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 homogeneity. 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 protein 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 delta-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 delta-N-monomethylarginines.
Chern
Ming-Kai
MK
Institute of Molecular Biology, Academia Sinica, NanKang Taipei, Taiwan 11529, Republic of China.
Chang
Kwang-Ning
KN
Liu
Li-Fan
LF
Tam
Tsuey-Chyi S
TC
Liu
Yi-Chen
YC
Liang
Yi-Lin
YL
Tam
Ming F
MF
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D011484:Protein-Arginine N-Methyltransferases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; C016700:ribosomal protein L7-L12; D012436:S-Adenosylmethionine; D008780:Methyltransferases; D011484:Protein-Arginine N-Methyltransferases; C116910:RMT2 protein, S cerevisiae