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pubs.acs.org/BiochemistryPublished on Web 05/19/2010r 2010 American Chemical Society
Biochemistry 2010, 49, 5225–5235 5225
DOI: 10.1021/bi100428x
Identification of Protein N-Terminal Methyltransferases in Yeast and Humans†
Kristofor J. Webb,‡ Rebecca S. Lipson,‡ Qais Al-Hadid,‡ Julian P. Whitelegge,‡,§ and Steven G. Clarke*,‡
‡Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, 607 CharlesE. Young Drive East, Los Angeles, California 90095, and §Pasarow Mass Spectrometry Laboratory, NPI-Semel Institute for
Neuroscience and Human Behavior, University of California, Los Angeles, Los Angeles, California 90024
Received March 22, 2010; Revised Manuscript Received May 19, 2010
ABSTRACT: Protein modification by methylation is important in cellular function. We show here that theSaccharomyces cerevisiaeYBR261C/TAE1 gene encodes an N-terminal protein methyltransferase catalyzingthe modification of two ribosomal protein substrates, Rpl12ab and Rps25a/Rps25b. The YBR261C/Tae1protein is conserved across eukaryotes; all of these proteins share sequence similarity with known seven!-strand class I methyltransferases. Wild-type yeast cytosol and mouse heart cytosol catalyze the methylationof a synthetic peptide (PPKQQLSKY) that contains the first eight amino acids of the processedN-terminus ofRps25a/Rps25b. However, no methylation of this peptide is seen in yeast cytosol from a !YBR261C/tae1deletion strain. Yeast YBR261C/TAE1 and the human orthologue METTL11A genes were expressed asfusion proteins in Escherichia coli and were shown to be capable of stoichiometrically dimethylating theN-terminus of the synthetic peptide. Furthermore, the YBR261C/Tae1 and METTL11A recombinantproteins methylate variants of the synthetic peptide containing N-terminal alanine and serine residues.However, methyltransferase activity is largely abolished when the proline residue in position 2 or the lysineresidue in position 3 is substituted. Thus, the methyltransferases described here specifically recognize theN-terminal X-Pro-Lys sequence motif, and we suggest designating the yeast enzyme Ntm1 and the humanenzyme NTMT1. These enzymes may account for nearly all previously described eukaryotic proteinN-terminal methylation reactions. A number of other yeast and human proteins also share the recognitionmotif and may be similarly modified. We conclude that protein X-Pro-Lys N-terminal methylation reactionscatalyzed by the enzymes described here may be widespread in nature.
Posttranslational and cotranslational modification of theN-terminus of proteins is well established, but the biologicalfunction is known only in a few cases (1-6). Acetylation, themost extensive modification, occurs on approximately 77% ofcytosolic proteins in mammals and about 50% of cytosolicproteins in Saccharomyces cerevisiae (3). Methylation of theN-terminus has also been observed for a variety of proteins inboth prokaryotes and eukaryotes (2). A major chemical conse-quence of such modification is controlling the charge at theN-terminus. For acetylated proteins, the partial positive charge onthe amino group is neutralized; for fully methylated N-terminalresidues, the positive charge is fixed. In both cases, these mod-ifications abolish the nucleophilicity of the R-amino nitrogen (2).
Eukaryotic N-terminal methylation has been detected to dateon one large subunit ribosomal protein in fungal and plantspecies, one small subunit ribosomal protein in S. cerevisiae, onelarge subunit ribosomal protein in higher plant chloroplasts, thesmall subunit of Rubisco in higher plant chloroplasts, histoneH2B proteins in several invertebrates, cytochrome c-557 speciesfrom one protozoan genus, myosin light chain proteins from
mammals, and the RCC1 protein of humans (Table 1). Interest-ingly, with the exception of the two chloroplast species, theN-terminal tetrapeptide sequences of all of these proteins aresimilar. All contain an N-terminal methionine residue that ispredicted to be cleaved (24), followed by a proline, alanine, orserine residue in the second position, and apparently invariantproline and lysine residues in the third and fourth positions,respectively. Most of these species are fully methylated(dimethylated proline, trimethylated alanine) to give a quatern-ary R-amino group. It has been proposed that a single enzymethat recognizes anX-Pro-LysN-terminal sequence could accountfor all of these methylation reactions (2). However, no geneproduct has been associated to date with this activity.
We have been interested in the modification of ribosomalproteins in the yeast S. cerevisiae and in identifying the genes thatencode the enzymes that catalyze these reactions. We have takenthe approach of analyzing intact ribosomal proteins by massspectrometry from extracts of mutant yeast lacking putativemethyltransferases to detect unmodified or undermodified spe-cies. Using this method, we have identified four SET domain-containing protein side-chain lysine methyltransferases, Rkm1,Rkm2, Rkm3, and Rkm4, and their large ribosomal proteinsubstrates Rpl23ab, Rpl12ab, and Rpl42ab (9, 25, 26) as well asRmt1-dependent protein arginine methylation of Rps2 (27). Thedistinct methylation of N-terminal proline residues has recentlybeen shown in the fission yeast (7) and plant (8) homologues ofbudding yeast Rpl12ab; we later confirmed the presence of thismodification in S. cerevisiae (9).
†This work was supported by National Institutes of Health GrantGM026020. R.S.L. was supported by the UCLA Cellular and Molec-ular BiologyTraining Program funded byNIHGrantGM007185.Massspectrometry was performed in the UCLA Molecular InstrumentationCenter supported byGrant S10RR024605 from the National Center forResearch Resources. The purchase of the LTQ FT Ultra mass spectro-meter was made possible by NIH/NCRR Grant S10 RR023045.*To whom correspondence should be addressed. Tel: 310-825-8754.
Fax: 310-825-1968. E-mail: [email protected].
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In this work, we have used the approach of intact massspectrometric profiling of ribosomal proteins from yeast cellsdeficient in putative methyltransferases (28) to identify the genenecessary for protein N-terminal methylation. We demonstratethat the loss of the S. cerevisiaeORF YBR261c/TAE1, encodinga candidate seven !-strand methyltransferase, results in the lossof the N-terminal methylation of both Rpl12ab and Rps25a/Rps25b. The YBR261C/TAE1 product had been previouslylinked to a translational function by a chemical genetic profileanalysis (29), but its specific function was not identified. Wedemonstrate that recombinant fusion proteins YBR261C/Tae1and METTL11A (the human homologue) are active methyl-transferases with specificity for stoichiometric modification ofX-Pro-Lys N-terminal sequences.
