Amplification and detection of substrates for protein carboxyl methyltransferases in PC12 cells.

A strategy that facilitates the identification of substrates for protein carboxyl methyltransferases that form "stable" methyl esters, i.e., those that remain largely intact during conventional polyacrylamide gel electrophoresis is described. Rat PC12 cells were cultured in the presence of adenosine dialdehyde (a methylation inhibitor) to promote the accumulation of hypomethylated proteins. Nonidet P-40 cell extracts were then incubated in the presence of S-[methyl-3H]adenosyl-L-methionine to label methyl-accepting sites via endogenous methyltransferases. After labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel slices were incubated in 4 N methanesulfonic acid or 6 N HCl to hydrolyze methyl esters. The resulting [3H]methanol was detected by trapping in liquid scintillation fluid. Seven carboxyl methylated proteins were observed with masses ranging from 18 to 96 kDa. Detection of five of these proteins required prior treatment of cells with adenosine dialdehyde, while methyl incorporation into one protein at 18 kDa was substantially enhanced by the treatment. The use of acidic conditions for methyl ester hydrolysis has an important advantage over assays that utilize alkaline hydrolysis conditions. In PC12 cells, and possibly other cell types where there are significant levels of arginine methylation, the methanol signal becomes obscured by high levels of volatile methylamines generated under the alkaline conditions. Carrying out diffusion assays under acidic conditions eliminates this interference. Adenosine dialdehyde, by virtue of increasing the methyl-accepting capacity of substrates for protein carboxyl methyltransferases, in combination with a more selective assay for carboxyl methylation, should prove useful in the isolation and characterization of new protein carboxyl methyltransferases and their substrates.     

Protein N-arginine methylation in subcellular fractions of lymphoblastoid cells

Arginine methylation in RNA-binding proteins containing arginine- and glycine-rich RGG motifs is catalyzed by specific protein arginine N-methyltransferase in cells. We previously showed that lymphoblastoid cells grown in the presence of an indirect methyltransferase inhibitor, adenosine dialdehyde (AdOx), accumulated high level of hypomethylated protein substrates for the endogenous protein methyltransferases or recombinant yeast arginine methyltransferase [Li, C. et al. (1998) Arch. Biochem. Biophys. 351, 53-59]. In this study we fractionated the lymphoblastoid cells to locate the methyltransferases and the substrates in cells. Different sets of hypomethylated methyl-accepting polypeptides with wide range of molecular masses were present in cytosolic, ribosomal, and nucleus fractions. The methylated amino acid residues of the methyl-accepting proteins in these fractions were determined. In all three fractions, dimethylarginine was the most abundant methylated amino acid. The protein-arginine methyltransferase activities in the three fractions were analyzed using recombinant fibrillarin (a nucleolar RGG protein) as the methyl-accepting substrate. Fibrillarin methylation was strongest in the presence of the cytosolic fraction, followed by the ribosomal and then the nucleus fractions. The results demonstrated that protein-arginine methyltransferases as well as their methyl-accepting substrates were widely distributed in different subcellular fractions of lymphoblastoid cells.     

Carboxyl methylation of platelet rap1 proteins is stimulated by guanosine 5'-(3-O-thio)triphosphate

Carboxyl methylation of platelet ras-related proteins, known as rap proteins, was investigated in this study. Platelet membrane proteins of Mr 23,000 incorporated radioactivity in the presence of S-[methyl-3H]adenosylmethionine and platelet cytosol. About 97% of the radioactivity present in the Mr 23,000 proteins was liberated as volatile methanol under basic (1 M sodium hydroxide) conditions. Cycloheximide, an inhibitor of protein synthesis, inhibited incorporation of S-[methyl-3H]adenosylmethionine by 25%. These results suggest that at least 75% of the radioactivity present in the Mr 23,000 proteins is due to carboxyl methylation and not due to the incorporation of S-[methyl-3H]adenosylmethionine into proteins or due to the incorporation of base-stable methyl groups into side chains of arginine, histidine, or lysine residues. Protein methylation did not occur if membranes or cytosol alone was incubated with S-[methyl-3H]adenosylmethionine. Guanosine 5'(3-O-thio)triphosphate increased methylation of the Mr 23,000 proteins in a time- and concentration-dependent manner. Acetyl-farnesylcysteine, a synthetic substrate for carboxyl methyltransferases, completely blocked methylation of the Mr 23,000 membrane proteins. On the basis of one- and two-dimensional Western blots using rap-specific antisera, the Mr 23,000 methylated proteins were identified as rap1 proteins. The existence of the carboxyl-terminal CAAX motif in rap1 proteins, similar to the CAAX motif present in p21ras as well as in the yeast mating factors, leads us to suggest that methylation of rap1 proteins possibly occurs at the alpha-carboxyl-terminal cysteine.     

