Effects of deprival of glucose or individual amino acids on polyribosome distribution and rate of protein synthesis in cultured mammalian cells

Venrooij, W.J.W. van; Henshaw, Edgar C.; Hirsch, Carl A.

doi:10.1016/0005-2787(72)90480-7

Cited by 128 publications

(30 citation statements)

References 30 publications

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“…The results reported in this paper are reminiscent of the effects of a number of other conditions which regulate cell growth, such as serum or growth factor deprivation [23,[29][30][31], amino acid starvation [27,28,32,33] and growth of cells to confluence [34]. In all Vol.…”

Section: Regulation Of Protein Synthesis By Interferonsmentioning

confidence: 86%

Inhibition of cell proliferation by interferons. Relative contributions of changes in protein synthesis and breakdown to growth control of human lymphoblastoid cells

McNurlan¹,

Clemens²

1986

Biochemical Journal

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Treatment of the Daudi line of human lymphoblastoid cells with concentrations of human interferons within the physiological range progressively inhibits cell proliferation over 1-4 days. Rigorous measurement of the overall rate of protein synthesis during this period, using a concentration of [3H]phenylalanine sufficient to equalize the specific radioactivity of intracellular and extracellular precursor pools, shows that protein synthesis becomes progressively inhibited as the growth inhibition develops. There is a strong correlation between inhibition of amino acid incorporation and inhibition of cell proliferation. In contrast, we find no evidence for any increase in protein degradation rate under these conditions. These results suggest that interferon treatment of susceptible cells can inhibit protein synthesis even in the absence of virus infection and that this inhibition is of a sufficient magnitude to account for the anti-proliferative effect. INTRODUCTIONThe ability of interferons to inhibit viral replication in infected cells through effects on [4][5][6]. On the other hand,-inhibition of incorporation of labelled amino acids has been observed in some systems in which cell proliferation is inhibited [7][8][9]. The human lymphoblastoid Daudi cell line is extremely sensitive in exhibiting a marked suppression of growth when human interferons are added in culture [10][11][12]. We have investigated, using this system, the extent to which the changes in growth rate can be correlated with changes in protein synthesis. In order to do this it is necessary to quantify both processes rigorously. Such an approach also allows indirect estimates to be made of the rate of protein breakdown, and of the relative contributions of changes in synthesis versus breakdown to the inhibition of cell growth caused by interferon treatment.The rate of incorporation of a radioactive amino acid into protein reflects both the actual rate at which protein is synthesized and the specific radioactivity of the precursor which is being incorporated. If the values of the latter are different between two populations of cells then spurious conclusions can be drawn. In an attempt to overcome any such problems we have adapted procedures developed for measurement of protein synthesis rates in perfused organs [13] and in tissues of

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Section: Regulation Of Protein Synthesis By Interferonsmentioning

confidence: 86%

Inhibition of cell proliferation by interferons. Relative contributions of changes in protein synthesis and breakdown to growth control of human lymphoblastoid cells

McNurlan¹,

Clemens²

1986

Biochemical Journal

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“…One plausible mechanism to be considered for a defect in initiation and for defective polysome formation in mutant cells is that shown to occur in the cytosol of mammalian cells after amino acid starvation (44). Amino acid starvation produced by omission of an essential amino acid from the medium blocks initiation of protein synthesis by causing an increase in phosphorylated eIF-2 (45).…”

Section: Mitochondrial Trna Leu(uur) Mutationmentioning

confidence: 99%

The Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like Episode Syndrome-associated Human Mitochondrial tRNALeu(UUR) Mutation Causes Aminoacylation Deficiency and Concomitant Reduced Association of mRNA with Ribosomes

Chomyn

Enrı́quez²,

Micol³

et al. 2000

Journal of Biological Chemistry

187

108

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The pathogenetic mechanism of the mitochondrial tRNA Leu(UUR) A3243G transition associated with the mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome has been investigated in transmitochondrial cell lines constructed by transfer of mutant mitochondrial DNA (mtDNA)-carrying mitochondria from three genetically unrelated MELAS patients or of isogenic wild-type mtDNA-carrying organelles into human mtDNA-less cells. An in vivo footprinting analysis of the mtDNA segment within the tRNA Leu(UUR) gene that binds the transcription termination factor failed to reveal any difference in occupancy of sites or qualitative interaction with the protein between mutant and wild-type mtDNAs. Cell lines nearly homoplasmic for the mutation exhibited a strong (70 -75%) reduction in the level of aminoacylated tRNA Leu(UUR) and a decrease in mitochondrial protein synthesis rate. The latter, however, did not show any significant correlation between synthesis defect of the individual polypeptides and number or proportion of UUR codons in their mRNAs, suggesting that another step, other than elongation, may be affected. Sedimentation analysis in sucrose gradient showed a reduction in size of the mitochondrial polysomes, while the distribution of the two rRNA components and of the mRNAs revealed decreased association of mRNA with ribosomes and, in the most affected cell line, pronounced degradation of the mRNA associated with slowly sedimenting structures. Therefore, several lines of evidence indicate that the protein synthesis defect in A3243G MELAS mutation-carrying cells is mainly due to a reduced association of mRNA with ribosomes, possibly as a consequence of the tRNA Leu(UUR) aminoacylation defect.

