Protein synthesis in rabbit reticulocytes: A study of the mechanism of action of the protein factor RF that reverses protein synthesis inhibition in heme-deficient reticulocyte lysates

Authors

M. Grace, University of Nebraska - Lincoln
M. Bagchi, University of Nebraska at Lincoln
F. Ahmad, University of Nebraska at Lincoln
T. Yeager, University of Nebraska at Lincoln
C. Olson, University of Nebraska at Lincoln
I. Chakravarty, University of Nebraska at Lincoln
N. Nasrin, University of Nebraska at Lincoln
A. Banerjee, Calcutta University
N. K. Gupta, University of Nebraska at Lincoln

Date of this Version

1984

Comments

Published in Proc. Natl. Acad. Sci. USA Vol. 81, pp. 5379-5383, September 1984. Used by Permission.

Abstract

A eukaryotic initiation factor 2 (eIF-2)-ancillary protein factor Co-eIF-2 promotes displacement of GDP from eIF-2GDP and facilitates ternary complex (Met-tRNA_f• eIF-2-GTP) formation in the presence of Mg²⁺. Heme-regulated protein synthesis inhibitor, HRI, phosphorylates the α-subunit of eIF-2 and thus inhibits ternary complex formation as Co-eIF•2 does not displace GDP from eIF-2α(P)•GDP. RF, a high molecular weight cell supernatant factor, reverses protein synthesis inhibition in heme-deficient reticulocyte lysates and also reverses HRI inhibition of ternary complex formation. RF contains Co-eIF•2 activity. In addition, an active RF preparation contains excess α-subunit of eIF-2 in the free and unphosphorylated form and this α-subunit of eIF-2 is not phosphorylated by HRI and ATP. In this paper we report (i) an active RF preparation contains excess α-subunit of eIF-2 and this α-subunit can be phosphorylated by HRI and ATP in the presence of GDP; (ii) RF promotes ternary complex formation by elF- 2•[³H]GDP with accompanying GDP displacement; (iii) in the presence of HRI and ATP, RF promotes ternary complex formation by eIF-2•[³H]GDP without accompanying GDP displacement; (iv) in the presence of HRI and ATP, the ternary complex formed using RF is active in Met-tRNA_f 40S initiation complex formation; (v) both the ternary complex and the Met-tRNA_f 40S complex formation in the presence of HRI and ATP are completely inhibited by prior incubation of RF with GDP; (vi) upon further fractionation of an active RF fraction, a preparation can be obtained that contains HRI-sensitive CoeIF- 2 activity. However, this preparation does not efficiently reverse protein synthesis inhibition in heme-deficient reticulocyte lysates and does not contain excess a-subunit of eIF-2. Based on these observations, we have suggested (α) RF provides the unphosphorylated a-subunit to eIF-2a(P)-GDP and restores eIF-2 activity. This RF activity is inhibited as the α-subunit in the RF preparation becomes phosphorylated by HRI and ATP in the presence of GDP; (b) RF contains Co-eIF- 2 activity, which has dual functions: (i) stimulation of ternary complex formation by eIF-2 and (i) GDP displacement from eIF-2•GDP during ternary complex formation. In the presence of HRI and ATP, Co-eIF-2 still stimulates ternary complex formation by unphosphorylated eIF-2 but does not displace GDP from eIF-2α(P)•GDP.