EXPERIMENTAL PROCEDURES
Purification of S. cerevisiae Ribosomal Proteins fromWild-Type andMutant Strains. The strains used in this studywere obtained from the SaccharomycesGenomeDeletionProjectand included the parent strains BY4742 and BY4741 and theYBR261C/tae1 deletion strain in each background, as well as thedeletion strains listed in Supporting Information Table 1. Largeand small ribosomal proteins were isolated from wild-type and!YBR261C/tae1 strains as described previously (9).Liquid Chromatography with Electrospray Ionization
Mass Spectrometry of Intact Ribosomal Proteins. Riboso-mal proteins were fractionated using reverse-phase liquid chro-matography as described previously (9). The resulting effluentwas directed into a QSTAR Elite (Applied Biosystems/MDXSCIEX) electrospray mass spectrometer running in MS onlymode. The instrument was calibrated using external peptidestandards to yield a mass accuracy of 40 ppm or better.Localization of Methylation Sites by Top Down Mass
Spectrometry Using Collisionally Activated Dissociation
or Electron Capture Dissociation. Ribosomal proteinsRpl12ab andRps25a/Rps25b from the!YBR261C/tae1 deletionand wild-type strains were separated by HPLC as describedabove, and fractions containing the isolated intact proteins weredirectly infused on a hybrid linear ion-trap/FTICR1 mass spec-trometer (LTQ FT Ultra; Thermo Fisher, Bremen, Germany)and fragmented using collisionally activated dissociation orelectron capture dissociation as described previously (9). AllFTICR MSMS spectra were processed using ProSightPC soft-ware (Thermo Fisher, San Jose, CA) in single protein mode witha 15 ppm mass accuracy threshold. The root-mean-squaredeviations for assigned fragments were less than 5 ppm. Thereference database for the ProSightPC software included theS. cerevisiae proteome from Swiss-Prot. Intact mass measurementswere processed using the manual Xtract program, version 1.516(Thermo Fisher, San Jose, CA). All top down experimental dataobtained for this study are available online at http://www.proteomecommons.org.Preparation of Cytosolic Extracts. S. cerevisiae cytosol
was isolated from wild-type (BY4742) and !YBR261C/tae1strains. These strains were grown at 30 !C in 500 mL of YPDto an OD600 of 0.7. The cells were centrifuged at 5000g for5 min at 4 !C. The resulting pellet was resuspended in 4 mL ofphosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl,4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4) and 100 "Mphenylmethanesulfonyl fluoride. The cells were lysed by vortex-ing for 1 min in the presence of 0.2 g of baked zirconium beads(Biospec Products, Bartlesville, OK), followed by cooling on icefor 1 min, for a total of seven cycles. A crude cytosolic fractionwas then obtained by centrifuging for 15 min at 12000g at 4 !C.Mouse heart cytosol was obtained as previously described (30).
Table 1: Known Eukaryotic N-Terminally Methylated Proteins
protein name N-terminal encoded sequence organism known N-terminal modification
Rpl12 MPPKFDPNEV Schizosaccharomyces pombe N,N-dimethylproline (7)L12 MPPKLDPSQI Arabidopsis thaliana N,N-dimethylproline (8)Rpl12ab MPPKFDPNEV S. cerevisiae N,N-dimethylproline (9)Rps25a/Rps25b MPPKQQLSKA S. cerevisiae N,N-dimethylproline (10)histone H2B MPPKPSGKGQ Asterias rubens N,N-dimethylproline (11)histone H2B MPPKPSAKGA Sipunculus nudus N,N-dimethylproline (12)histone H2B MPPKPSGKGQ Asterina pectinifera N,N-dimethylproline (13)histone H2B MPPKSGKGQK Marthasterias glacialis N,N-dimethylproline (14)histone H2B MPPKTSGKAA Drosophila melanogaster N-methylproline (15)cytochrome c-557 MPPKAREPLP Crithidia oncopelti N,N-dimethylproline (16)cytochrome c-557 MPPKARAPLP Crithidia fasciculata N,N-dimethylproline (17)
histone H2B MAPKAEKKPA A. thaliana N-methylalanine, N,N-dimethylalanine,N,N,N-trimethylalanine (18)
histone H2B MAPKKAPAAA Tetrahymena thermophila N,N,N-trimethylalanine (19)histone H2B MAPKKAPAAA Tetrahymena pyriformis N,N,N-trimethylalanine (20)fast skeletal myosin light chain 2 MAPKKAKRRA Oryctolagus cuniculus N,N,N-trimethylalanine (21)myosin light chain 1,
skeletal muscle isoformMAPKKDVKKP O. cuniculus N,N,N-trimethylalanine (21)
myosin light chain 3 MAPKKPDPKK Bos taurus N,N,N-trimethylalanine (21)
regulator of chromosome condensation 1 MSPKRIAKRR Homo sapiens N-methylserine, N,N-dimethylserine,N,N,N-trimethylserine (5)
chloroplast ribosomal protein L2 MAIHLYKTST Spinacia oleracea N-methylalanine (22)chloroplast Rubisco small subunit MQVWPPIGKK Pisum sativum N-methylmethionine (23)
1Abbreviations: [3H]AdoMet, S-adenosyl-L-[methyl-3H]methionine;AdoMet, S-adenosyl-L-methionine; FTICR, Fourier-transform ioncyclotron resonance; MSMS, tandem mass spectrometry.
