Protein synthesis initiation factors from human HeLa cells and rabbit reticulocytes are similar: comparison of protein structure, activities, and immunochemical properties

Abstract

Five initiation factors, eIF-2, eIF-3, eIF-4A, eIF-4B, and eIF-5, were purified from human HeLa cells. Methods of protein fractionation and assays for initiation factors which had been developed for rabbit reticulocytes were found to be suitable for HeLa factors. The initiation factors from HeLa cells are similar to or indistinguishable from the corresponding rabbit reticulocyte factors with respect to specific activities, molecular weights as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subunit structure. The molecular weight of eIF-3 particles from both species is about 410000 as determined by equilibrium sedimentation analytical centrifugation. The partial protease fragmentation patterns of corresponding proteins also are similar and indicate that the primary sequences of the factors are related in the two species. Antisera raised in goats against rabbit eIF-3 and human eIF-2, eIF-4A, and eIF-4B cross-react with the cognate factors from both species. On the basis of immunoblotting techniques, eIF-4A is highly conserved, eIF-2 alpha, eIF-3, and eIF-4B are somewhat less conserved, and eIF-2 beta is the least conserved of the proteins examined. The functional, structural, and immunological results are all consistent with the view that initiation factors from different mammalian cells are very similar.

Identification of a Translation Initiation Factor 3 (eIF3) Core Complex, Conserved in Yeast and Mammals, That Interacts with eIF5

“…An important role for Sui1p has been suggested from the observation that eIF3 activity in the RSW fraction of a temperature-sensitive sui1 mutant grown at the nonpermissive temperature did not stimulate Met-puromycin synthesis (43), although an indirect effect of the mutation on one of the eIF3 subunits was not ruled out. More experimentation is required to resolve this question, particularly since the mammalian counterpart of Sui1p, eIF1, does not seem to be required for eIF3 activity in the Met-puromycin assay (5,11).…”

“…It also stimulates binding of mRNA to 40S subunits (9,56), presumably through its interactions with the cap-binding initiation factor eIF4F (36,38) or eIF4B (41). A mammalian eIF3 complex, purified by its ability to promote methionylpuromycin (Met-puromycin) synthesis by an 80S initiation complex in an assay containing purified eIF1A, eIF2, eIF5, eIF5A, and ribosomal subunits in addition to eIF3 (11), contained nine nonidentical polypeptides, called p170, p116, p110, p66, p48, p47, p44, p40, and p36. All nine polypeptides could be coimmunoprecipitated with affinity-purified antibodies against the p170 subunit (3)(4)(5)40), supporting the idea that they are subunits of the same complex.…”

Biochemical Characterization of Mammalian Translation Initiation Factor 3 (eIF3)

“…eIF3 was purified originally from rabbit reticulocyte lysates on the basis of its ability to stimulate translation of globin mRNA by a protein synthesizing system with partially purified reconstituted proteins (6,15,16). Subsequently the factor was purified from a variety of eukaryotic sources (17)(18)(19)(20)(21) including the yeast Saccharomyces cerevisiae (22,23). The subunit composition of purified eIF3 is, however, controversial.…”

“…The subunit composition of purified eIF3 is, however, controversial. eIF3 purified in different laboratories is reported to contain 7-11 polypeptides ranging in molecular masses between 26 and 170 kDa (15)(16)(17)(18)(19)(20)(21), although some of the subunits seem to be present in non-stoichiometric amounts, suggesting that eIF3 might be heterogeneous. Furthermore, the possibility also exists that at least some of the smaller polypeptides are artificially derived from larger ones by limited proteolysis.…”

Differential Role for Host Translation Factors in Host and Viral Protein Synthesis during Human Cytomegalovirus Infection

“…In particular, the eIF4F complex is a critical host translation initiation complex required for the efficient recruitment of ribosomes to mRNAs containing a 7-methylguanosine cap (m 7 G cap) on their 5= terminal nucleotide (1)(2)(3). Most cellular messages are capped (4), and therefore the eIF4F complex is thought to be required for the translation of the majority of cellular mRNAs.…”