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ABSTRACT: Modification of proteins of the translational apparatus is common in many organisms. In the yeast Saccharomyces cerevisiae, we provide evidence for the methylation of Rpl1ab, a well conserved protein forming the ribosomal L1 protuberance of the large subunit that functions in the release of tRNA from the exit site. We show that the intact mass of Rpl1ab is 14 Da larger than its calculated mass with the previously described loss of the initiator methionine residue and N-terminal acetylation. We determined that the increase in mass of yeast Rpl1ab is consistent with the addition of a methyl group to lysine 46 using top-down mass spectrometry. Lysine modification was confirmed by detecting (3)H-N-ε-monomethyllysine in hydrolysates of Rpl1ab purified from yeast cells radiolabeled in vivo with S-adenosyl-l-[methyl-(3)H]methionine. Mass spectrometric analysis of intact Rpl1ab purified from 37 deletion strains of known and putative yeast methyltransferases revealed that only the deletion of the YLR137W gene, encoding a seven-β-strand methyltransferase, results in the loss of the +14-Da modification. We expressed the YLR137W gene as a His-tagged protein in Escherichia coli and showed that it catalyzes N-ε-monomethyllysine formation within Rpl1ab on ribosomes from the ΔYLR137W mutant strain lacking the methyltransferase activity but not from wild-type ribosomes. We also showed that the His-tagged protein could catalyze monomethyllysine formation on a 16-residue peptide corresponding to residues 38-53 of Rpl1ab. We propose that the YLR137W gene be given the standard name RKM5 (ribosomal lysine (K) methyltransferase 5). Orthologs of RKM5 are found only in fungal species, suggesting a role unique to their survival.
Journal of Biological Chemistry 04/2011; 286(21):18405-13. · 4.77 Impact Factor
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ABSTRACT: We have shown that Rpl3, a protein of the large ribosomal subunit from baker's yeast (Saccharomyces cerevisiae), is stoichiometrically monomethylated at position 243, producing a 3-methylhistidine residue. This conclusion is supported by top-down and bottom-up mass spectrometry of Rpl3, as well as by biochemical analysis of Rpl3 radiolabeled in vivo with S-adenosyl-l-[methyl-(3)H]methionine. The results show that a +14-Da modification occurs within the GTKKLPRKTHRGLRKVAC sequence of Rpl3. Using high-resolution cation-exchange chromatography and thin layer chromatography, we demonstrate that neither lysine nor arginine residues are methylated and that a 3-methylhistidine residue is present. Analysis of 37 deletion strains of known and putative methyltransferases revealed that only the deletion of the YIL110W gene, encoding a seven β-strand methyltransferase, results in the loss of the +14-Da modification of Rpl3. We suggest that YIL110W encodes a protein histidine methyltransferase responsible for the modification of Rpl3 and potentially other yeast proteins, and now designate it Hpm1 (Histidine protein methyltransferase 1). Deletion of the YIL110W/HPM1 gene results in numerous phenotypes including some that may result from abnormal interactions between Rpl3 and the 25 S ribosomal RNA. This is the first report of a methylated histidine residue in yeast cells, and the first example of a gene required for protein histidine methylation in nature.
Journal of Biological Chemistry 11/2010; 285(48):37598-606. · 4.77 Impact Factor
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ABSTRACT: Eukaryotic elongation factor 1A (eEF1A) is an abundant cytosolic protein in Saccharomyces cerevisiae and is well conserved amongst species. This protein undergoes multiple posttranslational modifications, including the N-methylation of four side chain lysine residues. However, the enzyme(s) responsible for catalyzing these modifications have remained elusive. Here we show by intact protein mass spectrometry that deletion of either of two genes coding for putative methyltransferases results in a loss in mass of eEF1A. Deletion of the YHL039W gene, a member of the SET domain subfamily including cytochrome c and ribosomal protein lysine methyltransferases, results in an eEF1A mass loss corresponding to a single methyl group. Deletion in the YIL064W/SEE1 gene, encoding a well conserved seven beta strand methyltransferase sequence, has been shown previously to affect vesicle transport; in this work we show that deletion results in the loss of two methyl group equivalents from eEF1A. We find that deletion of thirty-five other putative and established SET domain and seven beta strand methyltransferases has no effect on the mass of eEF1A. Finally, we show that wild type extracts, but not YIL064W/SEE1 mutant extracts, can catalyze the S-adenosylmethionine-dependent in vitro methylation of hypomethylated eEF1A. We suggest that YHL039W (now designated EFM1 for elongation factor methyltransferase 1) and YIL064W/SEE1 encode distinct eEF1A methyltransferases that respectively monomethylate and dimethylate this protein at lysine residues.
