Protein synthesis in rabbit reticulocytes: Characteristics of a postribosomal supernatant factor that reverses inhibition of protein synthesis in heme-deficient lysates and inhibition of ternary complex (met-tRNA(f)Met.e IF-2.GTP) formation by heme-regulated inhibitor

R. O. Ralston, A. Das, M. Grace, H. Das, N. K. Gupta

Research output: Contribution to journalArticle

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Abstract

During heme deficiency in reticulocyte lysates, a translational inhibitor (heme-regulated inhibitor, HRI) that blocks polypeptide chain initiation is activated. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNAA(f)(Met) binding factor, eIF-2. Phosphorylation of eIF-2 by HRI prevents its interaction with at least two additional factors, resulting in a net reduction in formation of ternary complex(Met-tRNA(f)(Met)·eIF-2·CTP) and AUG-dependent transfer of Met-tRNA(f)(Met) to 40S ribosomal subunits. A factor (sRF) that reverse protein synthesis inhibition in heme-deficient lysates has been purified from reticulocyte postribosomal supernatant. sRF also reverses the inhibition of ternary complex formation by HRI in a fractionated system. The ternary complex inhibition reversal activity and the protein synthesis inhibition reversal activity cosediment at 12.5 S upon glycerol density gradient centrifugation, and both activities are sensitive to heat or N-ethylmaleimide. Purified sRF does not dephosphorylate eIF-2 whose phosphorylation has been catalyzed by HRI, nor does the sRF prevent the phosphorylation of eIF-2 by HRI in a fractionated system. sRF stimulates ternary complex formation by both phosphorylated and nonphosphorylated eIF-2. These observations suggest that the sensitivity of protein synthesis to phosphorylation of eIF-2 by HRI may be modulated by the concentration and activity of sRF.

Original languageEnglish
Pages (from-to)5490-5494
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume76
Issue number11
DOIs
StatePublished - 1979

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