Rabbit reticulocyte initiation factor 2 contains two polypeptide chains of molecular weights 48,000 and 38,000.

Abstract

Eukaryotic initiation factor 2 (eIF-2) purified from rabbit reticulocyte lysates consists of equimolar amounts of two polypeptide chains of Mr 48,000 and 38,000. Determination of the molecular weight of the native factor gave a value which is consistent with a Mr of 86,000, indicating that the factor is composed of one Mr 48,000 and one Mr 38,000 polypeptide. The purified factor exhibited all the binding activities characteristic of eIF-2. The factor formed ternary complexes with Met-tRNA? et and GTP; it bound GDP to form a binary complex; and it also possessed the property of binding a wide variety of RNA species, including reoviral mRNA, phage T3 mRNA, rRNAs, and tRNA. Furthermore, the ternary complex formed by purified eIF-2 interacted with the 40S ribosomal subunit in the presence of AUG codon to form a 40S initiation complex. These results indicate that all binding activities attributed to eIF-2 are contained in the 48,000-and 38,000-dalton polypeptides.We have shown that eukaryotic initiation factor 2 (eIF-2) purified from ribosomal salt wash proteins from calf liver is composed of one 48,000-and one 38,000-dalton polypeptide corresponding to a molecular mass of 86,000 for the native factor (1). These results were in contrast to numerous reports that eIF-2 from other eukaryotic sources, and in particular from rabbit reticulocytes, consisted of three nonidentical polypeptide chains with molecular masses in the ranges 55,000-52,000, 50,000-48,000, and 38,000-35,000 daltons (2-11). In order to resolve the anomaly in the subunit composition (and molecular mass) of eIF-2 prepared from these two mammalian sources, we purified eIF-2 from rabbit reticulocyte lysates by the procedure used for preparation of the factor from calf liver.In this paper we show that eIF-2 from rabbit reticulocytes that was purified by our rigorous purification procedure also produced only two polypeptide bands, of 48,000 and 38,000 daltons, on 15% polyacrylamide gels in the presence of sodium dodecyl sulfate. Similar to the eIF-2 from calf liver, our highly purified eIF-2 from rabbit reticulocytes exhibited all the activities known for eIF-2. MATERIALS AND METHODS Materials

Removal of beta subunit of the eukaryotic polypeptide chain initiation factor 2 by limited proteolysis.

“…The molecular weights of the two subunits, given as 35,000 and 48,500 for the pig liver factor (5) and as 38,000 and 48,000 for the factors from calf liver (6) and rabbit reticulocytes (7), are reasonably close to our values for protease-treated eIF-2. It has in fact been reported (22) that the two-subunit eIF-2 from rabbit reticulocytes (7) lacks the polypeptide that is specifically phosphorylated by reticulocyte casein kinase-i.…”

“…1 and 3). On the other hand, eIF-2 from pig (5) and calf (6) liver has been isolated as a protein consisting of only two subunits, and this has also recently been reported (7) for eIF-2 from rabbit reticulocytes.…”

Structure and function of peptide initiation factor 2: differential loss of activities during proteolysis and generation of a terminal fragment containing the phosphorylation sites of the alpha subunit.

“…The rapid and differential loss of activity for binding of MettRNAf to 40S ribosomal subunits is surprising in the light of reports that eIF-2 consisting ofonly the a and ysubunit is active for peptide initiation (15)(16)(17)(18). It is possible that the activity observed in these studies was due to a small amount ofthree-subunit eIF-2 that was present but undetected in preparations of the two-subunit factor.…”

“…The results appear to indicate that prior modification of eIF-2, possibly elimination of the /3 subunit, must occur before clipping of the a subunit can occur efficiently. Activity of trypsin-treated eIF-2 Although reticulocyte eIF-2 consisting ofonly the a and y subunits was found to be less stable to thermal denaturation than the native factor (18), eIF-2 lacking the 3 subunit was able to support peptide initiation (17,18) and reverse inhibition ofpeptide synthesis that was caused by hemin deficiency in cell lysates (18). These results were interpreted to indicate that the twosubunit eIF-2 is functionally active in the reactions of peptide initiation.…”

“…A form of eIF-2 containing only the a and y subunits but apparently active in peptide initiation has been isolated from liver (15,16) and reticulocytes (17). Recently, Mitsui et al (18) presented data indicating that the ( subunit of eIF-2 appears to be more sensitive to limited proteolysis than are the other two subunits, thus providing a possible mechanism by which the two-subunit form of eIF-2 may have been generated.…”