Methylation of ribosomal proteins in HeLa cells

Chang, F. N.; Navickas, I.J.; Chang, Chi-Hsiung; Dancis, B.M.

doi:10.1016/0003-9861(76)90117-x

Cited by 58 publications

(31 citation statements)

References 17 publications

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“…In one case it has been suggested that the N-methylation of cytoc4rome c (25). The N-methylation of ribosomal proteins that interact with polynucleotides has been shown to occur in both procaryotes and eucaryotes (4,5,10,23). Although there are mutations affecting the methylation of all three sites of protein Lll in E. coli, the methyl-deficient protein appears to participate in the formation of fully functional 50S ribosomes (6).…”

Section: Discussionmentioning

confidence: 99%

Regulation of protein synthesis factor EF-1 alpha in Mucor racemosus.

Fonzi¹,

Katayama²,

Leathers³

et al. 1985

Mol. Cell. Biol.

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The protein synthesis elongation factor EF-la of Mucor racemosus hyphae contained eight or nine methylated amino acids per molecule, whereas the factor from sporangiospores was nonmethylated. During the course of spore germination, the specific activity of the factor in crude extracts increased sixfold. This increase in activity was accompanied by a constant level of EF-let-specific mRNA and a constant level of EF-loa protein.Methylation of the protein, however, accelerated during the germination process, in parallel with the increase in specific activity of the factor. We propose that the activity of EF-loa is regulated during germination through methylation of the protein and does not involve transcriptional regulation.Mucor racemosus is a dimorphic fungus that grows as a budding yeast in CO2 and as a mycelium in air. These alternative forms of growth, as well as the process of spore germination, are accompanied by differences in the translational system. Among these are differences in the rate of peptide bond formation (18,19), the extent of phosphorylation of ribosomal protein S-6 (11, 12), and the methylation of the elongation factor EF-la (9). In the latter case, we have found that of the 44 lysine residues, 8 or 9 are methylated when the organism grows in the mycelial form (9). The extent of methylation is less in the yeast form, and spores contain an EF-la that is essentially nonmethylated.In an effort to understand the regulation of EF-la synthesis and activity and the role EF-la may play in changes in the rate of peptide bond formation, we studied the acquisition of EF-la activity during spore germination. These studies were aided by obtaining a cDNA clone which made it possible to measure mRNA levels. We show here that EF-la protein and mRNA are made at constant levels during a period that EF-la activity is rising dramatically. Our conclusion is that activity of EF-la in M. racemosus is regulated through the methylation of the protein after synthesis. MATERIALS AND METHODSOrganisms and culture conditions. M. racemosus (M. lusitanicus) ATCC 1216B was used in all experiments. Sporangiospores were prepared as described previously (20). Spores (2 x 105/ml) were germinated in YPG medium (2% glucose, 1% Bacto-Peptone, 0.3% yeast extract [pH 5.5] idodiphosphate was used in place of GTP, the reaction was stopped with 50 ,ul of 4% glutaraldehyde to stabilize the EF-1 complex bound to the ribosome. Polyuridylate-directed phenylalanine synthesis was conducted as described by Merrick (16), except that the buffer contained 50 mnM KCl and 0.8 mM spermidine.[3H]Phe-tRNA was prepared from yeast tRNA Phe (Boehringer Mannheim Biochemicals) and L-[2,6-3H]Phe (Amersham Corp.) (50 Ci/mmol) with Escherichia coli crude aminoacyl-tRNA synthetases (16). High-salt washed ribosomes were prepared as described by Larsen and Sypherd (11), except that NH4Cl was substituted for KCI. Elongation factor EF-la was prepared as previously described (9), and EF-2 was prepared from wheat germ as described by Legocki (13). Cell extracts for ...

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Section: Discussionmentioning

confidence: 99%

Regulation of protein synthesis factor EF-1 alpha in Mucor racemosus.

Fonzi¹,

Katayama²,

Leathers³

et al. 1985

Mol. Cell. Biol.

