Proteomic Characterization of Evolutionarily Conserved and Variable Proteins of Arabidopsis Cytosolic Ribosomes  

Abstract

Analysis of 80S ribosomes of Arabidopsis (Arabidopsis thaliana) by use of high-speed centrifugation, sucrose gradient fractionation, one-and two-dimensional gel electrophoresis, liquid chromatography purification, and mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization) identified 74 ribosomal proteins (r-proteins), of which 73 are orthologs of rat r-proteins and one is the plant-specific r-protein P3. Thirty small (40S) subunit and 44 large (60S) subunit r-proteins were confirmed. In addition, an ortholog of the mammalian receptor for activated protein kinase C, a tryptophan-aspartic acid-domain repeat protein, was found to be associated with the 40S subunit and polysomes. Based on the prediction that each r-protein is present in a single copy, the mass of the Arabidopsis 80S ribosome was estimated as 3.2 MD (1,159 kD 40S; 2,010 kD 60S), with the 4 single-copy rRNAs (18S, 26S, 5.8S, and 5S) contributing 53% of the mass. Despite strong evolutionary conservation in r-protein composition among eukaryotes, Arabidopsis 80S ribosomes are variable in composition due to distinctions in mass or charge of approximately 25% of the r-proteins. This is a consequence of amino acid sequence divergence within r-protein gene families and posttranslational modification of individual r-proteins (e.g. amino-terminal acetylation, phosphorylation). For example, distinct types of r-proteins S15a and P2 accumulate in ribosomes due to evolutionarily divergence of r-protein genes. Ribosome variation is also due to amino acid sequence divergence and differential phosphorylation of the carboxy terminus of r-protein S6. The role of ribosome heterogeneity in differential mRNA translation is discussed.The ribosome is a two-subunit ribonucleoprotein complex that catalyzes the peptidyl transferase reaction of polypeptide synthesis, an absolute requirement for cellular growth and differentiation. The structure and function of both prokaryotic and eukaryotic ribosomes have been investigated, with the eukaryotic emphasis on ribosomes of Baker's yeast (Saccharomyces cerevisiae) and rat (Rattus rattus and Rattus norvegicus). The cytosolic ribosomes of eukaryotes are composed of a large number of ribosomal proteins (r-proteins) and four distinct rRNAs, the 18S rRNA of the 40S subunit, and the 5S, 5.8S, and 23S-like (25-28S) rRNAs of the 60S subunit (Bielka, 1982). Early evaluation of the buoyant density and sedimentation coefficients of eukaryotic ribosomes predicted that the rat 80S ribosome has a higher mass (4.2-4.6 MD) than that of pea (Pisum sativum; 3.9 MD) due to distinctions in the 60S subunit (Cammarano et al., 1972). Further evidence that the plant ribosome is smaller than that of mammals was provided by the three-dimensional reconstruction of wheat (Triticum aestivum) and rabbit (Oryctolagus cuniculus) ribosomes by use of cryoelectron microscopy (Verschoor et al., 1996). Despite an overall similarity in architecture, the 60S subunit of wheat appeared approximately 20% smaller than that...

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).…”

New Evidence for Differential Roles of L10 Ribosomal Proteins from Arabidopsis

“…2). Proteomic studies of the 80S cytosolic ribosome using different Arabidopsis tissues by 2D electrophoresis followed by spectrometric mass showed that both RPL10A and RPL10C were components of the 60S subunit, while RPL10B was not identified (Chang et al, 2005;Carroll et al, 2008;Turkina et al, 2011). However, our complementation studies in yeast suggest that RPL10B, as well as RPL10A and RPL10C, may fulfill the function of yeast RPL10 and, consequently, are all probably involved in translation.…”

Determination of in vivo RNA structure in low-abundance transcripts

“…3b) indicating that the structure of the RNA in vivo is very different from in vitro. Much of the weak correlation, in this instance, is likely due to the presence of intermolecular RNA-RNA interactions in the cellular environment, as part of 5.8S rRNA is known to base pair with 25S rRNA 24 (see the phylogenetic structure in Fig. 3c).…”

“…(Yeast ribosomal proteins are evolutionarily conserved with A. thaliana and other eukaryotes 24 ). We identified several ribosomal proteins near H18-H20 (Fig.…”