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“…Such a rate-limiting step has been observed in such diverse tissues as cells in mitosis (1), muscles of diabetic animals (2), livers of fasted animals (3,4), as well as in cell cultures exposed to aminoacid-deficient medium (5,6), temperature changes (7), and alterations in sodium-potassium ratios (8). Other studies in both prokaryotes and eukaryotes indicate that the sequence of events leading to the formation of an 80S initiation complex is mediated by the stepwise participation of specific ribosome factors (9)(10)(11)(12)(13)(14).…”

mentioning

confidence: 99%

Protein Initiation in Eukaryotes: Formation and Function of a Ternary Complex Composed of a Partially Purified Ribosomal Factor, Methionyl Transfer RNA _f , and Guanosine Triphosphate

Levin¹,

Kyner²,

Acs³

1973

Proc. Natl. Acad. Sci. U.S.A.

122

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A protein factor contained in a 1 M KCI extract of L-cell ribosomes and partially purified by chromatography on DEAE-cellulose forms a specific ternary complex with rat-liver Met-tRNAf and GTP. The complex is measured by its quantitative retention on nitrocellulose membranes. Complex assembly is optimal at 100 mM KC1 and 0.2 mM MgCl2, and is independent of mRNA and of ribosomes. The GTP requirement can be replaced over 65% by its methylene analogue GDPCH2P, indicating that GTP hydrolysis is not involved. Complex formation is inhibited by 10 MM aurintricarboxylic acid, but is unaffected by 100 MM pactamycin, 100 MM fusidic acid, or by excess uncharged methionine tRNAf. The ternary complex is relatively stable and appears at the void volume during filtration on Sephadex G-100. At 1-3 mM MgCl2 and in the presence of other factors, the ternary complex is implicated in protein initiation by (i) its capacity to bind to the 40S ribosomal subunit to form a 48S complex; and (ii) the subsequent association of the 48S complex with a 60S subunit to form a functional "80S complex."There is considerable evidence to suggest that in eukaryotes, the rate-determining step in protein synthesis occurs during the initiation phase of translation. Such a rate-limiting step has been observed in such diverse tissues as cells in mitosis (1), muscles of diabetic animals (2), livers of fasted animals (3, 4), as well as in cell cultures exposed to aminoacid-deficient medium (5, 6), temperature changes (7), and alterations in sodium-potassium ratios (8). Other studies in both prokaryotes and eukaryotes indicate that the sequence of events leading to the formation of an 80S initiation complex is mediated by the stepwise participation of specific ribosome factors (9)(10)(11)(12)(13)(14). Consequently, the first steps in the initiation sequence are of particular importance if it is assumed that they represent logical sites for the operation of regulatory controls.Recent studies inI prokaryotic systems have rel)orted that one of the earliest steps in the initiation sequence is the formation of a ternary complex containing the ribosome initiation factor and GTP (15,16).In this report, we describe the in vitro formation of a eukaryotic ternary complex with similar properties, composed of a partially purified ribosome factor from L-929 mouse fibroblasts (L-cells), rat-liver Met-tRNAf, and GTP. Preliminary data were presented in a previous communication (17

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Effects of deprival of glucose or individual amino acids on polyribosome distribution and rate of protein synthesis in cultured mammalian cells

Cited by 128 publications

References 30 publications

Inhibition of cell proliferation by interferons. Relative contributions of changes in protein synthesis and breakdown to growth control of human lymphoblastoid cells

Inhibition of cell proliferation by interferons. Relative contributions of changes in protein synthesis and breakdown to growth control of human lymphoblastoid cells

The Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like Episode Syndrome-associated Human Mitochondrial tRNALeu(UUR) Mutation Causes Aminoacylation Deficiency and Concomitant Reduced Association of mRNA with Ribosomes

Protein Initiation in Eukaryotes: Formation and Function of a Ternary Complex Composed of a Partially Purified Ribosomal Factor, Methionyl Transfer RNA _f , and Guanosine Triphosphate

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Effects of deprival of glucose or individual amino acids on polyribosome distribution and rate of protein synthesis in cultured mammalian cells

Cited by 128 publications

References 30 publications

Inhibition of cell proliferation by interferons. Relative contributions of changes in protein synthesis and breakdown to growth control of human lymphoblastoid cells

Inhibition of cell proliferation by interferons. Relative contributions of changes in protein synthesis and breakdown to growth control of human lymphoblastoid cells

The Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like Episode Syndrome-associated Human Mitochondrial tRNALeu(UUR) Mutation Causes Aminoacylation Deficiency and Concomitant Reduced Association of mRNA with Ribosomes

Protein Initiation in Eukaryotes: Formation and Function of a Ternary Complex Composed of a Partially Purified Ribosomal Factor, Methionyl Transfer RNA f , and Guanosine Triphosphate

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Protein Initiation in Eukaryotes: Formation and Function of a Ternary Complex Composed of a Partially Purified Ribosomal Factor, Methionyl Transfer RNA _f , and Guanosine Triphosphate