrated overnight at room temperature in 240 l of 8.5 M urea, 2.25% CHAPS, 0.5% (v/v) Pharmalyte 3-10, and 15 mM dithiothreitol (DTT). Proteins in samples for isoelectric focusing were quantified by the Bradford assay (20) and then precipitated with 2 volumes of 20% trichloroacetic acid and washed at least three times with cold acetone to remove the acid. Pellets were dissolved in 100 l of isoelectric focusing sample buffer (9 M urea, 4% CHAPS, 0.8% (v/v) Pharmalyte 3-10, and 15 mM DTT), loaded onto the DryStrips with a sample cup, and focused for 32,000 V-h for the pH 3-10 DryStrips. An additional 0.5% (v/v) Pharmalyte 9 -11 was included in the sample buffer for runs with pH 6 -11 DryStrips. After focusing, the DryStrips were equilibrated in 10 ml of isoelectric focusing equilibration buffer (50 mM Tris-HCl, pH 6.8, 6 M urea, 4% SDS, 30% glycerol, 100 mM DTT, and 0.01% bromphenol blue) and layered on top of a vertical 15% SDS-polyacrylamide gel for the second dimension separation.
Amino Acid Analysis of Proteins and Peptides Methylated in Vitro-Proteins from the methylation assays were transferred to 6 ϫ 50-mm glass vials and precipitated with 2 volumes of 20% trichloroacetic acid. The protein pellets were hydrolyzed with gaseous HCl at 110°C for 24 h in a Waters (Milford, MA) Pico-Tag work station. Amino acids released were analyzed on an AA511 cation exchanger at 0.5 ml/min driven by a Waters model 510 HPLC pump. Eluents were collected for ninhydrin test or scintillation counting. Base treatment of the candidate ␦-Nmonomethylarginine peak was carried out according to the method of Zobel-Thropp et al. (3). The ␦-N-monomethylornithine used as a standard was a generous gift from Dr. Steven Clarke (UCLA, Los Angeles, CA).
N-terminal biotinyl peptides captured on streptavidin-agarose beads as described above for the protein-arginine methytransferase assay were further washed with water to replace buffer VII. The beads were then resuspended in 10% (v/v) acetic acid and boiled for 10 min. The supernatant was then passed through a filtration unit equipped with a 10-kDa cut-off membrane, and the filtrate was dried in vacuo to completion. The peptides thus collected were hydrolyzed and analyzed as outlined above.
Amino Acid Analysis of Proteins Methylated in Vivo-The SEY6210 and ⌬RMT2 yeast cells were labeled in vivo with [ 3 H]AdoMet for 60 min as described (16). The cells were harvested and resuspended in 50 l of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 2 g/ml of leupeptin and pepstatin) and then lysed by vortexing with acid-washed glass beads (425-600 m; Sigma). The lysate was collected, and the glass beads were washed repeatedly with buffer VIII (50 mM Tris-HCl, pH 7.4, 50 mM KCl, and 5 mM Mg(OAc) 2 ). These fractions were combined and cleared by centrifugation at 12,000 ϫ g for 15 min. The ribosomes in the combined fractions were pelleted by spinning at 427,300 ϫ g for 2 h. The ribosomes were then resuspended in 150 l of buffer IX (50 mM Tris-HCl, pH 7.4, 500 mM KCl, 5 mM Mg(OAc) 2 , and 1 mM DTT). Two volumes of glacial acetic acid were added, and the magnesium ion concentration was raised to 0.1 M for rRNA precipitation. The rRNA was removed by centrifugation, and the proteins in the supernatant were dialyzed overnight at 4°C against 66.7% acetic acid. Proteins in the sample was then dried down in vacuo, separated by SDS-PAGE, and visualized by Coomassie Blue dye staining and fluorography. Recombinant L12 was loaded on the same gel to facilitate the localization of the corresponding in vivo labeled protein. L12 is one of the two major methylated ribosomal proteins on the fluorograph (data not shown). The in vivo methylated L12 was excised from the gel and eluted overnight by diffusion in 100 l of 0.3 M (NH 4 )HCO 3 and 0.5% SDS. The eluted protein was then dried in vacuo and hydrolyzed for amino acid analysis as described above.
