The regulation of protein synthesis in mammalian cells by amino acid supply

In many bacterial systems chloramphenicol has been shown to inhibit protein synthesis (Hahn & Wisseman, 1951; Gale & Folkes, 1953). The precise mechanism of this inhibition is not clear, although the evidence suggests that the interaction of the soluble RNA-amino acid complex with the ribosomes is prevented because the attachment of the messenger RNA to the ribosomes is itself impaired (Lacks & Gros, 1959; Nathans & Lipman, 1961; Jardetsky & Julian, 1964; Julian & Jardetsky, 1964). In contrast to its effect on bacterial systems, chloramphenicol has been reported to have little or no action on the protein synthesis by cell-free extracts of mammalian cells (Rendi, 1959; Ehrenstein & Lipmann, 1961). A basis for this resistance has been proposed by Vazquez (1964), who finds that whereas bacterial ribosomes bind chloramphenicol, ribosomes from other organisms do not. Nevertheless, it cannot be stated with any confidence that chloramphenicol has no effect on the protein synthesis of animal cells.

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Deprivation of a single amino acid induces protein synthesis-dependent increases in c-jun, c-myc, and ornithine decarboxylase mRNAs in Chinese hamster ovary cells

Molecular and Cellular Biology ◽

10.1128/mcb.10.11.5814-5821.1990 ◽

1990 ◽

Vol 10 (11) ◽

pp. 5814-5821

Author(s):

P Pohjanpelto ◽

E Hölttä

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Ornithine Decarboxylase ◽

Mammalian Cells ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Amino Acid Starvation ◽

Cellular Growth ◽

Single Amino Acid ◽

Mrna Levels

Genes of higher eucaryotic cells are considered to show only a limited response to nutritional stress. Here we show, however, that omission of a single essential amino acid from the medium caused a marked rise in the mRNA levels of c-myc, c-jun, junB and c-fos oncogenes and ornithine decarboxylase (ODC) in CHO cells. There was no general accumulation of mRNAs in amino acid-starved cells, since the gamma-actin, beta-tubulin, protein kinase C, RNA polymerase II, and glyceraldehyde-3-phosphate dehydrogenase mRNAs and the total poly(A)+ mRNA were not increased. The levels of c-myc, ODC, and c-jun mRNAs were elevated more by amino acid starvation than by inhibition of protein synthesis with cycloheximide, which is known to increase the levels of these mRNAs. Importantly, however, cycloheximide present during amino acid starvation reduced the rise in the levels of the mRNAs down to the level obtained with cycloheximide alone. This implies that protein synthesis is required for the accumulation of c-myc, ODC, and c-jun mRNAs in amino acid-deprived cells. The junB and c-fos mRNAs, instead, were increased to the same extent or less by amino acid starvation than by cycloheximide treatment. The accumulation of the c-myc mRNA in amino acid-starved cells was due to both stabilization of the mRNA and increase of its transcription. The rise in the c-jun mRNA level seemed to be caused merely by stabilization of the mRNA. Further, despite the inhibition of general protein synthesis, amino acid starvation led to an increase in the synthesis of c-myc polypeptide. The results suggest that mammalian cells have a specific mechanism for registering shortages of amino acids in order to make adjustments compatible with cellular growth.

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Pools and protein synthesis in mammalian cells

Biochemical Journal ◽

10.1042/bj1780699 ◽

1979 ◽

Vol 178 (3) ◽

pp. 699-709 ◽

Cited By ~ 31

Author(s):

J H Robertson ◽

D N Wheatley

Keyword(s):

Amino Acids ◽

Protein Synthesis ◽

Amino Acid ◽

Mammalian Cells ◽

External Medium ◽

Essential Amino Acids ◽

Poor Correlation ◽

Experimental Conditions ◽

Linear Kinetics ◽

Kinetics Of

From the kinetics of incorporation into protein shown by four amino acids and one amino acid analogue in suspension cultured HeLa S-3 cells, two distinctly different patterns were observed under the same experimental conditions. An initial slow exponential incorporation followed by linear kinetics was characteristic of the two non-essential amino acids, glycine and proline, whereas the two essential amino acids studied, phenylalanine and leucine, showed linear kinetics of incorporation with no detectable delay. The analogue amino acid, p-fluorophenylalanine also showed immediate linear kinetics of incorporation. There was a poor correlation between the rate of formation of acid-soluble pools and incorporation kinetics. However, the rate of formation of the freely diffusible pool of amino acids correlated more closely with incorporation kinetics. The lack of direct involvement of the acid-soluble pool in protein synthesis was also demonstrated by pre-loading of pools before treatment of cells with labelled amino acids. The results partially support the hypothesis that precursor amino acids for protein synthesis come from the external medium rather than the acid-soluble pool, but suggest that the amino acid which freely diffuses into the cell from the external medium could also be the source.

