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.
Highlights
This protein is a member of the ribosomal L1 family that is conserved in most organisms and forms the “L1 protuberance” that is involved in tRNA exit from the E site of the large subunit (20 –22)
The YLR137W gene product, designated Rkm5, joins four other protein lysine methyltransferases of the seven--strand superfamily: the Dot1 methyltransferase acting on histone H3 [25, 26], the CaM KMT acting on calmodulin [27], the See1 methyltransferase acting on elongation factor 1A [17], and the PrmA methyltransferase acting on prokaryotic ribosomal protein L11 [28]
The YLR137W Gene Is Required for the Modification of the Yeast Ribosomal Protein Rpl1ab—In an effort to identify new protein methyltransferases, yeast strains with deletions of known and putative methyltransferase genes were screened for loss of ribosomal protein methylation
Summary
Isolation of Ribosomal Proteins from S. cerevisiae Wild-type and Mutant Strains—The wild-type BY4742 and the ⌬YLR137W/rkm deletion strain in the BY4742 background, as well as the deletion strains listed in supplemental Table S1, were obtained from the Saccharomyces Genome Deletion Project via Open Biosystems (Huntsville, AL). Liquid Chromatography with Electrospray Ionization Mass Spectrometry of Intact Ribosomal Proteins—Ribosomal proteins extracted from the large subunit were fractionated using reverse phase liquid chromatography as described previously [9]. The YLR137W/ RKM5 open reading frame was cloned into the pET100/DTOPO Escherichia coli expression vector as instructed (Invitrogen). The vector pET100/D-TOPO encodes a His-tagged N-terminal linker sequence (MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDHPFT) that is followed by the complete amino acid sequence of the YLR137W/RKM5 open reading frame, including the initiator methionine residue. This plasmid was transformed into E. coli BL21(DE3) cells (Invitrogen). The resulting supernatant was loaded onto a 5-ml HisTrap HP nickel affinity column (GE Healthcare part number 17-5248-1), and the fusion protein was purified using a gradient of 30 –500 mM imidazole per the manufacturer’s specifications
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