Identification of the Methylated Protein-Overlaying the fluorograph with the two-dimensional polyacrylamide gel identified the methylated protein. The protein was excised from the gel with a pipette tip. In gel digestion was performed essentially as described by Li et al. (21) with minor modifications. The gel plug was washed extensively with destaining solution (10% acetic acid and 50% methanol in water). It was then incubated in the digestion buffer (0.05 M Tris-HCl, pH 8.0, and 20 mM CaCl 2 ) for 10 min. The buffer was removed, and the gel plug was dehydrated by adding acetonitrile followed by vacuum centrifugation. Trypsin solution (0.0375 mg/ml in digestion buffer) was then added to restore the gel plug to its original volume. Digestion was carried out at 25°C overnight. Resulting peptides in the gel plug were extracted sequentially with 60% acetonitrile in digestion buffer, 60% acetonitrile in water with 0.1% trifluoroacetic acid and acetonitrile. The solutions were combined, and the acetonitrile was removed by vacuum centrifugation. The solution was further cleaned up with a ZipTip pipette tip before MALDI-TOF analysis.
Peptide mass mapping was performed on a Bruker (Bruker-Daltonics, Bremen, Germany) REFLEX III time-of-flight mass spectrometer equipped with the SCOUT source and delayed extraction. Detection was set in positive ion reflector mode with each mass determination being an average of 100 spectra. Samples for mass measurement were prepared using the solution-phase nitrocellulose method (22)

RMT2 Methylates Yeast Ribosomal Protein L12
against a comprehensive nonredundant protein sequence data base (NCBInr) using the Mascot program (23) for protein identification. The postsource decay fragment ion spectra were acquired with adrenocorticotropic hormone fragment 18 -39 as a standard.

RESULTS AND DISCUSSION
Expression and Purification of RMT2 and RMT1-Yeast ORF YDR465c was amplified with PCR and heterologously expressed in E. coli with a His tag at the N terminus (His-RMT2). His-RMT2 was isolated on a Ni 2ϩ affinity column, and the preparation was ϳ60% pure. Recombinant proteins in the fractions eluted off of the affinity column were highly concentrated, and approximately half of the proteins were precipitated out of solution. The purity of the soluble protein can be improved to Ͼ90% by passing through a preparative Superose 12 gel filtration column (1.6 ϫ 50 cm). His-RMT2 thus obtained has a molecular mass of 49,503 Ϯ 2 Da (predicted molecular mass ϭ 49,501 Da) by electrospray ionization mass spectrometry. We obtained ϳ15 mg of purified His-RMT2 from a liter of cell culture.
To overcome the precipitation problem of His-RMT2 on the affinity column, we expressed RMT2 without the His tag. We put the gene under the control of the phoA promoter that turns on upon phosphate starvation (24). The recombinant protein was purified on two consecutive anion exchangers, and we obtained ϳ38 mg of protein per liter of cell culture. The protein preparation is estimated at 95% pure with a molecular mass of 47,339 Ϯ 2 Da (predicted molecular mass ϭ 47,338 Da), as determined by electrospray ionization mass spectrometry.
PRMTs have been reported to exist in multimeric forms (12,25). In contrast, RMT2 exists mainly as a monomer. The chromatographic property of the purified RMT2 (9 mg/ml) was examined on an analytical Superdex 75 column. The RMT2 sample exhibited two UV (280 nm)-absorbing peaks that eluted before aldolase (158 kDa) and after bovine serum albumin (67 kDa) (data not shown). Both peaks gave a single protein band of identical electrophoretic mobility on a denaturing gel (data not shown). Therefore, they represent the monomeric and multimeric forms of RMT2. However, the amount of monomeric RMT2 is at least 10-fold higher than that of the multimeric form (data not shown). Among PRMTs, only the rat PRMT3 has been reported to exist in a monomeric form (6).
His-RMT1 was used in methylation assays as control. The recombinant protein was expressed from a pET vector in E. coli and was more than 90% pure after passing through a Ni 2ϩ affinity column. We routinely obtain over 110 mg of protein/ liter of cell culture. His-RMT1 exists as a dimeric protein as detected by gel filtration chromatography (data not shown). We could not detect the monomeric or other multimeric forms of this enzyme by gel filtration chromatography.