Modification of yeast ribosomal proteins. Methylation.

Two-dimensional polyacrylamide-gel electrophoretic analysis of yeast ribosomal proteins uniformly labelled in vivo with [methyl-3H]methionine and [1-14C]methionine revealed that four ribosomal proteins are methylated, i.e. proteins S31, S32, L15 and L41. Lysine and arginine appear to be the predominant acceptors of the methyl groups. The degree of methylation ranges from 0.09 to 0.20 methyl group per modified ribosomal protein species.     

Identification of methylated proteins in the yeast small ribosomal subunit: a role for SPOUT methyltransferases in protein arginine methylation

We have characterized the posttranslational methylation of Rps2, Rps3, and Rps27a, three small ribosomal subunit proteins in the yeast Saccharomyces cerevisiae, using mass spectrometry and amino acid analysis. We found that Rps2 is substoichiometrically modified at arginine-10 by the Rmt1 methyltransferase. We demonstrated that Rps3 is stoichiometrically modified by ω-monomethylation at arginine-146 by mass spectrometric and site-directed mutagenic analyses. Substitution of alanine for arginine at position 146 is associated with slow cell growth, suggesting that the amino acid identity at this site may influence ribosomal function and/or biogenesis. Analysis of the three-dimensional structure of Rps3 in S. cerevisiae shows that arginine-146 makes contacts with the small subunit rRNA. Screening of deletion mutants encoding potential yeast methyltransferases revealed that the loss of the YOR021C gene results in the absence of methylation of Rps3. We demonstrated that recombinant Yor021c catalyzes ω-monomethylarginine formation when incubated with S-adenosylmethionine and hypomethylated ribosomes prepared from a YOR021C deletion strain. Interestingly, Yor021c belongs to the family of SPOUT methyltransferases that, to date, have only been shown to modify RNA substrates. Our findings suggest a wider role for SPOUT methyltransferases in nature. Finally, we have demonstrated the presence of a stoichiometrically methylated cysteine residue at position 39 of Rps27a in a zinc-cysteine cluster. The discovery of these three novel sites of protein modification within the small ribosomal subunit will now allow for an analysis of their functional roles in translation and possibly other cellular processes.     

Uncovering the Protein Lysine and Arginine Methylation Network in Arabidopsis Chloroplasts

Post-translational modification of proteins by the addition of methyl groups to the side chains of Lys and Arg residues is proposed to play important roles in many cellular processes. In plants, identification of non-histone methylproteins at a cellular or subcellular scale is still missing. To gain insights into the extent of this modification in chloroplasts we used a bioinformatics approach to identify protein methyltransferases targeted to plastids and set up a workflow to specifically identify Lys and Arg methylated proteins from proteomic data used to produce the Arabidopsis chloroplast proteome. With this approach we could identify 31 high-confidence Lys and Arg methylation sites from 23 chloroplastic proteins, of which only two were previously known to be methylated. These methylproteins are split between the stroma, thylakoids and envelope sub-compartments. They belong to essential metabolic processes, including photosynthesis, and to the chloroplast biogenesis and maintenance machinery (translation, protein import, division). Also, the in silico identification of nine protein methyltransferases that are known or predicted to be targeted to plastids provided a foundation to build the enzymes/substrates relationships that govern methylation in chloroplasts. Thereby, using in vitro methylation assays with chloroplast stroma as a source of methyltransferases we confirmed the methylation sites of two targets, plastid ribosomal protein L11 and the β-subunit of ATP synthase. Furthermore, a biochemical screening of recombinant chloroplastic protein Lys methyltransferases allowed us to identify the enzymes involved in the modification of these substrates. The present study provides a useful resource to build the methyltransferases/methylproteins network and to elucidate the role of protein methylation in chloroplast biology.     

