Protein synthesis in rabbit reticulocytes: Mechanism of protein synthesis inhibition by heme-regulated inhibitor

A. Das, R. O. Ralston, M. Grace, R. Roy, P. Ghosh-Dastidar, H. K. Das, B. Yaghmai, S. Palmieri, N. K. Gupta

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Abstract

Partially purified Met-tRNA(f) binding factor, eIF-2, was phosphorylated by using heme-regulated inhibitor (HRI). Phosphorylated eIF-2 was freed from HRI by phosphocellulose column chromatography Analysis by isoelectric focusing showed 100% phosphorylation of the 38,000-dalton subunit of eIF-2. Both eIF-2 and eIF-2(P) formed ternary complexes with Met-tRNA(f) and GTP with almost the same efficiency, and in both cases the ternary complex formation was drastically inhibited by prior addition of Mg 2+. However, whereas the ternary complexes formed with eIF-2 could be stimulated by Co-eIF-2C at 1 mM Mg 2+ and dissociated by Co-eIF-2B at 5 mM Mg 2+, the ternary complexes formed with eIF-2(P) were unresponsive to both Co-eIF-2B and Co-eIF-2C. Also, under conditions of eIF-2 phosphorylation, HRI drastically inhibited AUG-dependent Met-tRNA(f) binding to 40S ribosomes. However, HRI (in the presence of ATP) had no effect on the joining of preformed Met-tRNA(f) x 40S x AUG complex to the 60S ribosomal subunit to form Met-tRNA(f) x 80S x AUG complex. These studies suggest that HRI inhibits protein synthesis initiation by phosphorylation of the 38,000-dalton subunit of eIF-2. HRI-phosphorylated eIF-2 does not interact with at least two other protein factors, Co-eIF-2B and Co-eIF-2C, and is thus inactive in protein synthesis initiation.

Original languageEnglish
Pages (from-to)5076-5079
Number of pages4
JournalProceedings of the National Academy of Sciences of the United States of America
Volume76
Issue number10
DOIs
StatePublished - 1 Dec 1979

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Reticulocytes
Heme
Rabbits
Proteins
Phosphorylation
Eukaryotic Large Ribosome Subunits
Protein Synthesis Inhibitors
Isoelectric Focusing
Guanosine Triphosphate
Ribosomes
Chromatography
Adenosine Triphosphate
formylmethionine-tRNA

Cite this

Das, A. ; Ralston, R. O. ; Grace, M. ; Roy, R. ; Ghosh-Dastidar, P. ; Das, H. K. ; Yaghmai, B. ; Palmieri, S. ; Gupta, N. K. / Protein synthesis in rabbit reticulocytes : Mechanism of protein synthesis inhibition by heme-regulated inhibitor. In: Proceedings of the National Academy of Sciences of the United States of America. 1979 ; Vol. 76, No. 10. pp. 5076-5079.
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title = "Protein synthesis in rabbit reticulocytes: Mechanism of protein synthesis inhibition by heme-regulated inhibitor",
abstract = "Partially purified Met-tRNA(f) binding factor, eIF-2, was phosphorylated by using heme-regulated inhibitor (HRI). Phosphorylated eIF-2 was freed from HRI by phosphocellulose column chromatography Analysis by isoelectric focusing showed 100{\%} phosphorylation of the 38,000-dalton subunit of eIF-2. Both eIF-2 and eIF-2(P) formed ternary complexes with Met-tRNA(f) and GTP with almost the same efficiency, and in both cases the ternary complex formation was drastically inhibited by prior addition of Mg 2+. However, whereas the ternary complexes formed with eIF-2 could be stimulated by Co-eIF-2C at 1 mM Mg 2+ and dissociated by Co-eIF-2B at 5 mM Mg 2+, the ternary complexes formed with eIF-2(P) were unresponsive to both Co-eIF-2B and Co-eIF-2C. Also, under conditions of eIF-2 phosphorylation, HRI drastically inhibited AUG-dependent Met-tRNA(f) binding to 40S ribosomes. However, HRI (in the presence of ATP) had no effect on the joining of preformed Met-tRNA(f) x 40S x AUG complex to the 60S ribosomal subunit to form Met-tRNA(f) x 80S x AUG complex. These studies suggest that HRI inhibits protein synthesis initiation by phosphorylation of the 38,000-dalton subunit of eIF-2. HRI-phosphorylated eIF-2 does not interact with at least two other protein factors, Co-eIF-2B and Co-eIF-2C, and is thus inactive in protein synthesis initiation.",
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Protein synthesis in rabbit reticulocytes : Mechanism of protein synthesis inhibition by heme-regulated inhibitor. / Das, A.; Ralston, R. O.; Grace, M.; Roy, R.; Ghosh-Dastidar, P.; Das, H. K.; Yaghmai, B.; Palmieri, S.; Gupta, N. K.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 76, No. 10, 01.12.1979, p. 5076-5079.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Protein synthesis in rabbit reticulocytes