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Plasmid Construction and Preparation of the Recombi-nant Yeast YBR261C/Tae1 and Human METTL11AFusion Proteins. The BG1805-A vector containing the yeastYBR261C/TAE1 gene and the pSPORT1 vector containing thefull-length human cDNA for METTL11A (accession numberNM_014064) were purchased fromOpenBiosystems (Huntsville,AL). The two open reading frameswere cloned into the pET-100/D-TOPO Escherichia coli expression vector according to themanufacturer’s instructions (Invitrogen, Carlsbad, CA). Properinsertion into the pET100D vector was verified by full DNAsequencing of both constructs (GENEWIZ,NJ) using the standardT7 forward and T7 reverse primers. The vector pET-100/D-TOPO carries a His6 tag in the linker region MRGSHHHHHH-GMASMTGGQQMGRDLYDDDDKDHPFT which is incor-porated before the initiator methionine residue of the clonedprotein. The constructed plasmids were transformed into E. coliBL21(DE3). The recombinant proteins were overexpressed bygrowing 2 L of the transformed E. coli cells at 37 !C in LBmediawith 100 "g/mL ampicillin to an OD600 of 0.7. Recombinantprotein production was induced for 4 h by the addition ofisopropyl !-D-thiogalactopyranoside to a final concentration of0.4 mM. Washed cells were resuspended in 20 mL of PBS and100 "M phenylmethanesulfonyl fluoride and subsequently bro-ken by ten 30 s sonicator pulses (50%duty; setting 4) on icewith aSonifier cell disruptorW-350 (SmithKline Corp.). The lysate wascentrifuged for 50 min at 13000g at 4 !C. The resulting super-natant was loaded onto a 5 mL HisTrap HP nickel affinitycolumn (GE Healthcare part number 17-5248-1), and the fusionproteins were purified using a gradient of 30-500 mM imidazoleper the manufacturer’s specifications.Protein Concentration Determination. A modified Lowry
procedure was used to determine protein concentrations ofrecombinant proteins and cell cytosol following precipitationwith 1.0 mL of 10% (w/v) trichloroacetic acid (31). A stocksolution of bovine serum albuminwas used as a protein standard.In Vitro Methylation of Peptides. Synthetic nonapeptides
were prepared by Biosynthesis, Inc. (Lewisville, TX). Thesepeptides were based on the sequence of the first eight aminoacids of the N-terminus of the S. cerevisiae ribosomal proteinRps25a/Rps25b with an additional tyrosine residue added at theC-terminus for UV detection. Peptides were prepared withN-terminal proline (PPKQQLSKY), alanine (APKQQLSKY),and serine (SPKQQLSKY) residues. A peptide mixture was syn-thesized where equal amounts of each of the 20 protected aminoacid derivatives were used for the first position (X20PKQ-QLSKY). A similarly prepared mixture was made except thatproline and alanine were not included (X18PKQQLSKY). Twoadditional peptide mixtures were prepared: one designed tocontain equal amounts of each of the 20 amino acids at thesecond position except proline (PX19KQQLSKY), and onedesigned to contain equal amounts of the 20 amino acids at thethird position except lysine (PPX19QQLSKY). Methylationradiolabeling experiments were carried out in the presence of0.5 "M S-adenosyl-L-[methyl-3H]methionine ([3H]AdoMet; 70.8Ci/mmol from a 7.8 "Mstock solution in 10mMH2SO4/ethanol,9:1; PerkinElmer, Inc.). Nonradiolabeled methylation reactionswere carried out in 200 "M S-adenosyl-L-methionine p-toluene-sulfonate salt (Sigma; from a 10 mM stock solution in 10 mMHCl). Allmethylation reactionswere performed in 100mMNaCland 100 mM sodium phosphate buffer at pH 7.0 in a totalreaction volume of 200 "L. Reactions were terminated by theaddition of 20 "L of 10% trifluoroacetic acid. After centrifuga-
tion at 20000g for 10 min at room temperature, the supernatantwas fractionated by HPLC. A PLRP-S polymeric column wasused with a pore size of 300 A, bead size of 5 "m, and dimensionsof 150 ! 2 mm (Polymer Laboratories, Amherst, MA). Thecolumn was maintained at 50 !C and initially equilibrated insolvent A (0.1% trifluoroacetic acid in water) at 95% and solventB (0.1% trifluoroacetic acid in acetonitrile) at 5%. Peptides wereeluted at a flow rate of 200 "L/min using a program of 5 min at5% B, followed by a 20 min gradient from 5% to 60% B; half-minute fractions were collected.
The incorporation of [3H]methyl radioactivity into peptideswas determined either by direct counting or using an inlinescintillation counter as described in the figure legends. For thenon-isotopically labeled reaction mixtures, methylated peptideswere analyzed by MALDI (matrix-assisted laser desorptionionization) mass spectrometry. An aliquot of the HPLC peptidepeak fraction or fractions (1 "L) was mixed with 1 "L of 70%acetonitrile/0.1% trifluoroacetic acid containing 1 mg/mLR-cyano-4-hydroxycinnamic acid as the MALDI matrix andspotted onto a MALDI target plate. The samples were analyzedusing a Voyager-DE STR MALDI-TOF mass spectrometer(Applied Biosystems).
RESULTS
Yeast YBR261C/Tae1 Is Required for the Methylationof Two Ribosomal Proteins. After the loss of the initiatormethionine residue (24),S. cerevisiae ribosomal proteinsRpl12aband Rps25a/Rps25b share the common N-terminal sequence Pro-Pro-Lys. Rpl12ab represents the identical polypeptides encodedby the yeast RPL12A and RPL12B genes; Rps25a and Rps25bare encoded by the yeast RPS25A and RPS25B genes and differonly at a single amino acid residue. The N-terminal prolineresidue in each of these proteins has been shown to bemodified attheR-amino group by dimethylation (9, 10), although the enzymeor enzymes that catalyze the methylation reactions have not beenidentified. The Rpl12ab protein is modified by methylation attwo additional sites: trimethylation at lysine 3 catalyzed by theRkm2 enzyme (9, 26) and #-monomethylation at arginine 66catalyzed by the Rmt2 enzyme (32). All of these modificationsappear to be stoichiometric, and no evidence for additional typesof modifications has been observed (9).
To identify the enzymes responsible for the N-terminal prolinemodification, we measured the intact mass of Rpl12ab andRps25a/Rps25b from large and small ribosomal proteins pre-pared fromwild-type yeast cells and from 37 yeast deletion strainslacking known or putative methyltransferase genes (28, 33-35)(Supporting Information Table 1). In this screen, purified ribo-somal proteins from small or large subunits were fractionated byHPLC and analyzed by electrospray mass spectrometry as de-scribed in Experimental Procedures. We found that Rpl12ab fromthe wild-type strain (Figure 1A) and from 34 of the 37 deletionstrains contained the full complement of methyl groups (data notshown; Supporting Information Table 1). As expected, the intactmass of the Rpl12ab protein from the rkm2 deletion strain wasreduced by 42 Da, consistent with the loss of trimethylation atLys-3 (9). Furthermore, the intact mass of Rpl12ab from the rmt2deletion strain was reduced by 14 Da, consistent with the loss ofmonomethylation at Arg-66 (data not shown). However, in theRpl12ab protein prepared from theYBR261C/tae1 deletion strain,we did not detect the mass of the fully modified protein (averagemass 17775) but instead found a series of products differing by the
5228 Biochemistry, Vol. 49, No. 25, 2010 Webb et al.
replacement of three to five methyl groups with hydrogen atoms at17733Da (-3CH2), 17719Da (-4CH2), and 17705Da (-5CH2)(Figure 1A). This result suggests that YBR261C/TAE1 encodes agene needed for the methylation of Rpl12ab at the N-terminalproline residue. These losses were verified in an independent
YBR261C/tae1 deletion strain in the BY4741 background (datanot shown). The appearance of a mixture of species lacking three,four, and five methyl groups suggests that the affinity of Rkm2 forLys-3 is decreased when theN-terminal modification of the prolineresidue is not present.
