S-Adenosylmethionine-dependent Methylation inSaccharomyces cerevisiae

Abstract

We used sequence motifs conserved in S-adenosylmethionine-dependent methyltransferases to identify 26 putative methyltransferases from the complete genome of the yeast Saccharomyces cerevisiae. Seven sequences with the best matches to the methyltransferase consensus motifs were selected for further study. We prepared yeast disruption mutants of each of the genes encoding these sequences, and we found that disruption of the YJL125c gene is lethal, whereas disruptions of YCR047c and YDR140w lead to slow growth phenotypes. Normal growth was observed when the YDL201w, YDR465c, YHR209w, and YOR240w genes were disrupted. Initial analysis of protein methylation patterns of all mutants by amino acid analysis revealed that the YDR465c mutant has a defect in the methylation of the ␦-nitrogen atom of arginine residues. We propose that YDR465c codes for the methyltransferase responsible for this recently characterized type of protein methylation, and we designate the enzyme as Rmt2 (protein arginine methyltransferase). In addition, we show that the methylation of susceptible residues in Rmt2 substrates is likely to take place on nascent polypeptide chains and that these substrates exist in the cell as fully methylated species. Interestingly, Rmt2 has 27% sequence identity over 138 amino acids to the mammalian guanidinoacetate N-methyltransferase, an enzyme responsible for methylating the ␦-nitrogen of the small molecule guanidinoacetate.The recent explosion in the number of uncharacterized genes resulting from sequencing of entire genomes presents a new challenge in biology. Although several living organisms can now be completely defined in terms of their genetic codes, understanding the functions and interactions of macromolecules encoded by the multitude of genes is in its infancy. Methods are needed to assign function to new gene products in order to come closer to understanding what makes up a living system. With this goal in mind, we wanted to assign function to a fraction of the new sequences, specifically to S-adenosylmethionine-dependent methyltransferases in the yeast Saccharomyces cerevisiae, for which the complete genome has recently been reported (1). A large class of AdoMet 1 -dependent methyltransferases have been found to share a conserved catalytic domain structure due to the interaction of the enzymes with a common cofactor, S-adenosylmethionine (2). To date, the structures of nine AdoMet-dependent methyltransferases have been solved, and they show a similar folding pattern with a central parallel ␤-sheet surrounded by ␣-helces (3-11). The common three-dimensional structure of these enzymes is reflected in sequence motifs that are conserved among a large number of AdoMet-dependent methyltransferases (12-17). The conserved regions, which we have previously designated as motifs I, post-I, II, and III (15), are always found in the same order on the polypeptide chain and are separated by comparable intervals. The three-dimensional structures of AdoMet-dependent methyltransferases have revealed that motif I and ...

Human Fibrillarin Forms a Sub-complex with Splicing Factor 2-associated p32, Protein Arginine Methyltransferases, and Tubulins α3 and β1 That Is Independent of Its Association with Preribosomal Ribonucleoprotein Complexes

“…Thus, given that FIB is involved in ribosome biogenesis and is itself an RNA methyltransferase, our results provide biochemical evidence that these three different types of methyltransferases physically associate and therefore may function in concert. Given that, during ribosome biogenesis, not only are pre-rRNAs methylated but several nucleolar and ribosomal proteins as well (37)(38)(39), three types of methylation reactions may be required to coordinate certain stages of ribosome biogenesis and/or related processes.…”

The methylator meets the terminator

“…But for many methyltransferases (including the HemK methyltransferase seen here), it doesn't seem to be so simple. For example, sequence analysis of the 13 best methyltransferase candidates in yeast led to probably incorrect identifications of 12 of these gene products and only one useful ''hit,'' where a novel protein arginine methyltransferase was matched with a guanidinoacetate methyltransferase of higher cells (22).…”

“…These enzymes have the very simple function of transferring a methyl group from the sulfonium atom of AdoMet to a variety of nucleophiles, including oxygen, sulfur, nitrogen, and carbon atoms on proteins, nucleic acids, carbohydrates, lipids, and small molecules in all organisms. Evolution has been kind to the workers in this field because most of these enzymes have retained four ''signature motifs,'' short stretches of sequence that have been apparently conserved over billions of years of evolution and that can allow the identification of candidate methyltransferases with some success (21,22). It was originally hoped that examination of sequence similarity outside of the signature motifs would allow one to start characterizing families of methyltransferases specific for N-DNA, C-DNA, protein carboxyl, protein amino, protein guanidino, etc., methyl-accepting substrates.…”

The Biosynthesis of Methylated Amino Acids in the Active Site Region of Methyl-coenzyme M Reductase

“…It has been shown recently that stable heavy isotope labels can be followed by mass spectrometry down to the level of purified peptides and that it is possible to evaluate the spectra thus obtained quantitatively (5). 3 ]Methionine (purity Ͼ99%) was purchased from ICN Biomedicals, and L-[methyl- 14 C]methionine (0.1 mCi/ml; 54 mCi/mmol) was from Moravek Biochemicals. Tosyl-L-lysine-chloromethylketone-treated chymotrypsin (EC 3.4.21.1) was from Roche Molecular Biochemicals.…”

“…Methyl-coenzyme M reductase was purified from Methanobacterium thermoautotrophicum strain Marburg as described earlier (6). The methanogenic archaeon was cultivated at 65°C on standard medium (7) or on standard medium supplemented with either 2.5 mM L-[methyl-D 3 ]methionine or 2.5 mM [methyl- 14 C]methionine (10,000 dpm/ml) (4).…”