In Vitro Methylation of Fractionated Proteins from Yeast Extracts-Proteins from the wild type and the RMT1 and RMT2 deletion strains were enriched by fractionation before the methylation assay. Membranes and associated proteins were discarded, and the ribosomal fractions were separated from the cytosolic proteins through two sequential centrifugation steps. The pellet thus obtained was incubated in high salt (0.5 M NH 4 Cl) to dissociate loosely bound proteins from the ribosome. A further centrifugation step through a sucrose cushion was used to separate the ribosome from the ribosome salt wash fraction. The protein profiles of these three fractions from the ⌬RMT2 strain on a denaturing gel are shown in Fig. 1A, and they are similar to those from the wild type and the ⌬RMT1 strains (data not shown).
The cytosolic, ribosomal, and ribosome salt wash fractions from the wild type, ⌬RMT1, and ⌬RMT2 strains were used as methyl acceptors for His-RMT2. Proteins from the methylation assay were separated on SDS-PAGE and visualized by fluorography. The results presented in Fig. 1B represent an x-ray film that has been exposed for 37 h. Protein(s) with a molecular mass of ϳ20 kDa on denaturing polyacrylamide gel from the S fraction of the ⌬RMT2 strain can act as a methyl acceptor for His-RMT2. Small peptides (Ͻ5 kDa) in the ribosomal fraction of the wild type and ⌬RMT2 strains can also be methylated. They probably represent degraded proteins and were not further investigated. Upon prolonged exposure (72 h), a band at ϳ18 kDa can be detected in the cytosolic fraction of the wild type yeast (data not shown). We have used RMT2 and His-RMT2 in these experiments. We have not noticed any difference in the labeling pattern or band intensity. We concluded that the His tag has no influence on the enzymatic activity of RMT2. His-RMT2 and RMT2 are used interchangeably hereafter in this investigation.
Methylation of the protein(s) that we observed was not catalyzed by endogenous methyltransferases, in that proteins from ⌬RMT2 were not methylated in the absence of exogenous recombinant His-RMT2 (data not shown). The protein(s) la- FIG. 1. RMT2 specifically methylates a yeast ribosomal protein. Cytosols from SEY6210 (WT), yeast strain with YBR034c/RMT1 deletion (⌬RMT1), or yeast strain with YDR465c/RMT2 deletion (⌬RMT2) were separated into the C, R, and S fractions. The 4 -20% SDS-polyacrylamide gel protein profiles of these fractions from ⌬RMT2 before the methylation assay are presented in A and visualized with Coomassie Blue staining. These fractions from wild type, ⌬RMT1, and ⌬RMT2 were used as methyl acceptors for the recombinant His-RMT2 (B). To examine the effect of endonuclease treatment, these three fractions from ⌬RMT2 were preincubated with (ϩ) or without (Ϫ) Benzonase (300 units) at 30°C for 30 min prior to the methylation assay (C). After the methylation reaction, one-twentieth of each sample (17 g) was separated on a 4 -20% SDSpolyacrylamide gel, and the tritium-labeled proteins were visualized by fluorography. The fluorographs represent a 37-h exposure. beled by His-RMT2 differs from that methylated by His-RMT1; His-RMT1 methylated predominantly two proteins of 40 and 10 kDa in the ribosome salt wash fraction from ⌬RMT2 (data not shown).
RNase pretreatment of yeast and mammalian cell extracts can affect the in vitro methylation pattern of type I PRMTs (26). To test whether a similar phenomenon can be observed for RMT2, we pretreated the fractionated yeast extracts with endonuclease (Benzonase; Sigma) at 30°C for 30 min prior to the methylation assay. The proteins were separated on SDS-PAGE, and the isotopically labeled proteins were visualized by fluorography. Fractions from ⌬RMT1 were also treated with endonuclease and methylated with His-RMT1 as controls. As reported by Frankel and Clarke (26), the intensity of some of the protein bands methylated by His-RMT1 was either enhanced or diminished upon endonuclease treatment (data not shown). As for the protein band labeled by His-RMT2 in the ribosome salt wash fraction of ⌬RMT2, we observed a slight increase in intensity upon endonuclease treatment (Fig. 1C). In addition, a protein at approximately the same molecular weight was methylated in the ribosomal fraction of the ⌬RMT2 extracts.