We additionally observed that the intact masses of the smallribosomal proteinsRps25a (averagemass 11936Da) andRps25b(average mass 11906 Da) were consistent with the presence of anN-terminal dimethylproline residue (10) in the wild-type strain(Figure 1B). Rps25a and Rps25b differ only at position 104; athreonine residue is present in the former and an alanine residuein the latter. These forms were also detected in 36 of the 37deletion strains examined (data not shown; Supporting Informa-tion Table 1). However, Rps25a/Rps25b was found to have anintact mass reduced by 28 Da (loss of two methyl groups) in theYBR261C/tae1 deletion strain (Figure 1B). This loss was alsoverified in the independent YBR261C/tae1 deletion strain in theBY4741 background (data not shown). The results obtained withRpl12ab and Rps25a/Rps25b suggest that the YBR261C/TAE1product is necessary for the formation of the dimethylprolineresidue in each of these ribosomal proteins.YBR261C/Tae1-Dependent N-TerminalMethylation of
Rpl12ab and Rps25a/Rps25b. To identify the residue(s) whereloss of methylation occurs in the YBR261C/tae1 deletion strain,we performed intact mass top down fragmentation analysis. Thisapproach allows an unbiased assignment of posttranslationalmodifications across the entire polypeptide chain (36). Riboso-mal proteins were fractionated using reverse-phase HPLC, andpurified fractions containing Rpl12ab and Rps25a/Rps25b werecollected for offline top down analysis. In Rpl12ab isolated fromthe YBR261C/tae1 deletion strain we found that the majorspecies present was monomethylated (Figure 1A). When thisspecies was fragmented using collisionally activated dissociation,we localized the site ofmethylation to the region between residues35 and 73 containing the known monomethylation site atarginine 66 (Figure 2A). Additionally, we observed that theN-terminal region was unmodified (Figure 2A). The intactdimethylated Rpl12ab species (average mass 17719) isolatedfrom the YBR261C/tae1 deletion strain was fragmented, and ab20 ion was detected with the addition of one methyl group, sug-gesting partial methylation of lysine 3 (data not shown). Neither ofthese species was dimethylated at the N-terminal residue.
Wild-type Rps25a/Rps25b was isolated and subjected to thesame top down analysis to verify the location and extent ofmethylation. Our data are in agreement with the observations ofMeng and colleagues (10), isolating the dimethylation to theN-terminal region ofRps25a/Rps25b (Figure 2B).We also foundno evidence for unmethylated or monomethylated species ofRps25a/Rps25b. Rps25a/Rps25b isolated from the YBR261C/tae1 deletion strain is 28 Da less than the wild type, and thefragmentation data indicate nomethylation is present (Figure 2C).The intact mass data from Rpl12ab and Rps25a/Rps25b inFigure 1 and the fragmentation data in Figure 2 from wild-typeand YBR261C/tae1 deletion strains confirm that the YBR261C/Tae1 protein is necessary for the stoichiometric methylation ofthe N-terminus of both Rpl12ab and Rps25a/Rps25b.YBR261C/Tae1 and the N-Terminus of Ribosomal Pro-
teins Rpl12ab and Rps25a/Rps25b Are Conserved acrossEukaryotes. Previously the YBR261C/TAE1 locus was identi-fied as encoding a hypothetical methyltransferase (28, 33-35)containing the seven !-strand class I conserved AdoMet-depen-dent methyltransferase motif I, post-I, motif II, and motif III
FIGURE 1: Loss of methylation of Rpl12ab and Rps25a/Rps25b inyeast !YBR261C/tae1 mutants. Ribosomal proteins were isolatedand separated by reverse-phase HPLC fromwild type (BY4742) anda YBR261C/tae1 deletion mutant described in the ExperimentalProcedures section. The HPLC effluent was directed to the sourceof an electrospray mass spectrometer (QSTAR Elite). The resultingdeconvoluted MS spectra are displayed with the average masses ofthe significant peaks shown. Changes in mass of the proteins frommutant and wild-type cells are indicated by double arrows.
Article Biochemistry, Vol. 49, No. 25, 2010 5229
(Figure 3). A BLAST search revealed homologues to theYBR261C/TAE1 gene in multiple eukaryotic, but not prokaryotic, organisms.Twohumanhomologueswere found,METTL11AandMETTL11B(Figure 3). Additionally, we noted that human ribosomal pro-teins L12 and S25 (the homologues of Rpl12ab and Rps25a/Rps25b) share the same N-terminal tripeptide sequence as the
yeast proteins. Human S25 has been reported to be modified bythe addition of two methyl groups, although their location in thepolypeptide chain has not been established (37).The YBR261C/TAE1 and Human METTL11A Genes
Encode N-Terminal Protein Methyltransferases. The re-sults above suggest that YBR261C/Tae1 is the methyltransferasethat directly modifies the ribosomal proteins. However, thepossibility remains that YBR261C/Tae1 may indirectly controlmethylation, for example, as a necessary noncatalytic subunit ofa methyltransferase complex. To address this issue, we deve-loped an in vitro assay for N-terminal methyltransferase activity(Figure 4). We observed the presence of an activity in yeast andmammalian cytosolic extracts capable of methylating a syntheticpeptide constructed using the sequence of the first eight aminoacids of the Rps25a/Rps25b protein and a C-terminal tyrosineresidue, PPKQQLSKY. Here, the cytosolic extracts were incu-bated with this peptide and [3H]AdoMet. The reaction mixturewas fractionated by HPLC, and the [3H]methyl groups in thepeptide were determined by scintillation counting (Figure 4A,B).In the wild-type cytosolic extract radioactivity migrated withthe synthetic peptide, indicating the presence of an AdoMet-dependentmethyltransferase. In the cytosol from the!YBR261C/tae1 strain no radioactivity migrated with the peptide. This resultshows that the synthetic peptide is a good substrate for the endo-genous methyltransferase. We also detected methylation of thepeptidewith the cytosolic extract ofmouse heart, a tissue containinghigh transcript levels of the METTL11A gene (Figure 4C).
Using this validated assay, we next directly tested whether thepurified recombinant forms of YBR261C/Tae1 and the humanhomologue METTL11A prepared as fusion proteins in E. colicould independentlymethylate the synthetic peptide. In a control,we showed no activity with an unrelated fusion protein preparedin parallel from E. coli. As shown in Figure 4D for the yeastprotein and in Figure 4E for the human protein, both werecapable of rapidly transferring methyl groups from [3H]AdoMetto the peptide. This result now clearly demonstrates thatYBR261C/Tae1 and METTL11A are methyltransferases thatdirectly catalyze the methylation reaction in the absence of otherprotein species. We have not been able to prepare a stablerecombinant form of human METTL11B; whether this proteinis also a similar methyltransferase remains to be seen.
To determine the extent of methylation catalyzed by theseenzymes, the PPKQQLSKY peptide was incubated withrecombinant YBR261C/Tae1 or recombinant METTL11A foran extended time in the presence of a high concentration ofAdoMet to drive methylation to completion (Figure 5). Bothrecombinant YBR261C/Tae1 and recombinant METTL11Awere found to completely dimethylate the PPKQQLSKYpeptide(Figure 5A-C). The N-terminal location of the methylation wasverified by MSMS analysis of the peptide product, localizing thedimethylation site within the first two proline residues (data notshown).