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“…The methylation of arginine residues is catalyzed by at least two different classes of protein arginine methyltransferase (PRMT) 2 enzymes. The Type I enzymes catalyze the formation of asymmetric N G ,N G -dimethylarginine residues and the Type II enzyme catalyzes the formation of symmetric N G ,NЈ G -dimethylarginine residues.…”

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confidence: 99%

“…The major pools of protein that are arginine methylated are the heterogeneous nuclear ribonucleoproteins, histones, and ribosomal proteins. Thirty years ago, HeLa cell ribosomal proteins were shown to be heavily lysine-and arginine-methylated (2), and further two-dimensional gel electrophoresis studies of purified ribosomes demonstrated that at least six prominent proteins are arginine methylated (3). PRMT3 is a ribosome-associated protein (4,5) and may be responsible for much of the arginine methylation that occurs in this molecular machine.…”

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confidence: 99%

Ribosomal Protein rpS2 Is Hypomethylated in PRMT3-deficient Mice

Swiercz

Cheng

Kim

et al. 2007

Journal of Biological Chemistry

121

101

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PRMT3 is a type I arginine methyltransferase that resides in the cytoplasm. A large proportion of this cystosolic PRMT3 is found associated with ribosomes. It is tethered to the ribosomes through its interaction with rpS2, which is also its substrate. Here we show that mouse embryos with a targeted disruption of PRMT3 are small in size but survive after birth and attain a normal size in adulthood, thus displaying Minute-like characteristics. The ribosome protein rpS2 is hypomethylated in the absence of PRMT3, demonstrating that it is a bona fide, in vivo PRMT3 substrate that cannot be modified by other PRMTs. Finally, the levels 40 S, 60 S, and 80 S monosomes and polyribosomes are unaffected by the loss of PRMT3, but there are additional as yet unidentified proteins that co-fractionate with ribosomes that are also dedicated PRMT3 substrates.Arginine methylation is a common posttranslational modification that occurs in both the nucleus and the cytoplasm (1). The methylation of arginine residues is catalyzed by at least two different classes of protein arginine methyltransferase (PRMT) 2 enzymes. The Type I enzymes catalyze the formation of asymmetric N G ,N G -dimethylarginine residues and the Type II enzyme catalyzes the formation of symmetric N G ,NЈ G -dimethylarginine residues. In mammals, there are five Type I enzymes (PRMT1,3,4, 6 and 8).Proteins that are substrates for the PRMTs usually contain glycine and arginine-rich patches, GAR motifs, that are the sites of methylation. The major pools of protein that are arginine methylated are the heterogeneous nuclear ribonucleoproteins, histones, and ribosomal proteins. Thirty years ago, HeLa cell ribosomal proteins were shown to be heavily lysine-and arginine-methylated (2), and further two-dimensional gel electrophoresis studies of purified ribosomes demonstrated that at least six prominent proteins are arginine methylated (3). PRMT3 is a ribosome-associated protein (4, 5) and may be responsible for much of the arginine methylation that occurs in this molecular machine.PRMT3 was identified as a PRMT1 binding protein in a yeast two-hybrid screen (6). However, gel filtration analysis of RAT1 cells demonstrated that PRMT3 occurs as a monomer and is not complexed in vivo with PRMT1 (6). PRMT3 also interacts with the tumor suppressor DAL-1 (7). This interaction inhibits PRMT3 enzymatic activity, both in in vitro reactions and in cell lines. At the organ level, PRMT3 is ubiquitously expressed (6), but in the brain it displays higher expression in neurons than in glial cells (8). PRMT3 is the only type 1 arginine methyltransferase that is localized exclusively to the cytoplasm (6, 9). Another unique property of PRMT3 is that it harbors a zinc finger domain at its N terminus. It has been proposed that this domain may play a role in the regulation of PRMT3 activity or in the recognition of PRMT3 substrates (6, 10). Deletion analysis studies demonstrated that PRMT3 lacking the zinc finger domain is still active in vitro, however the zinc finger minus enzyme loses its abil...

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“…Although it was claimed [5] that methylated amino acids in E. coli ribosomal proteins were confined predominantly to 50 S particles, the possibility of methylation in proteins S10 and S16 was conceded -although data for these proteins were unconvincing. In HeLa cells dimethylarginine and di-and trimethyllysine could be detected in both ribosomal subunits although the proteins involved were not identified [9]. Methylation was, however, higher in the 60 S particle and in fact exposure of 40 S particles to high salt (0.6 M KC1) led to a loss of some of the arginine-associated methyl groups.…”

Section: Introductionmentioning

confidence: 92%

“…Methylated amino acids have been detected in ribosomal proteins from Escherichia coil Blastocladiella emersoniL Euglena gracilis, cultured muscle cells and HeLa cells [1][2][3][4][5][6][7][8][9][10][11]. Several of these studies have indicated that methylated amino acids occur in the larger of the two ribosomal subunits.…”

Section: Introductionmentioning

confidence: 99%