Analyses of the RMT2 Methylation Products-Proteins from the methylation assay were precipitated with trichloroacetic acid and collected by centrifugation. The pellet was hydrolyzed with HCl, and the amino acids were analyzed on a cation exchanger. Part of the chromatogram of the amino acids from the ribosome salt wash fraction of ⌬RMT2 methylated with His-RMT2 is shown in Fig. 2A. The radioisotopes were eluted between -N G ,N G -dimethylarginine (ADMA) and -N Gmonomethylarginine (MMA). As a control, hydrolysate from the ribosome salt wash fraction of ⌬RMT1 methylated with His-RMT1 was analyzed, and the chromatogram is presented in Fig. 2B. The retention times of the radioisotope peaks coincide with those of the ADMA and the MMA standards. The results suggest strongly that RMT2 is not a type I arginine methyltransferase. Note also that the scale of the y axis in Fig.  2B is 5-fold larger than that of Fig. 2A. With our assay conditions, RMT1 has a much higher enzymatic activity than that of RMT2.
To confirm the identity of the 3 H-labeled residue, the radioisotopic peak from Fig. 2A was collected and base-hydrolyzed. After acidification and dilution with water, the radioisotopes were mixed with ␦-N-monomethylornithine and rechromatographed on the same column. The radioisotopes co-eluted with the ␦-N-monomethylornithine (Fig. 2C). Therefore, RMT2 is a ␦-N-monomethylarginine transferase as suggested by Niewmierzycka and Clarke (16).
Identification of in Vitro Methylated Protein-The ⌬RMT2 ribosome salt wash fraction was incubated with His-RMT2 and [ 3 H]AdoMet. The proteins were separated on a linear pH 3-10 Immobiline DryStrip in the first dimension, followed by SDS-PAGE on a 15% homogeneous gel in the second dimension. The silver-stained pattern of 170 g of proteins thus separated is shown in Fig. 3A. The fluorograph of the same gel is presented in Fig. 3B and shows clearly that a single spot with high pI and molecular mass around 20 kDa was labeled.
In order to resolve this methylated protein from other contaminants, the mixture after labeling was separated by twodimensional electrophoresis with a pH 6 -11 Immobiline DryStrip in the first dimension. Comparing the silver-stained gel with the fluorograph identified the methylated protein as marked in Fig. 4A. The spot was then excised from the gel and digested with trypsin. The molecular masses of the resulting peptides were determined on a MALDI-TOF mass spectrometer and searched against the nonredundant data base of NCBI using the MASCOT program (available on the World Wide Web at www.matrix-science.com). The masses of nine peptides covering 99 residues can be matched with those deduced from the amino acid sequence of yeast ribosomal protein L12 (Fig. 5; accession number gi 6320625). The deviations between the experimental results and theoretical calculations are 47-130 ppm. The most abundant ion (m/z 1240.8) on the spectrum was generated from a peptide with nonspecific proteolytic cleavage covering residues 61-71 (QLKIQNRQAAA) of L12. The partial sequence of this peptide was deduced from a postsource decay spectrum (data not shown) and affirms that the protein spot represents ribosomal protein L12.
Careful examination of the L12 protein spot revealed that there is a protein located right next to it on the basic side of the gel. We identified this protein as the mitochondrial HMG-1 homolog (accession number gi 6323717). Other proteins that were located near to L12 (Fig. 4) were identified as ribosomal proteins S19 (accession number gi 6324027) and S20 (accession number gi 6321772). Interestingly, in our gel system, S19 has FIG. 2. Amino acid analysis of methylated proteins. A, the S fraction of ⌬RMT2 was incubated with His-RMT2 and [ 3 H]AdoMet. The proteins were precipitated with trichloroacetic acid and then hydrolyzed with HCl. The released amino acids in 0.2 N sodium citrate, pH 2.0, were mixed with MMA and asymmetric ADMA as internal standards and then analyzed on an AA511 column that had been equilibrated in 0.35 N sodium citrate, pH 5.28, and 60°C. The column was eluted at 0.5 ml/min, and fractions were collected for scintillation counting (OE--OE) and ninhydrin assay (--). B, the S fraction of ⌬RMT1 was incubated with His-RMT1 and [ 3 H]AdoMet. The resulting proteins were hydrolyzed and analyzed similarly. C, the radioactive peak from A was collected, and NaOH was added to give a final concentration of 2 M. The mixture was incubated at 55°C for 24 h and then acidified with onefifth volume of 12 N HCl. The mixture was then diluted 10-fold with water, and ␦-N-monomethylornithine was added as internal standard. The sample was then analyzed on an AA511 column as outlined for A. a faster electrophoretic mobility than S20. Whether the slower migration rate of S20 is due to post-translational modification or other factors remains to be investigated.
