Abstract
The characteristics of component activities in Co-eIF-2 (where eIF is eukaryotic initiation factor) protein complex have been studied. (i) At limiting concentrations, Co-eIF-2 promoted rapid GDP binding to eIF-2 and also GDP displacement from eIF-2 X GDP during ternary complex formation in the presence of GTP and Mg2+ (Co-eIF-2C activity) but did not significantly stimulate ternary complex formation by eIF-2. (ii) At higher concentrations, Co-eIF-2 significantly enhanced ternary complex formation by eIF-2 and also rendered the complex stable to aurintricarboxylic acid presumably as Co-eIF-2 became physically bound to the ternary complex (Co-eIF-2A activity). (iii) Ternary complex preformed in the presence of Co-eIF-2 and without Mg2+ dissociated upon subsequent addition of Mg2+ (Co-eIF-2B activity). This dissociation reaction was presumably due to loss of interaction of the Co-eIF-2A component in Co-eIF-2 with the ternary complex (reversal of Co-eIF-2A activity) as the complex became increasingly sensitive to aurintricarboxylic acid with increasing Mg2+ concentration. In another study, purified eIF-2 was freed of bound GDP by treatment with alkaline phosphatase and the characteristics of native and GDP-free eIF-2 were compared. (i) One mM Mg2+ inhibited (60%) ternary complex formation by native eIF-2 but not by GDP-free eIF-2. Addition of exogenous GDP rendered GDP-free eIF-2 sensitive to Mg2+ indicating that Mg2+ inhibition is due to eIF-2-bound GDP. (ii) In the presence of Mg2+, Co-eIF-2 stimulated similarly ternary and Met-tRNAf X 40 S X AUG complex formation by both native and GDP-free eIF-2. Such stimulatory activity in each case was strongly inhibited by prior phosphorylation of eIF-2 alpha subunit by heme-regulated translational inhibitor. (iii) Ternary complexes preformed using either native and GDP-free eIF-2 and excess Co-eIF-2A80 in the absence of Mg2+ did not form Met-tRNAf X 40 S X AUG complex. They required trace amounts of Co-eIF-2 for such activity.
Highlights
Tein complex have been studied. (i)At limiting concen- and which is required for ternary complex formation by eIF
2 and GDP displacement from eIF-Z*GDP during protein complexes have been isolated from reticulocyte cell ternary complex formation in thperesence of GTP and supernatant (7-13) and from ascites ribosomal salt wash (14, M$+ (Co-eIF-2C activity)butdid not significantly 15) and have been termed SP (7), GEF (8, 9, 11, 14, 15), RF
Was presumably due to loss of interaction of the Co- Our laboratory has previously reported that Co-eIF-2 proeIF-2A component in Co-eIF-2 with the ternarycom- tein complex contains threeactivities described in thefollowplex as the complex ing paragraphs
Summary
Addition of exogenous GDP rendered GDP- activities and provided evidence that the 80,000 polypeptide free eIF-2 sensitive tMo g2+indicating that M 8 + inhi- (Co-eIF-2AsO)is a component of Co-eIF-2 protein complex bition is due to eIF-2-bound GDP. Met-tRNAf-40S-AUG complex formation by both na- lower molecular weight polypeptides, Co-eIF-2Amand Cotive and GDP-freeeIF-2. Such stimulatory activity in eIF-2AZ5,are possibly protease degradation products of Coeach case was strongly inhibitebdy prior phosphoryla- eIF-2AS0or other higher molecular weight polypeptides in Cotion of eIF-2 a subunit by heme-regulated translational eIF-2 protein complexes. (iii) Ternary complexes preformed using reported to be present in widely divergent eukaryotic cells either native and GDP-free eIF-2 andexcess Co-eIF- such as mouse ascites tumor cells (21), pig liver (22), wheat. The mechanism of Reticulocyte ribosomal salt wash contains a high molecular
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