Analysis of the Arabidopsis Cytosolic Ribosome Proteome Provides Detailed Insights into Its Components and Their Post-translational Modification

Abstract

Finding gene-specific peptides by mass spectrometry analysis to pinpoint gene loci responsible for particular protein products is a major challenge in proteomics especially in highly conserved gene families in higher eukaryotes. We used a combination of in silico approaches coupled to mass spectrometry analysis to advance the proteomics insight into Arabidopsis cytosolic ribosomal composition and its post-translational modifications. In silico digestion of all 409 ribosomal protein sequences in Arabidopsis defined the proportion of theoretical genespecific peptides for each gene family and highlighted the need for low m/z cutoffs of MS ion selection for MS/MS to characterize low molecular weight, highly basic ribosomal proteins. We undertook an extensive MS/MS survey of the cytosolic ribosome using trypsin and, when required, chymotrypsin and pepsin. We then used custom software to extract and filter peptide match information from Mascot result files and implement high confidence criteria for calling gene-specific identifications based on the highest quality unambiguous spectra matching exclusively to certain in silico predicted gene-or gene family-specific peptides. This provided an in-depth analysis of the protein composition based on 1446 high quality MS/MS spectra matching to 795 peptide sequences from ribosomal proteins. These identified peptides from five gene families of ribosomal proteins not identified previously, providing experimental data on 79 of the 80 different types of ribosomal subunits. We provide strong evidence for gene-specific identification of 87 different ribosomal proteins from these 79 families. We also provide new information on 30 specific sites of co-and post-translational modification of ribosomal proteins in Arabidopsis by initiator methionine removal, N-terminal acetylation, N-terminal methylation, lysine N-methylation, and phosphorylation. These sitespecific modification data provide a wealth of resources for further assessment of the role of ribosome modification in influencing translation in Arabidopsis. Molecular & Cellular Proteomics 7:347-369, 2008.Ribosomes are large ribonucleoprotein complexes that catalyze the peptidyltransferase reaction in polypeptide synthesis and are thus responsible for the translation of transcripts encoded in cellular genomes. These complexes play the most fundamental role of any protein complex in the generation of the cellular proteome as a whole. Ribosomes consist of two subunits, large and small, but the internal composition of these subunits and their macromolecular size varies between bacteria, animals, fungi, and plants. Both these subunits are composed on rRNA and protein (r-protein) 1 components. Among eukaryotes the 80 S cytosolic ribosomes of the yeast (Saccharomyces cerevisiae), rat (Rattus norvegicus), and human (Homo sapiens) have been the most extensively investigated. These studies have revealed four distinct rRNAs, the 18 S rRNA of the 40 S subunit and 5, 5.8, and 23 S rRNAs of the 60 S subunit. Up to 79 distinct proteins are part ...

The ribosome: A hot spot for the identification of new types of protein methyltransferases

“…By 2006, our laboratory had found evidence for five added methyl groups on the large ribosomal subunit yeast protein Rpl12ab near the N-terminus; modification at Lys-3 by a SET-domain methyltransferase designated Rkm2 and by a yet unidentified enzyme at Lys-10 (44). However, these results were called into question by reports on the modifications of the orthologs of Rpl12ab in Arabidopsis thaliana (51) and Schizosaccharomyces pombe (52); both reports suggested that our mass spectrometry data was more consistent with the dimethylation of the N-terminal proline residue and the trimethylation of Lys-3 in line with the similar N-terminal modification of the Arabidopsis and S. pombe proteins. Further analysis in our laboratory (45) confirmed the N-terminal modification and the major methylation of Lys-3 as opposed to Lys-10 (53).…”

Plant L10 Ribosomal Proteins Have Different Roles during Development and Translation under Ultraviolet-B Stress

“…We conclude that the RPL10B protein has a specific and essential participation during plant growth, which cannot be replaced by the other RPL10 proteins. Moreover, the presence of the RPL10B isoform has not yet been confirmed in the Arabidopsis 80S ribosome, as no specific tryptic peptide from this protein has been identified by MS/MS in survey experiments (Chang et al, 2005;Giavalisco et al, 2005;Carroll et al, 2008). Alternatively, it cannot be ruled out that RPL10B has extraribosomal functions during development.…”

“…Identification of proteins from purified ribosomes by two-dimensional (2D) electrophoretic analyses followed by mass spectrometry (MS) demonstrated the presence of 79 families of ribosomal proteins in the 80S ribosome. Nearly half are represented by two or more protein spots on 2D gels, indicating that proteins are posttranslationally modified and/or present as different isoforms (Chang et al, 2005;Giavalisco et al, 2005;Carroll et al, 2008). It is hypothesized that this ribosomal heterogeneity fosters selective translation of specific mRNA under particular cell conditions (Barakat et al, 2001;Szick-Miranda and Bailey-Serres, 2001;Giavalisco et al, 2005;Carroll et al, 2008).…”

Cytoskeleton-Associated Large RNP Complexes in Tobacco Male Gametophyte (EPPs) Are Associated with Ribosomes and Are Involved in Protein Synthesis, Processing, and Localization

“…The specific function of individual cytoskeleton-associated and/or RNA-binding proteins in these processes can only be speculated, although a number of ribosomal and cytoskeletal proteins also belonged among the highest-ranked proteins. Presence of all identified proteins together with great extent of vigorous post-translational modifications of plant ribosomal proteins 71 suggests high regulatory dynamics within EPP complexes, especially during the progamic phase. mRNP particles arranged in higher-order complexes or granules were previously identified in many yeast and animal systems.…”