Dissociation of N<sub>2</sub> Gas-induced Monomeric Ribosomes and Functioning of the Derived Subunits in Protein Synthesis in Pea

Lin, C. Y.; Key, Joe L.

doi:10.1104/pp.48.5.547

Cited by 17 publications

(8 citation statements)

References 26 publications

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“…Similarly the putative pentamethylated, doubly charged b3 ion (observed at m/z 197.15 in the wild-type spectrum) is apparently missing in the ⌬ydr198c spectrum, whereas the relative intensity of a putative dimethylated, singly charged b3 ion is greatly increased at m/z 351. 23. In addition to the 42.06-Da mass shift, the shift in charge state from z ϭ 2 to z ϭ 1, observed for the b3 signal, is consistent with the loss of constitutive positive charge associated with the quaternary ammonium group of the trimethyllysine residue.…”

Section: Conservation Of Covalent Modifications Between Eukaryoticsupporting

confidence: 51%

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

Carroll¹,

Heazlewood²,

Ito³

et al. 2008

Molecular & Cellular Proteomics

184

250

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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 ...

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Section: Conservation Of Covalent Modifications Between Eukaryoticsupporting

confidence: 51%

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

Carroll¹,

Heazlewood²,

Ito³

et al. 2008

Molecular & Cellular Proteomics

184

250

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“…In some experiments (shown in Figs. 2,7,8,and 10) and in particular when derived monomeric ribosomes were being prepared, the pH of the initial extraction medium and the 1 M sucrose was 7.9 and 7.4, respectively.Monomeric ribosomes were prepared from Lemna which was gassed in the dark with nitrogen for 1 or 2 hr (8). Subunits were prepared from these derived monomers by suspending the ribosomal pellet in 0.7 ml of 0.05 M KCI, 0.05 mM MgCI,, and 10 mm tris (pH 7.4) and centrifuging on a 20-ml 10 to 35% sucrose gradient containing the above concentrations of KCI, tris, and MgCI, at 50,000g for 16 hr.…”

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confidence: 99%

The Phosphorylation of Ribosomal Protein in Lemna minor

Trewavas

1973

Plant Physiol.

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Sterile cultures of Lemna minor have been labeled with 32pl, and the ribosomal proteins have been examined for radioactivity. In relatively short term labeling a radioactive protein was found which ran as a single component in both urea/acetic acid and sodium lauryl sulfate gel electrophoresis. The same strain of Lemniiia miinlor was used as previously described (16). Growth conditions were identical except for the use of a 12-hr day instead of continuous light. This reduced growth rates slightly, the average fresh weight rate constant of increase being 0.30 day-' (16). All experiments were carried out under sterile conditions, and nonsterile experiments were discarded. Assays for sterility were carried out as previously described (16).Three media have been used for labeling Lemna with "'Pi: (a) for 5 hr labeling, 50 ml of sucrose-mineral salts but with KH2PO4 reduced to 4 itM and with 'Pi at 50 yc/ml; (b) for 24 hr labeling, 50 ml of sucrose-mineral salts but with KH2P04 reduced to 40 fM and containing "'Pi at 50 lc/ml; (c) for 12 days labeling, 100 ml of sucrose-mineral salts containing 4 ,tc/ml 3'P, (2).As far as can be determined, growth (measured as frond number) continues normally on these media for the duration of the labeling period. METHODSIsolation and Separation of Ribosomes and Subunits. Plants were ground in 5 volumes of ice-cold 14% sucrose; 0.05 M KCI; 0.05 M tris, pH 8.5; 10 mm MgCl,. After filtering through Mira-cloth, the homogenate was spun at 15,000g for 15 min, and the supernatant was collected and made 2% in Triton X-100. Ribosomes were then layered over 2 ml of 35% sucrose containing 10 mM tris, pH 8.5; 10 mm MgCl,; and either 20 mM KCl (for the experiments shown in Figs. 2, 7, 8, and 10) or 0.25 M KCI (for the experiments shown in Figs. 3-6 and 9). The latter concentration of KCl gave cleaner ribosome preparations, containing about 39% protein, while 20 mm KCl ribosomes were about 45% protein. The ribosomes were spun through the 35% sucrose at 125,000g for 2 hr.The pellet was resuspended in 0.7 ml of 0.05 M KCI, 10 mM tris (pH 7.4), and 10 mm MgCI]; layered onto a 20-ml 10 to 35% exponential sucrose gradient containing 0.05 M KCI, 10 mM MgCl, and 10 mm tris (pH 7.4); and spun for 2.5 hr at 80,000g.Fractions of either 15 or 20 drops were collected after the tube was punctured and counted by Cerenkov radiation in a Packard scintillation counter. The desired fractions were collected, and the ribosomes were precipitated with 0.7 volume of ethanol. In some experiments (shown in Figs. 2,7,8,and 10) and in particular when derived monomeric ribosomes were being prepared, the pH of the initial extraction medium and the 1 M sucrose was 7.9 and 7.4, respectively.Monomeric ribosomes were prepared from Lemna which was gassed in the dark with nitrogen for 1 or 2 hr (8). Subunits were prepared from these derived monomers by suspending the ribosomal pellet in 0.7 ml of 0.05 M KCI, 0.05 mM MgCI,, and 10 mm tris (pH 7.4) and centrifuging on a 20-ml 10 to 35% sucrose gradient containing the above co...

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“…Ammonium chloride dissociation of plant monoribosomes produced ribosomal subunits which upon recombination were inactive in in vitro protein synthesis; however, the N40S subunit was active in aminoacyl-tRNA binding (17,18) and was active in poly(U)-directed phenylalanine incorporation in combination with the K60S subunits. The loss in activity of the N60S subunit related directly to the selective loss of a single ribosomal protein, L5.…”

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“…Dissociation of monoribosomes in the absence of Mg2`results in 40S "subunits" practically devoid of protein complement. In a previous report (17), 40S subunits prepared in the absence of Mg`were shown to sediment significantly slower on sucrose gradients. The detachment of the ribosomal proteins is sufficient to account for both the lowered sedimentation rate and the loss in in vitro poly(U)-directed phenylalanine incorporation.…”

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Studies on Pea Ribosomal Proteins

Lin¹,

Chia²,

Travis³

et al. 1975

Plant Physiol.

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Dissociation of N₂ Gas-induced Monomeric Ribosomes and Functioning of the Derived Subunits in Protein Synthesis in Pea

Cited by 17 publications

References 26 publications

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

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

The Phosphorylation of Ribosomal Protein in Lemna minor

Studies on Pea Ribosomal Proteins

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Dissociation of N2 Gas-induced Monomeric Ribosomes and Functioning of the Derived Subunits in Protein Synthesis in Pea

Cited by 17 publications

References 26 publications

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

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

The Phosphorylation of Ribosomal Protein in Lemna minor

Studies on Pea Ribosomal Proteins

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Dissociation of N₂ Gas-induced Monomeric Ribosomes and Functioning of the Derived Subunits in Protein Synthesis in Pea