The rate of initiation of protein synthesis appears to be controlled at the level of recycling of eIF-2. In this process a new factor, designated eRF, plays an important role. The factor has been purified from the postribosomal supernatant and has been called formerly anti-HRI and anti-inhibitor [Amesz, H., Goumans, H., Haubrich-Morree, Th., Voorma, H. O., and Benne, R. (1979) Eur. J. Biochem. 98, Its effect on the initiation of protein synthesis has been studied in several assays: a small but distinct effect is found in the assay for the formation of a ternary complex between eIF-2, GTP and Met-tRNA; a 4-Sfold stimulation is obtained in assays for 40s preinitiation complex formation and in the methionyl-puromycin reaction. In the latter assay a catalytic use of eIF-2 occurs provided that eRF is present.eRF forms a complex with eIF-2 which results in a decrease of the affinity of eIF-2 for GDP, giving it the properties of a GDP/GTP exchange factor. The model stresses the catalytic use of eIF-2 in initiation provided that conditions are met for GDP/GTP exchange by a transient complex formation between eIF-2 and eRF. On the other hand, it is shown that phosphorylation of eIF-2 by the hemin-regulated inhibitor (HRI) abolishes the recycling of eIF-2, by the formation of another stable complex comprising eIF-2aP, GDP and eRF.In recent years, an impressive amount of data has accumulated on the components involved in the initiation of protein synthesis in mammalian cells (for reviews, see [1 -31). The sequence of events comprising the construction of an initiation complex requires the participation of at least nine protein factors called eIF-1, -2, -3, -4A, -4B, -4C, -4D, -4E, -4F and -5. Many papers have dealt with the detailed description of the intricate manner in which initiation factors contribute in the assembly of an 80s initiation complex [l -51. In brief, the pathway leads through (a) formation of a ternary complex between eIF-2, Met-tRNA and GTP, (b) subsequent combination of this complex with a preformed complex of the 40s ribosomal subunit, eIF-3 and eIF-4C 161, (c) binding of the messenger with the aid of eIF-1 [5, 71, 91, and (d) eIF-5-mediated junction of the 40s complex and the 60s ribosomal subunit [4, 51.Very little, however, was known about the way this process is regulated. Most of the information acquired is derived from reticulocyte lysates in which the absence of hemin leads to the cessation of protein synthesis [lo, 111. This cessation is caused by the action of an inhibitor called hemin-regulated inhibitor, HRI [12, 131, which phosphorylates the small (Mr 36000) asubunit of eIF-2 [14-171. A causal relation between the halt of globin synthesis and the phosphorylation of eIF-2 appears to exist, since the addition of purified eIF-2 to a hemindeprived lysate results in relief of the inhibition [18-201, but until recently no difference in biological activity between purified phosphorylated eIF-2 and control eIF-2 could be detected in model assay systems and the crude Iysate system [23 -231.We have...

We have purified and partially characterized a supernatant factor which reverses the effect of the heme‐regulated translational inhibitor on protein synthesis in rabbit reticulocyte lysates. The anti‐inhibitor restores protein synthesis activity in heme‐deficient lysates (and in lysates to which the inhibitor has been added) to the level observed in the presence of heme. The factor has no effect on the phosphorylation of eIF‐2 by the inhibitor nor on any reaction carried out with purified initiation factors. The anti‐inhibitor probably consists of three subunits with molecular weights of 81000, 60000 and 41000. The factor is isolated from the postribosomal supernatant of rabbit reticulocytes both free and complexed to eIF‐2. A possible mechanism of action is discussed.