Yeast ribosomal protein L12 (27) is also known as YL23 and L15 (28,29). It contains dimethyllysine(s), trimethyllysines(s) (30), and methylarginine(s) (31). It can complex with rRNA in the presence of 0.5 M LiCl (32). Incubating the ribosome with 0.5 M NH 4 Cl generated the ribosome salt wash fraction in this study. This incubation step has been routinely used to generate prokaryotic ribosome and usually does not result in removing ribosomal proteins (33). L12 and a group of acidic proteins can be removed from the ribosome by incubating the macromolecular complex in 1 M ammonium salt and 50% ethanol (34,35).
We compared the protein profiles of the ribosomal and ribosome salt wash fractions from wild type and ⌬RMT2 yeast strains of our preparations. We loaded equal amounts of protein (85 g) on each gel, and the proteins were visualized by silver staining. The regions covering L12 and the surrounding proteins (mitochondrial HMG-1 homolog, S19, and S20) are presented in Fig. 4, B-E. Apparently, L12 and the HMG-1 homolog are more abundant in the ribosome salt wash fraction of ⌬RMT2 than that of the SEY6210. In our gel system, we cannot detect the acidic proteins (P1B, P2A, and P2B; pI values from 3.8 to 3.9) that normally associate with L12. We speculate that L12 binds less tightly with the ribosome in the ⌬RMT2 cells.
Upon nuclease digestion, a protein from the ribosomal fraction of ⌬RMT2 was methylated (Fig. 1C). Further analyses with two-dimensional gel electrophoresis and mass spectrometry revealed that the methylated protein is also L12 (data not shown). Seemingly, rRNA and/or other ribosomal proteins protect the methylation site on L12. The same nuclease treatment of fractions from wild type yeast cells did not generate a new methylation site for His-RMT2 (data not shown). Probably, the endonuclease treatment of the wild type ribosome is insufficient in disrupting its structure and exposing the methylation site on L12. Niewmierzycka and Clarke (16) have shown that inhibition of protein synthesis prevents in vivo methylation of the RMT2 substrate. The data can be interpreted as methylation occurs either before protein folding or its assembly into the ribosome.
The methylation site on L12-PRMTs usually methylate substrates containing glycine-and arginine-rich sequences in Arg-Gly-Gly or Arg-Xaa-Arg contexts (1, 10, 36). Recently, Mowen et  (A and B), the ⌬RMT2 ribosomal (C), the SEY6210 ribosome salt wash (D), and the SEY6210 ribosomal (E) fractions were incubated with His-RMT2 and AdoMet. The resulting proteins (85 g) were separated first on a 13-cm pH 6 -11 Immobiline Drystrip and then a 15% SDS-polyacrylamide gel. The proteins were visualized with silver staining. The asterisk denotes the protein that co-migrated with the tritium label when [ 3 H]AdoMet was used in the methylation assay. B-E represent the regions on twodimensional gel that are similar to that enclosed in A. Proteins that have been identified independently from each sample with MALDI-TOF mass spectrometry are labeled.
al. (37) reported that Arg 31 on the signal transducer and activator of transcription 1 (STAT1) can be methylated by PRMT1. The methylated arginine is situated N-terminally to a glutamine residue (PMEIRQYL). S. cerevisiae ribosomal protein L12 has 165 residues (including the initiator methionine) and eight arginines. Two of them (Arg 67 and Arg 114 ) have adjacent glutamine residues, and an RDR sequence is located at residues 90 -92. We constructed synthetic peptides (Table II)  A tyrosine residue was added to the C terminus of peptides 2, 3, 4, and 6 for quantification purpose. An additional leucine residue was added to the N-terminal of peptide 5, so that the penultimate aspartic acid would not convert into isoaspartic acid during peptide synthesis.
