CHARACTERISTICS OF THE EUKARYOTIC INITIATION FACTOR-II ASSOCIATED 67-KDA POLYPEPTIDE

A eukaryotic initiation factor 2 (eIF-2) associated 67-kDa polypeptide (p67) protects the eIF-2 a-subunit from eIF-2 kinase(s) catalyzed pho sphorylation, and this promotes protein synthesis in the presence of a ctive eIF-2 kinase(s), [Datta, B., et al. (1988) Proc. Natl. Acad. Sci . U.S.A. 85, 3324-3328]. This report presents the results of studies r elated to characteristics of p67 action and the mechanism of p67:eIF-2 interaction: (1) p67 antibodies inhibited protein synthesis in hemin- supplemented rabbit reticulocyte lysates, and such inhibition was reve rsed by preincubation of the antibodies, specifically with p67. (2) p6 7 inhibited HRI- and dsI-catalyzed phosphorylations of the eIF-2 alpha -subunit and histones, but it did not inhibit casein kinase catalyzed phosphorylation of the eIF-2 beta-subunit. (3) p67 bound specifically to the eIF-2 gamma-subunit. p67 co-immunoprecipitated with the eIF-2 s ubunits when a p67/eIF-2 mixture was treated with p67 or eIF-2 subunit antibodies and protein A agarose. However, when eIF-2 was preincubate d specifically with the eIF-2 gamma-subunit antibodies, subsequent co- immunoprecipitation of p67 with the eIF-2 subunits was completely inhi bited. Similarly, preincubation of p67 and p67 antibodies prevented su bsequent p67 binding to eIF-2. Preincubation of eIF-2, with either eIF -2 alpha- or beta-subunit antibodies, had no effect on p67 coimmunopre cipitation with the eIF-2 subunits. (4) p67:eIF-2 interaction is neces sary for p67 activity to protect the eIF-2 alpha-subunit from eIF-2 ki nase(s) catalyzed phosphorylation. p67 bound to eIF-2 only at low Mg2 concentrations (0.5-2 mM) and protected the eIF-2 alpha-subunit at th ese Mg2+ concentrations. High Mg2+ (3-5 mM) completely inhibited p67 b inding to eIF-2 and also inhibited p67 activity to protect the eIF-2 a lpha-subunit. These results suggest that p67 binding to eIF-2 is neces sary for p67 activity to protect the eIF-2 alpha-subunit from inhibito ry phosphorylation. Several factors, such as the p67 level in the cell , may affect the equilibrium between p67-bound eIF-2 and free eIF-2 an d thus regulate protein synthesis.