T2 - Mechanism of protein synthesis inhibition by heme-regulated inhibitor

AU - Das, A.

AU - Ralston, R. O.

AU - Grace, M.

AU - Roy, R.

AU - Ghosh-Dastidar, P.

AU - Das, H. K.

AU - Yaghmai, B.

AU - Palmieri, S.

AU - Gupta, N. K.

PY - 1979/12/1

Y1 - 1979/12/1

N2 - Partially purified Met-tRNA(f) binding factor, eIF-2, was phosphorylated by using heme-regulated inhibitor (HRI). Phosphorylated eIF-2 was freed from HRI by phosphocellulose column chromatography Analysis by isoelectric focusing showed 100% phosphorylation of the 38,000-dalton subunit of eIF-2. Both eIF-2 and eIF-2(P) formed ternary complexes with Met-tRNA(f) and GTP with almost the same efficiency, and in both cases the ternary complex formation was drastically inhibited by prior addition of Mg 2+. However, whereas the ternary complexes formed with eIF-2 could be stimulated by Co-eIF-2C at 1 mM Mg 2+ and dissociated by Co-eIF-2B at 5 mM Mg 2+, the ternary complexes formed with eIF-2(P) were unresponsive to both Co-eIF-2B and Co-eIF-2C. Also, under conditions of eIF-2 phosphorylation, HRI drastically inhibited AUG-dependent Met-tRNA(f) binding to 40S ribosomes. However, HRI (in the presence of ATP) had no effect on the joining of preformed Met-tRNA(f) x 40S x AUG complex to the 60S ribosomal subunit to form Met-tRNA(f) x 80S x AUG complex. These studies suggest that HRI inhibits protein synthesis initiation by phosphorylation of the 38,000-dalton subunit of eIF-2. HRI-phosphorylated eIF-2 does not interact with at least two other protein factors, Co-eIF-2B and Co-eIF-2C, and is thus inactive in protein synthesis initiation.

AB - Partially purified Met-tRNA(f) binding factor, eIF-2, was phosphorylated by using heme-regulated inhibitor (HRI). Phosphorylated eIF-2 was freed from HRI by phosphocellulose column chromatography Analysis by isoelectric focusing showed 100% phosphorylation of the 38,000-dalton subunit of eIF-2. Both eIF-2 and eIF-2(P) formed ternary complexes with Met-tRNA(f) and GTP with almost the same efficiency, and in both cases the ternary complex formation was drastically inhibited by prior addition of Mg 2+. However, whereas the ternary complexes formed with eIF-2 could be stimulated by Co-eIF-2C at 1 mM Mg 2+ and dissociated by Co-eIF-2B at 5 mM Mg 2+, the ternary complexes formed with eIF-2(P) were unresponsive to both Co-eIF-2B and Co-eIF-2C. Also, under conditions of eIF-2 phosphorylation, HRI drastically inhibited AUG-dependent Met-tRNA(f) binding to 40S ribosomes. However, HRI (in the presence of ATP) had no effect on the joining of preformed Met-tRNA(f) x 40S x AUG complex to the 60S ribosomal subunit to form Met-tRNA(f) x 80S x AUG complex. These studies suggest that HRI inhibits protein synthesis initiation by phosphorylation of the 38,000-dalton subunit of eIF-2. HRI-phosphorylated eIF-2 does not interact with at least two other protein factors, Co-eIF-2B and Co-eIF-2C, and is thus inactive in protein synthesis initiation.

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DO - 10.1073/pnas.76.10.5076

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