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Initiation of Protein Synthesis in Mammalian Cells with Codons Other Than AUG and Amino Acids Other Than Methionine

Molecular and Cellular Biology ◽

10.1128/mcb.18.9.5140 ◽

1998 ◽

Vol 18 (9) ◽

pp. 5140-5147 ◽

Cited By ~ 57

Author(s):

Harold J. Drabkin ◽

Uttam L. RajBhandary

Keyword(s):

Amino Acids ◽

Protein Synthesis ◽

Amino Acid ◽

Mammalian Cells ◽

Initiator Trna ◽

Cell Extracts ◽

Potential Applications ◽

Protein Synthesis Initiation ◽

Amino Acid Methionine ◽

The Many

ABSTRACT Protein synthesis is initiated universally with the amino acid methionine. In Escherichia coli, studies with anticodon sequence mutants of the initiator methionine tRNA have shown that protein synthesis can be initiated with several other amino acids. In eukaryotic systems, however, a yeast initiator tRNA aminoacylated with isoleucine was found to be inactive in initiation in mammalian cell extracts. This finding raised the question of whether methionine is the only amino acid capable of initiation of protein synthesis in eukaryotes. In this work, we studied the activities, in initiation, of four different anticodon sequence mutants of human initiator tRNA in mammalian COS1 cells, using reporter genes carrying mutations in the initiation codon that are complementary to the tRNA anticodons. The mutant tRNAs used are aminoacylated with glutamine, methionine, and valine. Our results show that in the presence of the corresponding mutant initiator tRNAs, AGG and GUC can initiate protein synthesis in COS1 cells with methionine and valine, respectively. CAG initiates protein synthesis with glutamine but extremely poorly, whereas UAG could not be used to initiate protein synthesis with glutamine. We discuss the potential applications of the mutant initiator tRNA-dependent initiation of protein synthesis with codons other than AUG for studying the many interesting aspects of protein synthesis initiation in mammalian cells.

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Alterations of transcription and translation in HeLa cells exposed to amino acid analogs.

Molecular and Cellular Biology ◽

10.1128/mcb.4.6.1063 ◽

1984 ◽

Vol 4 (6) ◽

pp. 1063-1072 ◽

Cited By ~ 27

Author(s):

G P Thomas ◽

M B Mathews

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Stress Response ◽

Mammalian Cells ◽

Stress Proteins ◽

Prolonged Exposure ◽

Stress Protein ◽

Electrophoretic Patterns ◽

Amino Acid Analogs ◽

Polypeptide Chains

Amino acid analogs, like other effectors of the stress response, induce in mammalian cells the same gene products that are induced upon heat shock; incorporation of the analog into protein is required for induction. We show here that induction by analogs involves controls operating at the levels of both transcription and translation. The electrophoretic patterns of newly made mRNAs simplify with time such that the putative stress protein mRNAs are the only species transported from the nucleus. Concomitantly, the patterns of protein synthesis simplify such that the stress proteins become nearly exclusive polypeptide products. Although the normal mRNAs are either not used or used with greatly reduced efficiency, they are not degraded and retain translatability when transferred to cell-free systems. Soon after the stress response has been induced, there follows a defect in the initiation of polypeptide chains, as evidenced by examination of polysome profiles. Upon prolonged exposure, polysomes are recovered, and although they give rise to stress proteins almost exclusively, the normal mRNAs are still present in these structures. Thus, in addition to the initiation defect, a lesion in elongation may also be involved. The extreme sensitivity of protein synthesis to the inhibition of RNA synthesis, together with the parallel simplifications in the patterns of newly made mRNAs and polypeptides, may imply that only newly made mRNAs are efficiently translated in analog-treated cells.

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Starvation-induced proteasome assemblies in the nucleus link amino acid supply to apoptosis

Nature Communications ◽

10.1038/s41467-021-27306-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Maxime Uriarte ◽

Nadine Sen Nkwe ◽

Roch Tremblay ◽

Oumaima Ahmed ◽

Clémence Messmer ◽

...

