pubmed.ncbi.nlm.nih.gov

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

  • ️Sun Jan 01 2012

. 2012 Jun 26;51(25):5091-104.

doi: 10.1021/bi300186g. Epub 2012 Jun 15.

Affiliations

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

Brian D Young et al. Biochemistry. 2012.

Abstract

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.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1

Rps3 is monomethylated at arginine-146. (A) HPLC-purified Rps3 from wild-type BY4742 cells grown in the presence of S-adenosyl-[methyl-3H]-L-methionine was acid hydrolyzed and the free amino acids were mixed with methylated standards and fractionated using high-resolution cation-exchange chromatography as described in the “Experimental Procedures” section. The 3H-radioactivity in the resulting fractions was quantified with a liquid scintillation counter and is marked by a solid line; the position of the added methylated amino acid standards detected with a ninhydrin assay are shown by the dotted line. 3H-radioactivity in Rps3 co-elutes just prior to the standard of ω-monomethylarginine, as expected for the tritium-labeled species (79). (B) Unlabeled HPLC-purified Rps3 was isolated from wild-type BY4742 cells and digested with cyanogen bromide prior to analysis of the resulting cleavage products with a hybrid linear ion trap/FTICR mass spectrometer. The spectra were deconvoluted and the resulting monoisotopic masses were searched against those predicted for Rps3 cyanogen bromide cleavage products. Matching unmodified peptides are boxed in light gray, while matching peptides containing one oxidation and one missed cleavage are underlined. Peptide matches are shown with the error. Matching unmethylated cleavage products were identified for all regions of Rps3 except between residues 127 and 149. The region spanning residues 127 and 149 contains the two arginine residues, arginine-143 and arginine-146, marked with gray circles. A detailed description of our mass spectrometry methods is given in the “Experimental Procedures” section. (C) Ribosomal proteins of the small subunit were isolated and analyzed by liquid chromatography-mass spectrometry from yeast strains expressing wild-type Rps3 and Rps3 containing arginine to lysine substitutions at residues 143 and 146, labeled as R143K Rps3 and R146K Rps3, respectively. Reconstructed spectra of Rps3 in each of these strains are shown. The R143K Rps3 strain has a mass for Rps3 that is 28 Da lower than the wild-type, which is consistent with an arginine to lysine substitution. The R146K Rps3 strain, however, has a mass for Rps3 that is 42 Da lower than the wild-type, indicating an arginine to lysine substitution and a loss of methylation. The generation of these strains is detailed in the “Experimental Procedures” section.

FIGURE 2
FIGURE 2

Arginine-146 in Rps3 likely forms close contacts with the 18S rRNA. The crystal structure of the small ribosomal subunit of S. cerevisiae (PDB IDs: 3U5B, 3U5C, 3U5F, 3U5G) (40) is explored to better understand the role of arginine-146 methylation in Rps3 from S. cerevisiae. (A) The structure of Rps3 alone is shown in yellow and arginine-146 is marked in orange for carbon atoms, red for oxygen atoms, and blue for nitrogen atoms. (B) The structure of Rps3 and arginine-146 (colored as in panel A) in the presence of other small ribosomal proteins (colored in green). Distances from arginine-146 in Rps3 to other nearby ribosomal proteins are shown. (C) Arginine-146 from Rps3 (colored as in panel A) and the 18S rRNA (purple) are shown. Adenine-1427, which has close interactions with arginine-146, is shown with carbon atoms in pink and nitrogen atoms in blue, and cytosine-1274, which interacts closely with adenine-1427, is shown with carbon atoms in teal, oxygen atoms in red, and nitrogen atoms in blue. (D) The distances between these bases and arginine-146 are indicated.

FIGURE 3
FIGURE 3

The amino acid identity at residue 146 in Rps3 is critical for optimal growth. Yeast strains expressing wild-type Rps3 and Rps3 containing amino acid substitutions at residues 143 and 146 were assayed for growth fitness. Serial dilutions of equal numbers of cells were plated and grown at 30 °C as described in the “Experimental Procedures” section. R143K, arginine to lysine substitution at position 143; R146K, arginine to lysine substitution at position 146; R143K + R146K, arginine to lysine substitutions at positions 143 and 146; R143A, arginine to alanine substitution at position 143; and R146A, arginine to alanine substitution at position 146.

FIGURE 4
FIGURE 4

Rps3 methylation is dependent on a SPOUT methyltransferase. Ribosomal proteins of the small subunit were isolated from wild-type and single-gene deletion strains and analyzed by liquid chromatography-mass spectrometry as described in the “Experimental Procedures” section. (A) Reconstructed spectra of Rps3 in BY4742 wild-type yeast and BY4742 yeast strains lacking Rmt1, Rmt2, and Hsl7 (labeled as BY4742 Δrmt1, BY4742 Δrmt2, BY4742 Δhsl7, respectively) are shown. (B) Reconstructed spectra of Rps3 in BY4741 wild-type yeast and a BY4741 strain lacking Yor021c (BY4741 Δyor021c) are shown. Rps3 in BY4741 Δyor021c has a mass that is approximately 14 Da lower than the wild-type strain, indicating a loss of methylation.