After the methylation reaction, the peptides were pulled down by streptavidin beads, and the tritium labels incorporated were detected by scintillation counting. Only peptide 2 was radioactively labeled (data not shown). We then combined peptides from 10 methylation assays for amino acid analysis. The data are presented in Fig. 6A and confirm that peptide 2 has a tritiated ␦-N-monomethylarginine. Peptide 4 was also submitted to amino acid analysis. The results show clearly that Arg 114 , although it has an adjacent glutamine, is not a methyl acceptor for RMT2 (Fig. 6A). This result also eliminates the possibility that the isotopes detected on peptide 2 were the consequence of arginine side chain tritium exchange. Therefore, using these synthetic peptides, we have shown that RMT2 methylates only Arg 67 and none of the other seven arginines in L12.
To affirm that Arg 67 on L12 is the methyl acceptor of RMT2, we expressed L12 and its R67K mutant in E. coli for in vitro assays. The total cell lysates from DH5␣, cells expressing L12, and cells expressing its mutant are presented in Fig. 7 (lanes  1-3). The identity of yeast ribosomal protein L12 was confirmed by N-terminal sequencing after the overexpressed protein was electrophoretically transferred onto polyvinylidene difluoride membrane (data not shown). L12 is clearly a methyl acceptor in the presence of RMT2 and [ 3 H]AdoMet (Fig. 7B, lane 4). The R67K mutant (Fig. 7B, lane 6) or the recombinant wild type L12 without RMT2 in the assay mixture (Fig. 7B, lane 5) was not labeled.
The methylated recombinant L12 was further subjected to amino acid analysis (Fig. 6B). The tritium labels have the same retention time on the ion exchange column as that of the hydrolysate from peptide 2 (Table II, Fig. 6A) or that of the S fraction of ⌬RMT2 that has been incubated with His-RMT2 and [ 3 H]AdoMet ( Fig. 2A). We collected the tritium labels from Fig.  6B and subjected them to base hydrolysis. Upon rechromatography on the same column, the tritium labels co-migrated with ␦-N-monomethylornithine (data not shown). Therefore, after reacting with RMT2, the modified amino acid on the recombinant L12 is ␦-N-monomethylarginine.
In Vivo Methylation of Yeast Ribosomal Protein L12-Kruiswijk et al. (31) have reported the presence of methylated arginine and lysine residues on L12. Although they could not distinguish whether the arginine residue(s) was modified at the ␦or -position, they estimated that there was only 0.09 -0.20 mol of methyl group/mol of modified ribosomal protein species.
To show that L12 is methylated in vivo, we grew wild type and ⌬RMT2 yeast cells in the presence of [ 3 H]AdoMet. L12 was then isolated electrophoretically and subjected to amino acid analysis. Tritium labels with retention time similar to that of ␦-N-  5. MALDI-TOF mass spectrum of the peptide digests generated from the spot marked with an asterisk in Fig. 4. Ions with masses that can match with those deduced from the yeast ribosomal protein L12 sequence are labeled. The amino acid residues that the ions encompass are given in parentheses. Part of the spectrum is enlarged 40-fold and presented as an inset. monomethylarginine were observed for the sample from the wild type yeast cells (Fig. 6C). This tritiated peak was absent from the L12 isolated from the ⌬RMT2 strain (Fig. 6C). We also observed tritium labels that co-eluted with the trimethyllysine standard. Therefore, L12 is methylated at both arginine and lysine residues as suggested by Kruiswijk et al. (31).
Type I and II methyltransferases have been shown to utilize peptides with a glycine-and arginine-rich region as substrates (1). In the case of signal transducer and activator of transcription 1, a methyl acceptor for PRMT1, the modified arginine is between residues with aliphatic and polar side chains (PMEIRQYL (37)). Peptide 4 (Table II) can also be a substrate for RMT1 (data not shown), although the amount of methyl group incorporated was at most half of that of a peptide having a sequence GGYGGRGGYG. The substrate specificity of PRMT1 is not as stringent as previously proposed (1). Conversely, the ␦-N-monomethylarginine on L12 is preceded and followed by polar residues (IQNRQA). Whether RMT2 can modify other proteins and the substrate specificity of this enzyme remain to be elucidated.