Taken together, these results indicate thatYBR261C/Tae1 andMETTL11A are N-terminal methyltransferases that catalyze theformation of N-terminal dimethylproline residues.Substrate Specificity of N-Terminal Protein Methyl-
transferases. Nonchloroplast eukaryotic proteins, includingRpl12ab and Rps25a/Rps25b, are modified by N-terminalmethylation on a common sequence motif (Table 1). In each ofthese proteins, an N-terminal proline, alanine, or serine residue isfollowed by a Pro-Lys sequence. The conservation of the X-Pro-Lys sequence has led to the postulation that one enzyme may be
FIGURE 2: Localization of methylation sites in intact ribosomalproteins. Fragmentation patterns for ribosomal protein Rpl12abfrom the wild-type (BY4742) strain and for the major form ofRpl12ab from the !YBR261C/tae1 strain are shown in panel A.Similar fragmentation patterns for ribosomal proteins Rps25a/Rps25b from wild-type (BY4742) and !YBR261C/tae1 strains areshown in panels B and C, respectively. “b” type cleavages are shownby the upper hash mark, “y” type cleavages are shown by the lowerhash mark, “c” type cleavages are indicated by the upper horizontalline, and “z” type cleavages are indicated by the lower horizontal line.Ribosomal proteinswere isolated by reverse-phaseHPLCas describedin Experimental Procedures. Fractions containing Rpl12ab andRps25a/Rps25b isolated from wild-type and !YBR261C/tae1 strainswere subjected to top down analysis where the intact protein wascollisionally activated to produce fragment ions. Dimethylation of theN-terminal proline residue in Rpl12ab and Rps25a/Rps25b is repre-sented by the circled P, trimethylation of lysine 3 of Rpl12ab isindicated by the circled K, and monomethylation of arginine 66 ofRpl12ab is indicated by the circled R.
5230 Biochemistry, Vol. 49, No. 25, 2010 Webb et al.
responsible for themodification of all of these proteins (2). In thisregard, recent results of the modification of the human RCC1protein containing a Ser-Pro-Lys N-terminal sequence are ofinterest (5). RCC1was found to be mono-, di-, and trimethylatedat the N-terminal serine residue. The authors expressed variantsof the N-terminal sequence in HeLa cells where proline andalanine residues replaced the serine residue. They found theproline formwas completely dimethylated; the alanine and serine
forms were found in states of methylation from unmodified totrimethylated (5). The authors also found that changing theresidues in the second and third position had a large effect onmethylation.
These results encouraged us to directly test the specificity ofthe YBR261C/Tae1 and METTL11A forms of the N-terminalmethyltransferase in both cytosolic extracts and as purifiedrecombinant proteins. When the N-terminal proline of the
FIGURE 3: Yeast YBR261C/Tae1 homologues in eukaryotes. The signature methyltransferase motifs common to all class I protein methyl-transferases are boxed. The accession numbers used in this alignment and the corresponding BLAST expect values related to the yeast YBR261C/Tae1 protein are as follows: humanMETTL11A (H. sapiens, NP_054783, expect value 6e-36), humanMETTL11B (H. sapiens, NP_001129579,expect value 6e-27),Drosophila (D. melanogaster, NP_610528, expect value 9e-37), C. elegans (Caenorhabditis elegans, NP_490660, expect value3e-35),S. cerevisiae (S. cerevisiae, NP_009820),S. pombe (S. pombe, NP_594227, expect value 1e-49),Arabidopsis (A. thaliana, NP_199258, expectvalue 2e-42).
Article Biochemistry, Vol. 49, No. 25, 2010 5231
Rps25a/Rps25b-derived peptide PPKQQLSKYwas replaced withan alanine or a serine residue and incubated with [3H]AdoMet andrecombinant YBR261C/Tae1 (Figure 4D) or recombinantMETTL11A (Figure 4E), peptide methylation was observedbut to a lower extent than with the N-terminal proline peptide.Methylation of the N-terminal variants APKQQLSKY andSPKQQLSKY was greatly reduced compared to PPKQQ-LSKY in cytosolic extracts of yeast (Figure 4A,B). Theseresults suggest that the yeast enzyme appears to prefer thePro-Pro-Lys N-terminal sequence over the Ala-Pro-Lys orSer-Pro-Lys sequence. The relative extent of methylation ofthe Ala-Pro-Lys and Ser-Pro-Lys peptides in Figure 4D maybe overestimated with recombinant enzyme because methyla-tion may have been limited by the amount of AdoMetpresent. The mammalian enzyme may be less specific becauserelatively more methylation of the Ala-Pro-Lys and Ser-Pro-Lys species is seen with the recombinant METTL11A enzymecompared to the yeast recombinant enzyme (Figure 4D,E).This trend is also observed when mouse heart cytosol is used asan enzyme source (Figure 4C), although it is possible thatother methyltransferase species (such as METT11B; seeFigure 3) may contribute to the modifications. The absenceof methylation of any of the peptides catalyzed by the yeastcytosol from the !YBR261c/tae1 deletion strain suggests thatonly oneN-terminal methyltransferase of this type is present inyeast.
The extent of methylation of the APKQQLSKY andSPKQQLSKY species was determined by incubating peptidewith recombinant YBR261C/Tae1 or recombinant METTL11Afor an extended time in order to drive methylation to completion,as described in the legend of Figure 5. Recombinant YBR261C/Tae1 and recombinant METTL11A nearly completely trimethy-lated the APKQQLSKY peptide, with only a small fractionremaining in the dimethylated form (Figure 5D-F). The methy-lation of SPKQQLSKYwas less extensive, with YBR261C/Tae1producing mono-, di-, and trimethylated forms (Figure 5H).METTL11A predominately trimethylated the SPKQQLSKYpeptide (Figure 5I). Taken together, these data suggest that thehuman METTL11A methyltransferase, and to a slightly lesserextent the YBR261c/Tae1 methyltransferase, can recognize spe-cies with N-terminal alanine and serine residues in addition tothose with proline residues.
