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Regulation of Protein Synthesis in Rabbit Reticulocyte Lysates. Phosphorylation of eIF-2 Does Not Inhibit Its Capacity to Recycle

1980; Wiley; Volume: 104; Issue: 2 Linguagem: Inglês

10.1111/j.1432-1033.1980.tb04452.x

1432-1033

Rob Benne, M M Salimans, Hans Coumans, Hans AMESZ, Harry O. Voorma,

RNA and protein synthesis mechanisms

eIF-2, phosphorylated in the α subunit (M, = 38000), was isolated from rabbit reticulocyte lysates in which protein synthesis was blocked by incubation in the presence of hemin-regulated translational inhibitor. Such eIF-2 was compared to non-phosphorylated eIF-2 from control lysates in three assay systems: (a)ternary complex formation with GTP and Met-tRNAYtmet, (b)methionyl-puromycin synthesis in a purified system and (c)rescue of a hemin-deficient rabbit reticulocyte lysate. No difference between the eIF-2 preparations could be detected in any of these assays. Addition of 1 pmol eIF-2 resulted in (a) the binding of 0.65 pmol [3H]Met-tRNAYtmet into a ternary complex in the first assay system, (b) the synthesis of 0.6 pmol methionyl-puromycin in the second reaction and (c) the synthesis of 2.5–3.0 pmol globin in the hemin-deficient lysate, with all eIF-2 preparations tested. In the first two assay systems no phosphorylation or dephosphorylation of added eIF-2 could be detected. However, in a hemin-deficient lysate added non-phosphorylated eIF-2 was readily phosphorylated (t1/2∼ 2.5 min), whereas the phosphate group of added phosphorylated eIF-2 was rapidly exchanged (t1/2∼ 2.5 min), without significantly affecting the overall level of phosphorylation of the factor. A protein factor, antagonistic in its mode of action to the inhibitor (therefore called anti-inhibitor) was isolated from the postribosomal supernatant of rabbit reticulocyte lysate [Amesz, H., Goumans, H., Haubrich-Morree, T., Voorma, H. O. and Benne, R. (1979) Eur. J. Biochem. 98, 513–520]. This factor induced the synthesis of 20 mol globin/mol eIF-2 present in the lysate, although no reduction of the level of phosphorylation of eIF-2 was observed. On the contrary, addition of the factor to the lysate system resulted in an almost twofold increase of phosphate incorporation into eIF-2α (from 0.7 mol to about 1.2 mol phosphate/mol eIF-2). These observations support the idea that phosphorylation of eIF-2α is not the sole cause (if any) of cessation of protein synthesis in rabbit reticulocyte lysates.