A GDP/GTP exchange factor essential for eukaryotic initiation factor 2 cycling in Ehrlich ascites tumor cells and its regulation by eukaryotic initiation factor 2 phosphorylation.

R Panniers,E C Henshaw

doi:10.1016/s0021-9258(20)82007-9

Abstract

Formation of the ternary complex Met-tRNAi X eukaryotic initiation factor (eIF) 2 X GTP from eIF-2 X GDP requires exchange of GDP for GTP. However, at physiological Mg2+ concentrations, GDP is released from eIF-2 exceedingly slowly (Clemens, M.J., Pain, V.M., Wong, S.T., and Henshaw, E.C. (1982) Nature (Lond.) 296, 93-95). However, GDP is released rapidly from impure eIF-2 preparations, indicating the presence of a GDP/GTP exchange factor. We have now purified this factor from Ehrlich cells and refer to it as GEF. CM-Sephadex chromatography of ribosomal salt wash separated two peaks of eIF-2 activity. GEF was found in association with eIF-2 in the first peak and co-purified with eIF-2 under low salt conditions. It was separated from eIF-2 in high salt buffers and further purified on hydroxylapatite and phosphocellulose. Gel electrophoresis of our purest preparations showed major bands at 85, 67, 52, 37, 27, and 21 kDa. Purified GEF increased the rate of exchange of [32P] GDP for unlabeled GDP 25-fold but did not function with phosphorylated eIF-2 (alpha subunit). The factor also stimulated markedly the rate of ternary complex formation using eIF-2 X GDP as substrate with GTP and Met-tRNAi but not using phosphorylated eIF-2 X GDP as substrate. eIF-2 is released from the 80 S initiation complex with hydrolysis of GTP. If eIF-2 X GDP is actually the complex released, then GEF is absolutely required for eIF-2 to cycle and it is therefore a new eukaryotic initiation factor. Furthermore, the inability of GEF to utilize eIF-2 (alpha P) X GDP explains how phosphorylation of eIF-2 can inhibit polypeptide chain initiation.

Highlights

Formation of theternary complex Met-tRNAi. eukaryotic initiation factor 2.GTP from eIF-2
We have,found thatGDPis released fromeIF-2.GDP complexes with a half-time of a t least 30 min so that ternary complex formation is potently inhibited by a preincubation of purified Ehrlich ascites tumor cell eIF-2 with GDP even showed majorbands at 85,67,52,37,27, and k2D1a. though phosphoenolpyruvate and pyruvate kinase are subse
We show below that peak I contains GEF, while peak I1 eIF-2 is an eIF-2.GDP complex and is very inhibitedin ternary complex formation in the absence of GEF

Summary

THE JOUHNAL OF BIOLOGICACLHEMISTRY

Vol 258, No 13, Issue of July 10, pp. 7928-7934,1983 Printed in U.S.A. A GDP/GTP Exchange Factor Essential for Eukaryotic Initiation Factor 2 Cycling in Ehrlich Ascites Tumor Cells and Its Regulation by Eukaryotic Initiation Factor 2 Phosphorylation*. The factor were not inhibited by preincubation with GDP under these stimulated markedly the rate of ternary complex for- conditions, and GDPcould be rapidly exchanged in the eIF-. Phosphorylation of eIF-2 is shown to inhibit the release of GDP by GEF, confirming ourprevious explanation for the inhibition of initiation by kinases of eIF-2 and indicating theprobable role of this modification in vivo. Regulation of polypeptide chaininitiationineukaryotic deficiency [17,18,19,20] Two such factors havbeeen shownto have cells is mediated mainlyvia the modulationof several complex GDP/GTP exchange activity with nonphosphorylated but not initiationfactors [1,2,3,4].One of thesefactors, e1F’-2, binds phosphorylated eIF-2 [21,29], anwde expect that the Ehrlich. Carboxymethyl-Sephadex; BND-cellulose, benzoylated naphthoylated diethylaminoethylcellulose; MOPS, 4-morpholinpropanesulfonic acid; BSA, bovineserum albumin; SDS, sodium dodecyl sulfate; HRI, heme-regulated inhibitor, an eIF-2 a-kinase; eIF-2(P), phosphorylated form of eIF-2

Radloactwe compounds

Purification of GEF

Characterization of GEF

DISCUSSION