Enzyme preference for the amino acid in the first position ofthe N-terminal X-Pro-Lys sequence was further tested by com-paring the methylation catalyzed by recombinant YBR261C/Tae1 or recombinant METTL11A with mixtures of syntheticpeptides designed to contain equal amounts of each of the 20amino acids at the first position (X20PKQQLSKY) or each of the20 amino acids at the first position except proline and alanine(X18PKQQLSKY) (Figure 6A). The substantial incorporation ofradioactivity into the mixed peptides, where each species ispresent at only 1/20 or 1/18 the concentration of the unmixedpeptides, suggests that these enzymes can recognize substrateswith a variety of amino acids in the first position. However, inyeast cytosol where the activity is present at lower levels, thehighest methylation levels are seen in the PPKQQLSKY peptide,suggesting that the proline residue in position 1 is a preferredsubstrate (Figure 6B). In mouse heart cytosol, each of thesepeptide mixtures is recognized to a similar level (Figure 6C). Asdescribed above, this may reflect the activity of additionalmethyltransferases such asMETT11B. The recombinant enzymereactions were repeated using the X20PKQQLSKY and
FIGURE 4: Specificity ofmethylation for synthetic peptides containingthe X-Pro-Lys N-terminal sequence. Nonapeptides (22 "g, 100 "M)containing the first eight amino acids of the N-terminus of Rps25a/Rps25b or with alanine or serine substituted at the N-terminalposition were incubated with cytosolic extracts containing 90 "g ofprotein in the presence of 0.5 "M [3H]AdoMet in a final volume of200 "L for 1 h and then subjected to reverse-phase HPLC asdescribed under Experimental Procedures. (A) Cytosol from yeastwild-type BY4742 cells was incubated at 30 !C; (B) cytosol fromyeast !YBR261C/tae1 cells was incubated at 30 !C; (C) cytosolfrom mouse heart was incubated at 37 !C. Radioactivity wasdetected by adding 75 "L from each HPLC fraction to 10 mL ofscintillation fluor and counted on a Beckman LS6500 instrument;the number of methyl groups was determined from a calculatedspecific radioactivity of 78000 cpm/pmol (50% counting efficiency).(D) Identical to (A) except the yeast extractwas replacedwith 1"gofrecombinant YBR261C/Tae1 fusion protein. (E) Identical to (A)except the yeast extract was replaced with 1 "g of recombinanthumanMETTL11A fusion protein and the incubation temperaturewas 37 !C.
5232 Biochemistry, Vol. 49, No. 25, 2010 Webb et al.
X18PKQQLSKYmixtures in the reaction conditions described inFigure 5 and subjected to MALDI analysis. Although theanalysis of the data was limited by overlapping signals in theMALDI spectrum, signals were detected for the methylation ofpeptides with alanine, proline, and glycine at the N-terminalposition (data not shown).
Finally, we investigated the effect of substituting other aminoacids for either the proline residue in the second position or thelysine residue in the third position of the X-Pro-Lys N-terminalsequence. With the recombinant yeast and human methyltrans-ferases, we found little or no methylation over background for apeptide mixture containing each of the 20 amino acids exceptlysine at the third position (Figure 6A; PPX19QQLSKY). A verysmall amount of methylation was observed for the peptidemixture containing each of the 20 amino acids except pro-line at position 2 (Figure 6A; PX19KQQLSKY). Similarly, weobserved little or no methylation over background for either ofthese peptide mixtures for yeast or mouse heart cytosolicpreparations (Figure 6B,C). These results suggest that themajor specificity factor for recognition of N-terminal polypep-tides by the yeast YBR261C/Tae1 or the human METTL11Aenzymes is the presence of a proline residue in position 2 and alysine residue in position 3. All of the known N-terminallymethylated species in eukaryotes contain this motif. We thusdesignate this enzymeNtm1 as theX-Pro-LysN-terminal proteinmethyltransferase.
DISCUSSION
Phenotypes of Yeast Lacking the X-Pro-Lys N-TerminalProtein Methyltransferase. A phenotype of increased sensiti-vity to paromomycin, an antibiotic from Streptomyces thattargets the ribosome, was previously found for the YBR261C/tae1 deletion strain in a functional genomics profile of yeastnonessential genes (29). This result suggests that themodificationmay be important in helping to protect yeast against microbialattack. Further analysis of this mutant showed an increasedsensitivity to the histidine biosynthesis inhibitor 3-amino-1,2,4-triazole, a reduction in polysomes, and a corresponding increasein the 80 and 60 S subunits as compared to thewild-type strain. Inaddition, the mutant demonstrated decreased translational effi-ciency and fidelity. For these reasons, the YBR261C gene wasdesignated TAE1 for “translational associated element 1” (29).However, we note that these phenotypes may not be a directeffect of loss of N-terminal methylation on ribosomal proteinsRpl12ab and Rps25a/Rps25b. In addition to these three riboso-mal proteins, there are 16 other yeast proteins with the (Pro/Ala/Ser)-Pro-Lys N-terminal methyltransferase recognition sequenceincluding the ribosomal associated protein Tma46, the Lsg1protein involved in 60S ribosomal subunit biosynthesis, and theLoc1 60S ribosomal subunit assembly protein (SupportingInformation Table 2). All of these proteins may be modified byN-terminal methylation, and their function(s) may be affected bythe loss of the YBR261C/Tae1 methyltransferase. It will be
FIGURE 5: Extent of modification of peptides with Pro-Pro-Lys, Ala-Pro-Lys, Ser-Pro-Lys N-terminal sequences by recombinant YBR261C/Tae1 andMETTL11A. Tenmicrograms of recombinant YBR261C/Tae1 or recombinantMETTL11Awas incubated with 10 "Mpeptide in thepresence of 200 "MAdoMet for 4.5 h at 30 and 37 !C, respectively. Shown are theMALDImass spectra results of PPKQQLSKY(A, B, and C),APKQQLSKY (D, E, and F), and SPKQQLSKY (G, H, and I) after incubation and HPLC purification with buffer only (no Enzyme, A, D,and G), yeast recombinant YBR261C/Tae1 (B, E, and H), or human recombinant METTL11A (C, F, and I).
Article Biochemistry, Vol. 49, No. 25, 2010 5233
important to determine whether some or all of these 16 yeastproteins with N-terminal (Pro/Ala/Ser)-Pro-Lys sequences are infact methylated at the N-terminus in reactions catalyzed byYBR261C/Tae1.Biological Significance of N-Terminal Methylation in
Other Cell Types. In Drosophila cells, evidence has beenpresented that the degree of monomethylation of the N-terminalproline residue of histone H2B can be increased by heat shocktreatment (15). This result opens the possibility that this methyla-tion reaction can be regulated as a response to stress. In humancell lines N-terminal methylation of a serine residue in theregulator of chromatin condensation 1 (RCC1) protein has beenobserved to yield mono-, di-, and trimethylated derivatives (5).RCC1 is a guanine nucleotide-exchange factor that plays roles inprotein transport through the nuclear envelope, nuclear envelopeassembly, and mitosis. In higher primates the N-terminal se-quence of RCC1 after methionine removal is Ser-Pro-Lys, and inother mammals it is Pro-Pro-Lys. N-Terminal serine residues areoften targets of acetylation (24), raising the possibility that theremay be competition between the acetylation and methylationreactions. However, at least in yeast, it is clear that none of thethree N-terminal acetyltransferases catalyze modification atN-terminal Ser-Pro (or Ala-Pro, Cys-Pro, Thr-Pro, or Val-Pro)sequences (24), thus making the R-amino group potentiallyavailable to the Ntm1 methyltransferase. RCC1 mutants with thelysine at position 3 substituted with a glutamine residue blockedN-terminal methylation and resulted in less efficient chromatinbinding during mitosis and increased spindle-pole defects ascompared to wild-type protein (5). In vitroDNA binding experi-ments usingmethylated and unmodified RCC1 peptides contain-ing the first 34 amino acids showed an increased affinity forDNAwhen the N-terminal methylation was present (5).
