Mitochondrial and cytoplasmic ribosomes. Distinguishing characteristics and a requirement for the homologous ribosomal salt-extractable fraction for protein synthesis.
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ABSTRACT:
SUBMITTER: Avadhani NG
PROVIDER: S-EPMC1167972 | biostudies-other | 1974 Apr
REPOSITORIES: biostudies-other
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Project description:Most ribosomal proteins play important roles in ribosome biogenesis and function. Here, we have examined the contribution of the essential ribosomal protein L40 in these processes in the yeast Saccharomyces cerevisiae. Deletion of either the RPL40A or RPL40B gene and in vivo depletion of L40 impair 60 S ribosomal subunit biogenesis. Polysome profile analyses reveal the accumulation of half-mers and a moderate reduction in free 60 S ribosomal subunits. Pulse-chase, Northern blotting, and primer extension analyses in the L40-depleted strain clearly indicate that L40 is not strictly required for the precursor rRNA (pre-rRNA) processing reactions but contributes to optimal 27 SB pre-rRNA maturation. Moreover, depletion of L40 hinders the nucleo-cytoplasmic export of pre-60 S ribosomal particles. Importantly, all these defects most likely appear as the direct consequence of impaired Nmd3 and Rlp24 release from cytoplasmic pre-60 S ribosomal subunits and their inefficient recycling back into the nucle(ol)us. In agreement, we show that hemagglutinin epitope-tagged L40A assembles in the cytoplasm into almost mature pre-60 S ribosomal particles. Finally, we have identified that the hemagglutinin epitope-tagged L40A confers resistance to sordarin, a translation inhibitor that impairs the function of eukaryotic elongation factor 2, whereas the rpl40a and rpl40b null mutants are hypersensitive to this antibiotic. We conclude that L40 is assembled at a very late stage into pre-60 S ribosomal subunits and that its incorporation into 60 S ribosomal subunits is a prerequisite for subunit joining and may ensure proper functioning of the translocation process.
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Project description:The aim of this study was to determine the antioxidant activity of the extractable and non-extractable phenolics of Terminalia. Sericea Burch. Ex DC. Free, ester bound, ether or glycoside bound and insoluble phenolics were extracted from the fruit, leaves, stem, and root samples. Follin Ciocalteu was used to estimate the phenolic content while DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was used to determine the antioxidant activity. The data obtained were subjected to multivariate analysis for relationships. The result indicated that the highest average total phenolic contents and antioxidant activities were found in the free (14.8 mgGAE/g; IC50 6.8 ?g/mL) and ester bound (15.1 mgGAE/g; IC50 6.4 ?g/mL) extractable phenolics. There was a strong negative correlation between TPC and DPPH (r = -0.828). Agglomerative hierarchical clustering revealed three clusters. Cluster one contained the insoluble and glycoside phenolics while cluster 2 contained only free phenolic acid of the root. The third cluster was predominantly free and ester bound phenolic extracts. The principal component analysis score plot indicated two major clusters with factor 1 (F1) explaining 61% of the variation. The nuclear magnetic resonance spectroscopy spectra indicated that gallic acid and resveratrol are the major phenolic compounds present in the root. This study has demonstrated that extractable phenolics contributed more to the antioxidant activities compared to the non-extractables.
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Project description:Solubilization of phosphatidylinositol (PtdIns) synthase (CDP-diacylglycerol: myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) from rat pituitary (GH3) tumours was investigated. PtdIns synthase activity was partially extracted from crude membranes by 3 M-KCl. Prior separation of membranes revealed that a greater proportion of plasma-membrane PtdIns synthase activity was salt-extractable than was endoplasmic reticulum activity. The activity of the salt-extracted enzyme was maximized by low concentrations of 3-(3-cholamidopropyl) dimethylammonio-1-propanesulphonate (CHAPS; 0.5 mM), Triton X-100 (0.1 mM) or a phospholipid mixture (0.05 mg/ml), but higher concentrations of detergents were inhibitory. The activity of salt-extracted PtdIns synthase was 0.25 +/- 0.08 nmol/min per mg of protein. Salt-extracted PtdIns synthase activity was dependent on Mg2+ (maximal at 0.1 mM) and Mn2+ (maximal at 5 mM), and its pH optimum was in the range 7.0-7.5. The apparent Km for myo-inositol (in the presence of 0.1 mM-CDP-diacylglycerol) was 0.06 mM, and that for CDP-diacylglycerol (at 0.1 mM-myo-inositol) was 0.21 mM. Salt-extracted PtdIns synthase activity was potently inhibited by Ca2+ (50% inhibition at 1 microM), with over 90% inhibition at 10 microM-Ca2+. These data imply the existence of two forms of membrane-associated PtdIns synthase, namely salt-extractable and salt-resistant, with different intracellular localizations. The salt-extractable form of this enzyme may be a useful preparation for further characterization and purification of mammalian PtdIns synthase.
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Project description:Structural studies have revealed that the core of the ribosome structure is conserved among ribosomes of all kingdoms. Kingdom-specific ribosomal proteins (r-proteins) are located in peripheral parts of the ribosome. In this work, the interactions between rRNA and r-proteins of eukaryote Saccharomyces cerevisiae ribosome were investigated applying LiCl induced splitting and quantitative mass spectrometry. R-proteins were divided into four groups according to their binding properties to the rRNA. Most yeast r-proteins are removed from rRNA by 0.5-1 M LiCl. Eukaryote-specific r-proteins are among the first to dissociate. The majority of the strong binders are known to be required for the early ribosome assembly events. As compared to the bacterial ribosome, yeast r-proteins are dissociated from rRNA at lower ionic strength. Our results demonstrate that the nature of protein-RNA interactions in the ribosome is not conserved between different kingdoms.
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