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•eIF-2•GTP) formation by heme-regulated inhibitor

Authors

• R. O. Ralston, University of Nebraska - Lincoln
• A. Das, University of Nebraska at Lincoln
• M. Grace, University of Nebraska - Lincoln
• H. Das, University of Nebraska - Lincoln
• N. K. Gupta, University of Nebraska - Lincoln

Document Type

Article

Date of this Version

1979

Comments

Published in Proc. Natl. Acad. Sci. USA Vol. 76, No. 11, pp. 5490-5494, November 1979. Used by Permission

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-tRNA_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•GTP) and AUG-dependent transfer of Met-tRNA_f^Met to 40S ribosomal subunits. A factor (sRF) that reverses protein synthesis inhibition in heme-deficient Iysates 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-ethylmaleirmide. 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.