N-Terminal methylation may also protect proteins fromattack by aminopeptidases (2). Interestingly, the humanX-prolylaminopeptidase, XPNPEP1, has a Pro-Pro-Lys N-terminal se-quence that may lead to its methylation and thus protectionagainst self-digestion. In Supporting Information Table 3, we listhuman proteins that have the N-terminal (Pro/Ala/Ser)-Pro-Lysrecognition sequence for the N-terminal methyltransferase: all ofthese proteins are candidates for modification.Prokaryotic Protein N-Methylation Reactions. The
E. coli large subunit ribosomal protein L11 is a distanthomologue of the yeast Rpl12ab protein. Interestingly, L11 isR-N-trimethylated at its N-terminal alanine residue and is $-N-trimethylated at Lys-3 and Lys-39 in reactions reportedly cata-lyzed by the PrmA methyltransferase as reviewed in ref 4. Thissituation is remarkably similar to the dimethylation of theN-terminal proline residue and the trimethylation of Lys-3 inyeast Rpl12ab. However, the N-terminal sequence of L11 is Ala-Lys-Lys, different from the Pro-Pro-Lys sequence seen in yeastand higher eukaryotes. Additionally, there is very little sequencesimilarity between PrmA and YBR261C/Tae1 or METTL11A.BLAST searches reveal that nonrelated methyltransferases inyeast and human are more similar to PrmA than the N-terminalmethyltransferases. The three methylated L11 residues in E. coliare apparently modified by the single PrmA methyltransferase,while N-terminal methylation in yeast Rpl12ab is dependent onthe YBR261C/Tae1 protein, the trimethylation at Lys-3 isdependent upon Rkm2, and the monomethylation at Arg-66 isdependent upon Rmt2.
N-Terminal methylation also occurs in four other E. coliribosomal proteins. Monomethylmethionine is found in L16
FIGURE 6: Importance of the N-terminal X-Pro-Lys sequence inmethyltransferase substrates. Recombinant YBR261c/Tae1, recombi-nantMETTL11A (A), wild-type yeast cytosol,!YBR261C/tae1 yeastcytosol (B), and mouse heart cytosol (C) were incubated at the sameconcentrations as in Figure 4 with 22 "g (!100 "M) of a syntheticpeptide (PPKQQLSKY or APPKQQLSKY) or synthetic peptidemixtures basedon these sequences. Themixtures includedone contain-ing all 20 amino acids at position 1 (denoted X20PKQQLSKY), onecontaining all 20 amino acids at position 1 except proline and alanine(denotedX18PKQQLSKY), one containing all aminoacids at position2 except proline (denoted PX19KQQLSKY), and one containing allamino acids at position 3 except lysine (denoted PXK19QQLSKY).Reactions were carried out in a final volume of 200 "L for 1 h in thepresence of 0.5 "M [3H]AdoMet at 37 !C for the human and mouseenzymes and at 30 !C for the yeast enzymes as described in theExperimental Procedures section. A portion (50 "L) of the reactionmixture was applied to the HPLC column as described in the Experi-mental Procedures section, and radioactivity was detected using aBeta-RAM model 5 inline scintillation counter under low flow modewith awindowof 2 s for all samples except themouse cytosolwhere thewindow was 5 s (LabLogic Inc.). Radioactivity was summed over theelution times of all peptides used (fromminute 12.5 tominute 20.5); thenumber of methyl groups was determined from a calculated specificradioactivity of 62400 cpm/pmol (40% counting efficiency).
5234 Biochemistry, Vol. 49, No. 25, 2010 Webb et al.
and L33, while monomethylalanine is found in L33 and S11 (4) .Monomethylmethionine is also found in the E. coli initiatorfactor IF3 and in the CheZ chemotaxis protein (2). Interestingly,monomethylalanine (22) and monomethylmethionine (23) havebeen found in eukaryotic chloroplast proteins, possibly reflectingthe prokaryotic origin of this organelle (Table 1). The enzymescatalyzing these reactions have not been described to date.However, the modification of pilin proteins from a number ofprokaryotic species by monomethylation at the N-terminal phenyl-alanine residues has been shown to be catalyzed by a bifunctionalenzyme that results in endopeptidic cleavage of a leader peptidefollowed by the methylation reaction (38). A similar enzymemay be present in chloroplasts (6). Importantly, none of theseproteins shares the X-Pro-Lys N-terminal sequence (2). Theseresults suggest that distinct enzymatic mechanisms are used forprokaryotic and chloroplast N-terminal methylation reactions.Finally, we note that major targets of N-terminal methylationreactions in both prokaryotes and eukaryotes include proteins intranslation.
ACKNOWLEDGMENT
We thank Drs. Joseph Loo, Jane Strouse, and Kym Faull forcontinuing support and helpful advice on mass spectrometry.
SUPPORTING INFORMATION AVAILABLE
A list of yeast strains used and lists of yeast and human openreading frames containing the X-Pro-Lys N-terminal methyl-transferase recognition motif. This material is available free ofcharge via the Internet at http://pubs.acs.org.
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SupplementalTable1Yeastgenedeletionstrainsusedinmethyltransferasedeletionscreen.AllstrainsareintheBY4742backgroundexpectforΔset1whichisintheKT1112background.