FIGURE 5
FIGURE 5

In vitro methyltransferase activity of recombinant Yor021c protein. (A) Yor021c was expressed as a His-tagged enzyme in E. coli as described in the “Experimental Procedures” section. This protein (40 μg) was incubated with ribosomes prepared from a yeast yor021c strain in a BY4742 background (200 μg protein) with 1 μM S-adenosyl-L-[methyl-3H]-L-methionine (75–85 Ci/mmol, from the stock described in the “Experimental Procedures” section) for 20 h at 30 °C in a buffer of 100 mM sodium phosphate, 100 mM NaCl, pH 7.0 in a final volume of 300 μl. After protein precipitation and acid hydrolysis as described in “Experimental Procedures,” 1 μmol of asymmetric NG,NG-dimethylarginine and 1 μmol of NG-monomethylarginine were added as standards and high-resolution cation-exchange chromatography was performed as described in “Experimental Procedures” but with a 8-cm column. Radioactivity (closed circles, solid lines) was measured in 500 μl of each column fraction with 5 ml of Safety Solve fluor. Amino acid standards were detected by mixing 50 μl of each fraction with 100 μl ninhydrin reagent as described in the “Experimental Procedures” section (path length = 0.4 cm) (open circles, dashed lines). Control reactions (B–E) were prepared and analyzed in a similar manner. (B) Wild-type ribosomes (strain BY4742) were substituted for the Δyor021c ribosomes. (C) Recombinant Yor021c was absent from the reaction mixture. (D) Wild-type ribosomes (strain BY4742) were substituted for the Δyor021c ribosomes and Yor021c was absent from the reaction mixture. (E) No ribosomes were included in the reaction mixture with the Yor021c enzyme. In panel (A), a peak of radioactivity elutes just prior to the standard of ω-monomethylarginine in the position expected for the tritium-labeled species (79). This peak is absent in all of the controls (panels B–E).

FIGURE 6
FIGURE 6

Rps27a is monomethylated at cysteine-39. (A) Ribosomal proteins of the small subunit were isolated from BY4742 wild-type yeast and analyzed by liquid chromatography-mass spectrometry, as described in the “Experimental Procedures” section. The reconstructed spectrum of Rps27a from wild-type yeast is shown. (B–E) HPLC-purified Rps27a from BY4742 wild-type yeast was fragmented and analyzed by top-down mass spectrometry. The resulting spectra were deconvoluted and the monoisotopic masses were searched with a 5.5-ppm error threshold against Rps27a with a theoretical N-terminal methyl group (B), Rps27b with two theoretical N-terminal methyl groups (C), Rps27a with a theoretical C-terminal methyl group (D), and Rps27b with two theoretical C-terminal methyl groups (E). Thick lines indicate matched fragments from electron capture dissociation fragmentation while thin lines mark matched fragments from collisionally activated dissociation fragmentation. Informative fragments indicating methylation at cysteine-39 are shown with gray lines. In (B), the c39 and b39 ions indicate methylation between residues 1–39. In (D–E), the c38 ion supports an absence of methylation from residues 1–38, suggesting that cysteine-39 is the site of methylation. The sole residue where Rps27a and Rps27b differ is encircled in gray, and cysteine-39, the putative methylation site, is boxed in gray.

FIGURE 7
FIGURE 7

Cysteine-39 in Rps27a is likely in a disrupted four-cysteine zinc cluster. (A) The crystal structure of the small ribosomal subunit of S. cerevisiae (PDB IDs: 3U5B, 3U5C, 3U5F, 3U5G) (40) is explored to better understand the role of cysteine-39 methylation in Rps27a from S. cerevisiae. The structures of Rps27a, other ribosomal proteins, and the 18S rRNA are shown in yellow, green, and purple, respectively. Cysteine-39 in Rps27a is marked in orange, while the cysteine residues at positions 36, 55, and 58 are colored pink. The latter cysteine residues form a roughly planar trigonal three-cysteine zinc cluster (zinc is shown as a gray sphere), while cysteine-39 appears to be displaced from the cluster (see text). (B) The structure of the N-terminal domain of Ada, an E. coli protein that repairs phosphotriester damage in DNA by transferring methyl groups to a cysteine residue (PDB ID: 1WPK) is shown in yellow. Cysteine-38, which is methylated, is shown in orange while the other cysteine residues forming the four-cysteine zinc cluster are colored pink. Zinc is represented by a gray sphere.

FIGURE 8
FIGURE 8

Rps2 is mono and dimethylated at arginine-10. HPLC-purified Rps2 isolated from BY4742 wild-type yeast cells was digested with chymotrypsin and analyzed by bottom-up mass spectrometry as detailed in the “Experimental Procedures” section. (A) Averaged full-scan spectra of Rps2, indicating the presence of unmethylated, monomethylated, and dimethylated peptides spanning residues 1–13 of Rps2. (B–D) Fragmentation spectra of the unmethylated (B), monomethylated (C), and dimethylated (D) N-terminal peptides of Rps2. Prominent fragments with <500-ppm mass errors are indicated.

Similar articles

Cited by

References

    1. Li E, Beard C, Jaenisch R. Role for DNA methylation in genomic imprinting. Nature. 1993;366:362–365. - PubMed
    1. Bonnerot C, Pintard L, Lutfalla G. Functional redundancy of Spb1p and a snR52-dependent mechanism for the 2′-O-ribose methylation of a conserved rRNA position in yeast. Mol Cell. 2003;12:1309–1315. - PubMed
    1. Walsh CT. Posttranslational Modification of Proteins: Expanding Nature’s Inventory. Roberts and Company Publishers; 2006.
    1. Ortega-Gutierrez S, Leung D, Ficarro S, Peters EC, Cravatt BF. Targeted disruption of the PME-1 gene causes loss of demethylated PP2A and perinatal lethality in mice. PLoS One. 2008;3:e2486. - PMC - PubMed
    1. Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell. 2006;127:635–648. - PubMed

Publication types

MeSH terms

Substances