A purification procedure is described for the initiation factors of protein synthesis from rabbit reticulocytes : (a) from the ribosomal wash and (b) from the postribosomal supernatant.A comparison is made between these preparations with respect to yield and specific activity. eIF-4A and eIF-4D occur mainly in the postribosomal supernatant; eIF-2, eIF-4C and eIF-5 are more evenly divided over both fractions, whereas eIF-1, eIF-3 and eIF-4B are found almost exclusively in the ribosomal wash. No significant difference in specific activity could be detected when factors from both sources were compared, with a possible exception of eIF-4A and eIF-4D.Several laboratories have reported the purification of initiation factors of protein synthesis from rabbit reticulocytes [l-71. As a first step the preparation of a crude ribosomal wash with high salt concentration was described. This procedure is based on the assumption that initiation factors are bound to ribosomes and are not present in the postribosomal supernatant. However, after completing the initiation sequence all of the initiation factors are released from the initiation complex into the cytoplasm, after which a new round of initiation is started [8 -101. Therefore, although detailed knowledge about the rate of protein synthesis and, more specifically, about the rate of attachment of each initiation factor to the ribosomes is lacking in vivo, the possibility arises that a certain amount of initiation factors exists free in the cytoplasm. Thus, the presence of initiation factors in the postribosomal supernatant might provide some information about their function in the process of initiation of protein synthesis.Another point of interest involves the question of whether supernatant factors differ from ribosomebound factors with respect to specific and total activity. It is possible that a pool of (inactivated?) factors exists, waiting for a signal to take part in another round of initiation. It has been shown that such a situation occurs in the case of certain mRNAs ~ Abbreviations. elF-I, eIF-2, elF-3, eIF-4A, eIF-4B, eIF-4C, eIF-4D and eIF-5 are eukaryotic initiation factors, see Anderson et al. (1977) FEBS Lett. 76, 1-10 and [14].De-finition. A260 unit; the quantity of material in 1 ml of a SOlution with an absorbance of 1 at 260 nm (2-cm path length).[ 1 1 -131. Therefore, useful information may be obtained by comparison of the specific activities and amounts of each initiation factor found in the postribosomal supernatant and on the ribosomes.We reported a procedure for the isolation of initiation factors from a crude lysate [14], in which an affinity chromatography step was used, i.e. binding of initiation factors to Sepharose-heparin. This paper describes a method to isolate initiation factors from the postribosomal supernatant utilizing chromatography on Sepharose-heparin at an early stage. Yield and specific activity of factors thus obtained were compared to those of factors isolated from the ribosomes via conventional methods. MATERIALS AND METHODS Mat...

Binding of eukaryotic initiation factor 4C (eIF-4C) to a 40-S initiation complex could be detected by analysis of assay mixtures for 40-S initiation complex formation on columns of Sepharose 6B and on sucrose gradients. On Sepharose 6B columns an almost stoichiometric binding of ['4C]eIF-4C was observed, provided that eIF-1, elF-2, eIF-3, Met-tRNAr and mRNA were included in the assay mixtures. On sucrose gradients the presence of 0.5 pM edeine was an additional prerequisite for stable binding of eIF-4C.The presence of eIF-4C on the 40-S initiation complex resulted in a pronounced increase of its stability, which might explain the observed stimulatory effect of the factor on the binding of MettRNAr into initiation complexes.The interaction between eIF-4C and the small ribosomal subunit presumably occurs at an early stage of the initiation process, since eIF-4C was capable of dissociating 80-S ribosomes into subunits. Protein synthesis in a 'pH-5' assay was almost totally dependent on the addition of eIF-4C, when the assay was performed with purified, nearly homogeneous, initiation factors.In the last ten years numerous papers have been published on the purification and characterization of eukaryotic initiation factors from rabbit reticulocytes [l-71. A considerable amount of work has been devoted to the elucidation of the mechanism of action of these factors, which has resulted in a better understanding of the specific contribution of each factor during the assembly of an initiation complex (for a review see [4]).However, a number of unanswered questions remain, which is in part due to the fact that no binding of the initiation factors (except for elF-2 and eIF-3) to nascent initiation complexes could be detected in the model assay systems used [7].One of the factors, eIF-4C (Mr 17500) stimulates the binding of the ternary complex between eIF-2, GTP and Met-tRNAf to the 40-S ribosomal subunit [3,5,8]. The factor is also thought to be implicated in inRNA binding [3,5], subunit joining [8] and dissociation of 80-S ribosomes [9]. All these effects of eIF-4C suggest its interaction, at some stage, with the 40-S ribosomal subunit, an interaction which could not be demonstrated ([7], see above).Recently it was shown that edeine inhibits initiation of protein synthesis in a crude reticulocyte lysate, causing the accumulation of heavy complexes between Ahhrei>iulion. eTF, eukaryotic initiation factor. 4 0 3 subunits and mRNA, containing eIF-4A and eIF-4B [lo-131. In this paper we show that the addition of edeine to a purified assay system leads to a stable binding of elF-4C to 40-S initiation complexes, which is detectable on sucrose gradients.A still better result could be obtained using Sepharose-6B columns as the aiialytic tool, showing almost stoichiometric amounts of eIF-4C to be present on 4 0 3 initiation complexes, even in the absence of edeine.These results are discussed in the context of the stimulatory effect of eIF-4C on the initiation of protein synthesis. It is feasible that all of the observed efTects ar...

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