WildTypeStrains SETDomainProteins SevenBeta‐StrandDomainProteins
WTBY4742 Δset1(KT1112) Δdot1WTKT1112 Δset2 Δhsl7
Δset3 Δrmt1Δset4 Δrmt2Δset5 ΔYBR141CΔset6 ΔYBR261CΔrkm1 ΔYBR271WΔrkm2 ΔYDR140WΔrkm3 ΔYDR316WΔrkm4 ΔYHR209WΔcmt1 ΔYIL064W
ΔYHL039W ΔYIL110WΔYJR129CΔYKL155CΔYKL162CΔYLR063WΔYLR137WΔYMR209CΔYMR228WΔYNL022CΔYNL024CΔYNL063WΔYNL092WΔYOR239WΔYPL009C
SupplementalTable2YeastopenreadingframescontainingtheX‐Pro‐LysN‐terminalmethyltransferaserecognitionmotif
ProteinName N‐TerminalSequence Description KnownN‐terminalModification
Rpl12ab MPPKFDPNEV Proteincomponentofthelarge(60S)ribosomalsubunit N,N‐dimethylprolineRef.1
Rps25ab MPPKQQLSKA Proteincomponentofthesmall(40S)ribosomalsubunit N,N‐dimethylprolineRef.2
TMA46 MPPKKGKQAQ Proteinofunknownfunctionthatassociateswithribosomes
RPT1 MPPKEDWEKY OneofsixATPasesofthe19Sregulatoryparticleofthe26Sproteasome
OLA1 MPPKKQVEEK P‐loopATPasewithsimilaritytohumanOLA1
LSG1 MPPKEAPKKW PutativeGTPaseinvolvedin60Sribosomalsubunitbiogenesis
NEW1 MPPKKFKDLN HomologoustomRNAexportfactorfromS.pombe
DPB4 MPPKGWRKDA SharedsubunitofDNApolymerase(II)epsilonandISW2/yCHRAC
DUPC MPPKQKAGDE PutativeuncharacterizedproteinDUPC
HOP2 MAPKKKSNDR Meiosis‐specificproteinthatlocalizestochromosomes
HHO1 MAPKKSTTKT HistoneH1,alinkerhistonerequiredfornucleosomepackagingatrestrictedsites
HSP31 MAPKKVLLAL PossiblechaperoneandcysteineproteasewithsimilaritytoE.coliHsp31
YIL054W MAPKAFFVCL Dubiousopenreadingframe;Uncharacterizedmembraneprotein
AFG2 MAPKSSSSGS ATPaseoftheCDC48/PAS1/SEC18(AAA)family;maybeinvolvedindegradationofaberrantmRNAs
YLR392C MAPKISISLN ORF,Uncharacterized
LOC1 MAPKKPSKRQ 60Sribosomalsubunitassembly/exportproteinLOC1
MRS1 MSPKNITRSV ProteinrequiredforthesplicingoftwomitochondrialgroupIintrons
BUD5 MSPKNKYVYI GTP/GDPexchangefactorforRsr1p
[1]WebbK.J.,LaganowskyA.,WhiteleggeJ.P.,andClarkeS.G.(2008)J.Biol.Chem.283,35561‐35568[2]Meng,F.,Du,Y.,Miller,L.M.,Patrie,S.M.,Robinson,D.E.,andKelleher,N.L.(2004)Anal.Chem.76,2852‐2858
SupplementalTable3HumanopenreadingframescontainingtheX‐Pro‐LysN‐terminalmethyltransferaserecognitionmotif
ProteinName/Accessionnumber N‐TerminalSequence Description KnownN‐terminalModification
RPL12 MPPKFDPNEI RibosomalproteinL12
RPS25 MPPKDDKKKK RibosomalproteinS25
CCDC153 MPPKNKEKGK Coiled‐coildomaincontaining
VRK2 MPPKRNEKYK Vacciniarelatedkinase2isoform1andisoform3
ZC3H15 MPPKKQAQAG Erythropoietin4immediateearlyresponse
MYL6B MPPKKDVPVK Smoothmuscleandnon‐musclemyosinalkalilightchain6B
RB1 MPPKTPRKTA Retinoblastoma1
VAMP4 MPPKFKRHLN Vesicle‐associatedmembraneprotein4
OLA1 MPPKKGGDGI Obg‐likeATPase1
XPNPEP1 MPPKVTSELL X‐prolylaminopeptidase
PIN4 MPPKGKSGSG Peptidyl‐prolylcis‐transisomeraseNIMA‐interacting4
XP_002348192 MPPKPGMPAA Hypotheticalprotein
XP_001713892 MPPKHYAGHE PREDICTED:similartovacuolarproteinsorting35
BAH13466 MPPKLLCAGR Chromosome22openreadingframe25
BAG60272 MPPKQELGSG Unnamedproteinproduct;growthhormoneinducibletransmembraneproteinGHITM
TXNIP MPPKHSLSHR Thioredoxininteractingprotein
XIRP1 MPPKKKPQLP Xinactin‐bindingrepeatcontaining1,IsoformC
EAX11699 MPPKQTPEIV hCG1646178,isoformCRA_b
EAW87558 MPPKDYKKK hCG1640659
EAW76268 MPPKNVNVNH hCG38498,isoformCRA_bandisoformCRA_e
EAW69186 MPPKCLSLLL hCG2045261
EAW63862 MPPKPESPAQ hCG1994237
MYL2 MAPKKAKKRA myosin,lightpolypeptide2,regulatory,cardiac,slow,isoformCRA_b
NP_001156386 MAPKKKRGPS coiled‐coildomaincontaining121‐like
BAH14246 MAPKKKGKKG cDNAFLJ55405,highlysimilartoGrowth‐arrest‐specificprotein8
FBF1 MAPKTKKGCK Fas(TNFRSF6)bindingfactor1
BAG61983 MAPKKLSCLR cDNAFLJ55394,highlysimilartoHomosapiensgalactosidase,beta1‐like(GLB1L)
BAG65204 MAPKGSSKQQ cDNAFLJ52061,highlysimilartoTranslocon‐associatedproteinsubunitgamma
BAG63505 MAPKFPEEES cDNAFLJ54989,highlysimilartoMitochondrialimportreceptorsubunitTOM34
BAG37206 MAPKRVVQLS Unnamedproteinproduct;peptidylargininedeiminase,typeI
SET MAPKRQSPLP Multitaskingprotein,involvedinapoptosis,transcription,nucleosomeassemblyandhistonebinding
PARP3 MAPKPKPWVQ Poly(ADP‐ribose)polymerasefamilyisoform1
SPAG17 MAPKKEKGGT spermassociatedantigen17
AAI41810 MAPKKSVSKA C9orf117protein
MORF4L1 MAPKQDPKPK Mortalityfactor4like1
EAX10229 MAPKIPEHLV hCG2045839
TMEM208 MAPKGKVGTR TMEM208protein
KIR3DX1 MAPKLITVLC Putativekillercellimmunoglobulin‐likereceptor‐likeproteinKIR3DX1
EAW51418 MAPKAKKEAP hCG2001000
RCC1 MSPKRIAKRR Regulatorofchromosomecondensation1 N‐methylserineRef.1
XP_002343201 MSPKGTTFST
VCY1B MSPKPRASGP Testis‐specificbasicproteinY1
VCX1 MSPKPRASGP VariablechargeX‐linkedprotein1
VCX2 MSPKPRASGP VCX/Ygenesencodesmallandhighlychargedproteinsofunknownfunction
VCX3 MSPKPRASGP VCX/Ygenesencodesmallandhighlychargedproteinsofunknownfunction
RIPK5 MSPKPGCRRE receptorinteractingproteinkinase5
EAW83010 MSPKSCHTQV hCG1815700
EAW60028 MSPKRESAKG hCG1820544
TMC1 MSPKKVQIKV Transmembranechannel‐likeprotein1
ZNF80 MSPKRDGLGT Zincfingerprotein80
Q5W0B8 MSPKESSRPQ PutativeuncharacterizedproteinC10orf136
PRR22 MSPKDKLCVI Proline‐richprotein22
[1]Chen,T.,Muratore,T.L.,Schaner‐Tooley,C.E.,Zhabanowitz,J.,Hunt,D.F.,andMacara,I.G.(2007)Nat.CellBiol.5,596‐603