A peptide that encompasses residues 61-71 of L12 (m/z 1240.8) was detected in the MALDI-TOF mass spectrum (Fig.  5). The methyl-accepting arginine, Arg 67 , is located in this fragment. Close scrutiny of the mass spectrum reveals an ion with an additional 14 daltons (m/z 1254.7; Fig. 5, inset). This ion possibly represents the methylated form of the peptide. Assuming the methylated and unmethylated forms of the peptide have the same ionization efficiency, ϳ1% of the peptide in the sample is methylated. This estimate is only slightly higher than that reported by Kruiswijk et al. (31). Histone H3 (38) and H4 (39) are substrates of coactivatorassociated arginine methyltransferase 1/PRMT4 and PRMT1, respectively. Amino acid analysis of histone H3 isolated from calf thymus revealed that only 3.7% of the molecules contain methylated arginine (38). N-terminal peptides with and without methylation at Arg 3 have been recovered from tryptic digests of calf thymus histone H3 (39). Therefore, the level of in vivo protein-arginine methylation can be rather low and not stoichiometric.
We have detected an ion at m/z 727.5 (Fig. 5). The mass and the postsource decay spectrum of this ion match with those of a peptide covering residues 12-16 of L12 (YLYLR). We have also electroblotted L12 from a two-dimensional gel onto a membrane for sequence determination. The protein is N-terminally blocked. Therefore, the mature L12 does not start from residue 17 as reported on the Saccharomyces Genome Data base (available on the World Wide Web at genome-www.standford.edu/ Saccharomyces).
The yeast ribosomal protein L12 is immunologically and functionally equivalent to the bacterial protein L11 (35). The prokaryotic L11 is located in the GTPase center of the ribosome and is the target for a family of thiazole antibiotics (40). The structure of L11 from T. maritima complexed with RNA is known (41). L11 has a two-domain structure: the N-terminal domain and the RNA binding C-terminal domain. Upon alignment, Arg 67 of L12 is located in the N-terminal domain and is not conserved among the L11 homologs from bacteria and yeast (41). However, yeast L12 has only 23% sequence identity with the T. maritima L11 and may not share similar structures. The consequence of Arg 67 methylation on the structures and functions of L12 and the ribosome at large remains to be investigated.
In summary, we have heterologously expressed the yeast arginine methyltransferase RMT2 in E. coli. With [ 3 H]AdoMet FIG. 6. Amino acid analyses of methylated peptides and proteins. A, peptide 2 (LKIQNRQAAASY; OE--OE) and 4 (IIEIAR-QMRDKSFGY; q--q) were incubated with His-RMT2 and [ 3 H]AdoMet. The resulting peptides from 10 assays were isolated for amino acid analysis as outlined under "Experimental Procedures" and in Fig. 2A. B, purified recombinant L12 (22.5 g) was incubated with RMT2 (1.5 g) and [ 3 H]AdoMet at 30°C for 2 h. After trichloroacetic acid precipitation, the protein mixture was subjected to acid hydrolysis, and the released amino acids were analyzed as in Fig. 2. C, L12 from wild type (q--q) and ⌬RMT2 (OE--OE) yeast cells that have been grown in the presence of [ 3 H]AdoMet were isolated and subjected to amino acid analysis. Trimethyllysine (TMK), ADMA, and MMA were included as internal standards and detected at 570 nm (--) after reacting with ninhydrin. were analyzed on a 4 -20% denaturating polyacrylamide gradient gel. The proteins were visualized with Coomassie Blue staining and presented in A. The same gel was subjected to fluorography, and the x-ray film that had been exposed for 18 h is presented in B.
as co-substrate, the purified recombinant protein can label yeast ribosomal protein L12. The enzymatic product is the ␦-N-monomethylarginine, and the methyl acceptor on L12 is the side chain of Arg 67 . We have proved that L12 from wild type yeast cells contains ␦-N-monomethylarginine. This modification is absent from yeast cells with RMT2 deletion. These results suggest that ribosomal protein L12 is the natural substrate of RMT2.