Analysis of erythrocyte protein methyl esters by two-dimensional gel electrophoresis under acidic separating conditions

A two-dimensional polyacrylamide gel electrophoresis system which is suitable for the analysis of protein methylation reactions in cells incubated with L-[methyl-3H]methionine is described. The procedure separates proteins under primarily acidic conditions by isoelectric focusing in the first dimension and by sodium dodecyl sulfate electrophoresis at pH 2.4 in the second dimension. The low pH is essential for preserving protein [3H]methyl esters, but it limits the effective separating range of this system to proteins with isoelectric points between 4 and 8. With this system, we have shown that most, if not all, erythrocyte membrane and cytosolic proteins can act as substoichiometric methyl acceptors for an intracellular S-adenosylmethionine-dependent carboxyl methyltransferase and that protein carboxyl methylation reactions may be the major methyl transfer reaction in erythrocytes. These results are most consistent with the generation of protein substrate sites for the carboxyl methyltransferase by spontaneous deamidation and racemization reactions.     

RNase treatment of yeast and mammalian cell extracts affects in vitro substrate methylation by type I protein arginine N-methyltransferases.

Type I protein arginine N-methyltransferases catalyze the formation of omega-NG-monomethylarginine and asymmetric omega-NG, NG-dimethylarginine residues using S-adenosyl-l-methionine as the methyl donor. In vitro these enzymes can modify a number of soluble methyl-accepting substrates in yeast and mammalian cell extracts including several species that interact with RNA. We treated normal and hypomethylated Saccharomyces cerevisiae and RAT1 cell extracts with RNase prior to in vitro methylation by recombinant protein N-arginine methyltransferases and found that the methylation of certain polypeptides is enhanced up to 12-fold whereas that of others is diminished. 2-D gel electrophoresis of RNase-treated yeast extracts allowed us to tentatively identify the glycine- and arginine-rich (GAR) domain-containing proteins Gar1, Nop1, Sbp1, and Npl3 as major methyl-acceptors based on their known isoelectric points and apparent molecular weights. These results suggest that the methylation and RNA-binding of GAR domain-containing proteins in vivo may regulate protein-nucleic acid or protein-protein interactions.     

Proteomic analysis of stable protein methylation in lymphoblastoid cells

We investigated the global distribution of methylaccepting proteins in lymphoblastoid cells by two-dimensional (2-D) gel electrophoresis. The 2-D electrophoreograms of normal and hypo-methylation (cells grown with a methyltransferase inhibitor adenosine dialdehyde) protein extracts did not exhibit significant differences. However, in vitro methylation of the hypomethylated extracts in the presence of the methyl-group donor S-adenosyl-[methyl-3H]-methionine revealed close to a hundred signals. Less than one-fifth of the signals could be correlated with protein stains, indicating that most of the methylaccepting proteins are low abundant ones. We analyzed six of the spots that can be correlated with protein stains and suggested their identities. Among these putative protein methylacceptors, three are heterogeneous nuclear ribonucleoproteins (hnRNPA2/B1 and hnRNP K) that are reportedly methylated in their arginine- and glycine-rich RGG motifs.     

Structural basis for substrate recognition in the salicylic acid carboxyl methyltransferase family

Recently, a novel family of methyltransferases was identified in plants. Some members of this newly discovered and recently characterized methyltransferase family catalyze the formation of small-molecule methyl esters using S-adenosyl-L-Met (SAM) as a methyl donor and carboxylic acid-bearing substrates as methyl acceptors. These enzymes include SAMT (SAM:salicylic acid carboxyl methyltransferase), BAMT (SAM:benzoic acid carboxyl methyltransferase), and JMT (SAM:jasmonic acid carboxyl methyltransferase). Moreover, other members of this family of plant methyltransferases have been found to catalyze the N-methylation of caffeine precursors. The 3.0-A crystal structure of Clarkia breweri SAMT in complex with the substrate salicylic acid and the demethylated product S-adenosyl-L-homocysteine reveals a protein structure that possesses a helical active site capping domain and a unique dimerization interface. In addition, the chemical determinants responsible for the selection of salicylic acid demonstrate the structural basis for facile variations of substrate selectivity among functionally characterized plant carboxyl-directed and nitrogen-directed methyltransferases and a growing set of related proteins that have yet to be examined biochemically. Using the three-dimensional structure of SAMT as a guide, we examined the substrate specificity of SAMT by site-directed mutagenesis and activity assays against 12 carboxyl-containing small molecules. Moreover, the utility of structural information for the functional characterization of this large family of plant methyltransferases was demonstrated by the discovery of an Arabidopsis methyltransferase that is specific for the carboxyl-bearing phytohormone indole-3-acetic acid.     