Archives of Biochemistry and Biophysics 08/2010; 500(2):137-43. · 2.93 Impact Factor
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ABSTRACT: Protein modification by methylation is important in cellular function. We show here that the Saccharomyces cerevisiae YBR261C/TAE1 gene encodes an N-terminal protein methyltransferase catalyzing the modification of two ribosomal protein substrates, Rpl12ab and Rps25a/Rps25b. The YBR261C/Tae1 protein is conserved across eukaryotes; all of these proteins share sequence similarity with known seven beta-strand class I methyltransferases. Wild-type yeast cytosol and mouse heart cytosol catalyze the methylation of a synthetic peptide (PPKQQLSKY) that contains the first eight amino acids of the processed N-terminus of Rps25a/Rps25b. However, no methylation of this peptide is seen in yeast cytosol from a DeltaYBR261C/tae1 deletion strain. Yeast YBR261C/TAE1 and the human orthologue METTL11A genes were expressed as fusion proteins in Escherichia coli and were shown to be capable of stoichiometrically dimethylating the N-terminus of the synthetic peptide. Furthermore, the YBR261C/Tae1 and METTL11A recombinant proteins 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 lysine residue in position 3 is substituted. Thus, the methyltransferases described here specifically recognize the N-terminal X-Pro-Lys sequence motif, and we suggest designating the yeast enzyme Ntm1 and the human enzyme NTMT1. These enzymes may account for nearly all previously described eukaryotic protein N-terminal methylation reactions. A number of other yeast and human proteins also share the recognition motif and may be similarly modified. We conclude that protein X-Pro-Lys N-terminal methylation reactions catalyzed by the enzymes described here may be widespread in nature.
Biochemistry 06/2010; 49(25):5225-35. · 3.42 Impact Factor
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ABSTRACT: Rps2/rpS2 is a well conserved protein of the eukaryotic ribosomal small subunit. Rps2 has previously been shown to contain asymmetric dimethylarginine residues, the addition of which is catalyzed by zinc-finger-containing arginine methyltransferase 3 (Rmt3) in the fission yeast Schizosaccharomyces pombe and protein arginine methyltransferase 3 (PRMT3) in mammalian cells. Here, we demonstrate that despite the lack of a zinc-finger-containing homolog of Rmt3/PRMT3 in the budding yeast Saccharomyces cerevisiae, Rps2 is partially modified to generate asymmetric dimethylarginine and monomethylarginine residues. We find that this modification of Rps2 is dependent upon the major arginine methyltransferase 1 (Rmt1) in S. cerevisiae. These results are suggestive of a role for Rmt1 in modifying the function of Rps2 in a manner distinct from that occurring in S. pombe and mammalian cells.
Biochemical and Biophysical Research Communications 01/2010; 391(4):1658-62. · 2.48 Impact Factor
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ABSTRACT: It has previously been shown that incubation of mammalian cell cytosolic extracts with the protein kinase inhibitor tyrphostin A25 results in enhanced transfer of methyl groups from S-adenosyl-[methyl-3H]methionine to proteins. These findings were interpreted as demonstrating tyrphostin stimulation of a novel type of protein carboxyl methyltransferase. We find here, however, that tyrphostin A25 addition to mouse heart cytosol incubated with S-adenosyl-[methyl-3H]methionine or S-adenosyl-[methyl-14C]methionine stimulates the labeling of small molecules in addition to proteins. Base treatment of both protein and small molecule fractions releases volatile radioactivity, suggesting labile ester-like linkages of the labeled methyl group. Production of both the base-volatile product and labeled protein occurs with tyrphostins A25, A47, and A51, but not with thirteen other tyrphostin family members. These active tyrphostins all contain a catechol moiety and are good substrates for recombinant and endogenous catechol-O-methyltransferase. Inhibition of catechol-O-methyltransferase activity with tyrphostin AG1288 prevents both base-volatile product formation and protein labeling from methyl-labeled S-adenosylmethionine in heart, kidney, and liver, but not in testes or brain extracts. These results suggest that the incorporation of methyl groups into protein follows a complex pathway initiated by the methylation of select tyrphostins by endogenous catechol-O-methyltransferase. We suggest that the methylated tyrphostins are further modified in the cell extract and covalently attached to cellular proteins. The presence of endogenous catechols in cells suggests that similar reactions can also occur in vivo.
Journal of Biological Chemistry 11/2007; 282(42):31094-102. · 4.77 Impact Factor