Keyword(s):

Protein Synthesis ◽

Phase Separation ◽

Amino Acid ◽

Liquid Phase ◽

Mammalian Cells ◽

Proteasome Inhibition ◽

Eukaryotic Cells ◽

Amino Acid Deprivation ◽

Common Response ◽

Liquid Liquid Phase Separation

AbstractEukaryotic cells have evolved highly orchestrated protein catabolic machineries responsible for the timely and selective disposal of proteins and organelles, thereby ensuring amino acid recycling. However, how protein degradation is coordinated with amino acid supply and protein synthesis has remained largely elusive. Here we show that the mammalian proteasome undergoes liquid-liquid phase separation in the nucleus upon amino acid deprivation. We termed these proteasome condensates SIPAN (Starvation-Induced Proteasome Assemblies in the Nucleus) and show that these are a common response of mammalian cells to amino acid deprivation. SIPAN undergo fusion events, rapidly exchange proteasome particles with the surrounding milieu and quickly dissolve following amino acid replenishment. We further show that: (i) SIPAN contain K48-conjugated ubiquitin, (ii) proteasome inhibition accelerates SIPAN formation, (iii) deubiquitinase inhibition prevents SIPAN resolution and (iv) RAD23B proteasome shuttling factor is required for SIPAN formation. Finally, SIPAN formation is associated with decreased cell survival and p53-mediated apoptosis, which might contribute to tissue fitness in diverse pathophysiological conditions.

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Deprivation of a single amino acid induces protein synthesis-dependent increases in c-jun, c-myc, and ornithine decarboxylase mRNAs in Chinese hamster ovary cells.

Molecular and Cellular Biology ◽

10.1128/mcb.10.11.5814 ◽

1990 ◽

Vol 10 (11) ◽

pp. 5814-5821 ◽

Cited By ~ 37

Author(s):

P Pohjanpelto ◽

E Hölttä

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Ornithine Decarboxylase ◽

Mammalian Cells ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Amino Acid Starvation ◽

Cellular Growth ◽

Single Amino Acid ◽

Mrna Levels

Genes of higher eucaryotic cells are considered to show only a limited response to nutritional stress. Here we show, however, that omission of a single essential amino acid from the medium caused a marked rise in the mRNA levels of c-myc, c-jun, junB and c-fos oncogenes and ornithine decarboxylase (ODC) in CHO cells. There was no general accumulation of mRNAs in amino acid-starved cells, since the gamma-actin, beta-tubulin, protein kinase C, RNA polymerase II, and glyceraldehyde-3-phosphate dehydrogenase mRNAs and the total poly(A)+ mRNA were not increased. The levels of c-myc, ODC, and c-jun mRNAs were elevated more by amino acid starvation than by inhibition of protein synthesis with cycloheximide, which is known to increase the levels of these mRNAs. Importantly, however, cycloheximide present during amino acid starvation reduced the rise in the levels of the mRNAs down to the level obtained with cycloheximide alone. This implies that protein synthesis is required for the accumulation of c-myc, ODC, and c-jun mRNAs in amino acid-deprived cells. The junB and c-fos mRNAs, instead, were increased to the same extent or less by amino acid starvation than by cycloheximide treatment. The accumulation of the c-myc mRNA in amino acid-starved cells was due to both stabilization of the mRNA and increase of its transcription. The rise in the c-jun mRNA level seemed to be caused merely by stabilization of the mRNA. Further, despite the inhibition of general protein synthesis, amino acid starvation led to an increase in the synthesis of c-myc polypeptide. The results suggest that mammalian cells have a specific mechanism for registering shortages of amino acids in order to make adjustments compatible with cellular growth.

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Alterations of transcription and translation in HeLa cells exposed to amino acid analogs

Molecular and Cellular Biology ◽

10.1128/mcb.4.6.1063-1072.1984 ◽

1984 ◽

Vol 4 (6) ◽

pp. 1063-1072

Author(s):

G P Thomas ◽

M B Mathews

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Stress Response ◽

Mammalian Cells ◽

Stress Proteins ◽

Prolonged Exposure ◽

Stress Protein ◽

Electrophoretic Patterns ◽

Amino Acid Analogs ◽

Polypeptide Chains

Amino acid analogs, like other effectors of the stress response, induce in mammalian cells the same gene products that are induced upon heat shock; incorporation of the analog into protein is required for induction. We show here that induction by analogs involves controls operating at the levels of both transcription and translation. The electrophoretic patterns of newly made mRNAs simplify with time such that the putative stress protein mRNAs are the only species transported from the nucleus. Concomitantly, the patterns of protein synthesis simplify such that the stress proteins become nearly exclusive polypeptide products. Although the normal mRNAs are either not used or used with greatly reduced efficiency, they are not degraded and retain translatability when transferred to cell-free systems. Soon after the stress response has been induced, there follows a defect in the initiation of polypeptide chains, as evidenced by examination of polysome profiles. Upon prolonged exposure, polysomes are recovered, and although they give rise to stress proteins almost exclusively, the normal mRNAs are still present in these structures. Thus, in addition to the initiation defect, a lesion in elongation may also be involved. The extreme sensitivity of protein synthesis to the inhibition of RNA synthesis, together with the parallel simplifications in the patterns of newly made mRNAs and polypeptides, may imply that only newly made mRNAs are efficiently translated in analog-treated cells.

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