The Saccharomyces cerevisiae Set1 complex includes an Ash2 homologue and methylates histone 3 lysine 4.

The SET domain proteins, SUV39 and G9a have recently been shown to be histone methyltransferases specific for lysines 9 and 27 (G9a only) of histone 3 (H3). The SET domains of the Saccharomyces cerevisiae Set1 and Drosophila trithorax proteins are closely related to each other but distinct from SUV39 and G9a. We characterized the complex associated with Set1 and Set1C and found that it is comprised of eight members, one of which, Bre2, is homologous to the trithorax-group (trxG) protein, Ash2. Set1C requires Set1 for complex integrity and mutation of Set1 and Set1C components shortens telomeres. One Set1C member, Swd2/Cpf10 is also present in cleavage polyadenylation factor (CPF). Set1C methylates lysine 4 of H3, thus adding a new specificity and a new subclass of SET domain proteins known to methyltransferases. Since methylation of H3 lysine 4 is widespread in eukaryotes, we screened the databases and found other Set1 homologues. We propose that eukaryotic Set1Cs are H3 lysine 4 methyltransferases and are related to trxG action through association with Ash2 homologues.     

Levansucrases from Pseudomonas syringae pv. tomato and P. chlororaphis subsp. aurantiaca: substrate specificity, polymerizing properties and usage of different acceptors for fructosylation

Levansucrases of Pseudomonas syringae pv. tomato DC3000 (Lsc3) and Pseudomonas chlororaphis subsp. aurantiaca (also Pseudomonas aurantiaca) (LscA) have 73% identity of protein sequences, similar substrate specificity and kinetic properties. Both enzymes produce levan and fructooligosaccharides (FOS) of varied length from sucrose, raffinose and sugar beet molasses. A novel high-throughput chip-based nanoelectrospray mass spectrometric method was applied to screen alternative fructosyl acceptors for levansucrases. Lsc3 and LscA could both transfructosylate d-xylose, d-fucose, l- and d-arabinose, d-ribose, d-sorbitol, xylitol, xylobiose, d-mannitol, d-galacturonic acid and methyl-α-d-glucopyranoside and heterooligofructans with degree of polymerization up to 5 were detected. The ability of d-sorbitol, xylobiose, d-galacturonic acid, d-mannitol, xylitol and methyl-α-d-glucopyranoside to serve as fructosyl acceptors for levansucrases is shown for the first time. Expectedly, site-directed mutagenesis of His321 in Lsc3 to Arg, Lys, Leu and Ser resulted in proteins with decreased catalytic activity, affinity for sucrose and polymerizing ability. Random mutagenesis yielded a Lsc3 mutant Thr302Pro with reduced synthesis of levan and long-chain FOS. Thr302 is located in conserved DQTERP region of levansucrases adjacent to predicted acid-base catalyst Glu303. Thr302 and His321 are predicted to belong to +1 subsite of the substrate binding region of Lsc3.     

Biosynthesis of methylphosphomannosyl residues in the oligosaccharides of Dictyostelium discoideum glycoproteins. Evidence that the methyl group is derived from methionine

