Previously, we have shown that phosphorylation of the eukaryotic initiation factor eIF-2a increases under several physiological stresses in which protein synthesis is inhibited in Ehrlich ascites tumor cells. As phosphorylated eIF-2 [eIF-2(aP)] is a potent inhibitor of guanine nucleotide exchange factor (GEF), it seemed likely that it was responsible for the inhibition. We have assayed GEF activity levels in extracts prepared from Ehrlich cells exposed to three such stresses, namely heat shock, serum deprivation and glutamine deprivation. Activity was estimated by the ability of GEF to enhance the release of [ N -~~P ] G D P from purified eIF-2 [a modification of the reticulocyte lysate assay of Matts, R. L. & London, I. M. (1984) J . Biol. Chem. 259, 67081. GEF activity was reduced from control values in extracts of heat-shocked cells and serum-deprived cells, concomitant with increased eIF-2a phosphorylation. Inhibition of GEF activity in heat-shocked and serumdeprived cells was reversed to control levels by increasing the concentration of purified eIF-2 . GDP added as substrate in the GEF assay. Since we have shown elsewhere that eIF-2(aP) . GDP inhibits GEF by competition with eIF-2 . GDP, the complete reversal of inhibition of GEF activity in heat-shocked and serum-deprived cells indicates that inhibition is due solely to phosphorylation of eIF-2a. In glutamine-deprived cells phosphorylation of eIF-2a was increased modestly and GEF activity was reduced but GEF activity could not be fully reversed by addition of eIF-2 . GDP, suggesting that GEF may also be regulated in other ways.There are greater amounts of GEF relative to eIF-2 in Ehrlich cells (approximately 50%) compared with rabbit reticulocytes (approximately 20%). This explains the efficient rates of protein synthesis in control Ehrlich cells even though they have 30% of their eIF-2 phosphorylated which is enough to inhibit GEF and initiation in reticulocytes completely but only enough to trap approximately 60% of the GEF in Ehrlich cells.The mechanism of heme regulation of translation in reticulocytes is well studied [l-31. In the absence of hemin, heme-controlled repressor (HCR), a protein kinase, is activated and phosphorylates the a subunit of eIF-2. eIF-2(aP) is a potent inhibitor of guanine-nucleotide-exchange factor, GEF (or eIF-2B), which is responsible for recycling eIF-2 . GDP after a round of initiation by exchanging GTP for the GDP. GEF function is virtually stoichiometrically inhibited by eIF-2(aP) which is a competitive inhibitor [4]. A similar mechanism is probably responsible for the inhibition of polypeptide chain initiation in non-erythroid cells that have been deprived of nutrients or serum or that have been heat-shocked. In each case, the reduced rate of initiation is accompanied by decreased 43s complex levels [5-91 and increased levels of eIF-2 phosphorylation [9 -111. In Chinese hamster ovary cells with a temperature-sensitive tRNA synthetase (a model for amino acid starvation), initiation is inhibited, eIF-2a phosphorylation is...
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