The phosphorylated oligosaccharides of Dictyostelium discoideum contain methylphosphomannosyl residues which are stable to mild-acid and base hydrolysis (Gabel, C. A., Costello, C. E., Reinhold, V. N., Kurtz, L., and Kornfeld, S. (1984) J. Biol. Chem. 259, 13762-13769). Here we present evidence that these methyl groups are derived from [methyl-3H]methionine, in vivo and [methyl-3H]S-adenosylmethionine in vitro. About 18% of the macromolecules secreted from vegetative cells labeled with [methyl-3H]methionine are released by digestion with preparations of endoglycosidase/peptide N-glycosidase F. The majority of the released molecules are sulfated, anionic high mannose-type oligosaccharides. Strong acid hydrolysis of the [3H]methyl-labeled molecules yields [3H]methanol with kinetics of release similar to those found for the generation of Man-6-P from chemically synthesized methylphosphomannose methylglycoside. Treatment of the [3H]methyl-labeled molecules with a phosphodiesterase from Aspergillus niger which is known to cleave this phosphodiester also releases [3H]methanol from a portion of the oligosaccharides. In vitro incorporation of [methyl-3H]S-adenosylmethionine into endogenous acceptors found in membrane preparations shows that the [3H]methyl group of the methylphosphomannose residues can be derived from this molecule.     

Novel Sm-like proteins with long C-terminal tails and associated methyltransferases

Sm and Sm-like proteins of the Lsm (like Sm) domain family are generally involved in essential RNA-processing tasks. While recent research has focused on the function and structure of small family members, little is known about Lsm domain proteins carrying additional domains. Using an integrative bioinformatics approach, we discovered five novel groups of Lsm domain proteins (Lsm12-16) with long C-terminal tails and investigated their functions. All of them are evolutionarily conserved in eukaryotes with an N-terminal Lsm domain to bind nucleic acids followed by as yet uncharacterized C-terminal domains and sequence motifs. Based on known yeast interaction partners, Lsm12-16 may play important roles in RNA metabolism. Particularly, Lsm12 is possibly involved in mRNA degradation or tRNA splicing, and Lsm13-16 in the regulation of the mitotic G2/M phase. Lsm16 proteins have an additional C-terminal YjeF_N domain of as yet unknown function. The identification of an additional methyltransferase domain at the C-terminus of one of the Lsm12 proteins also led to the recognition of three new groups of methyltransferases, presumably dependent on S-adenosyl-l-methionine. Further computational analyses revealed that some methyltransferases contain putative RNA-binding helix-turn-helix domains and zinc fingers.     

Rapid and convenient method for the assay of aminopropyltransferases.

A new method for the assay of aminopropyltransferase activity is described. The method measures the formation of [methyl-14C]methylthioadenosine from decarboxylated S-adenosyl[methyl-14C]methionine in the presence of an amine acceptor. When used with extracts from rat ventral prostate, kidney, liver or brain, and with putrescine or spermidine as amines, the method gave results in excellent agreement with those obtained by the much more time-consuming conventional method. It was found that 1,3-diamino-propane and 1,8-diamino-octane were not acceptors for the prostatic enzyme fraction, but 1,5-diaminopentane (cadaverine) was active and 1,9-diaminononane and 1,12-diaminododecane also lead to the production of [methyl-14C]methylthioadenosine.     

The human homologue of the yeast proteins Skb1 and Hsl7p interacts with Jak kinases and contains protein methyltransferase activity.

To expand our understanding of the role of Jak2 in cellular signaling, we used the yeast two-hybrid system to identify Jak2-interacting proteins. One of the clones identified represents a human homologue of the Schizosaccaromyces pombe Shk1 kinase-binding protein 1, Skb1, and the protein encoded by the Saccharomyces cerevisiae HSL7 (histone synthetic lethal 7) gene. Since no functional motifs or biochemical activities for this protein or its homologues had been reported, we sought to determine a biochemical function for this human protein. We demonstrate that this protein is a protein methyltransferase. This protein, designated JBP1 (Jak-binding protein 1), and its homologues contain motifs conserved among protein methyltransferases. JBP1 can be cross-linked to radiolabeled S-adenosylmethionine (AdoMet) and methylates histones (H2A and H4) and myelin basic protein. Mutants containing substitutions within a conserved region likely to be involved in AdoMet binding exhibit little or no activity. We mapped the JBP1 gene to chromosome 14q11.2-21. In addition, JBP1 co-immunoprecipitates with several other proteins, which serve as methyl group acceptors and which may represent physiological targets of this methyltransferase. Messenger RNA for JBP1 is widely expressed in human tissues. We have also identified and sequenced a homologue of JBP1 in Drosophila melanogaster. This report provides a clue to the biochemical function for this conserved protein and suggests that protein methyltransferases may have a role in cellular signaling.