Molecular cloning and functional expression of a human cDNA encoding translation initiation factor 6

Abstract

Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. In this paper, we devised a procedure for purifying eIF6 from rabbit reticulocyte lysates and immunochemically characterized the protein by using antibodies isolated from egg yolks of laying hens immunized with rabbit eIF6. By using these monospecific antibodies, a 1.096-kb human cDNA that encodes an eIF6 of 245 amino acids (calculated M r 26,558) has been cloned and expressed in Escherichia coli. The purified recombinant human protein exhibits biochemical properties that are similar to eIF6 isolated from mammalian cell extracts. Database searches identified amino acid sequences from Saccharomyces cerevisiae, Drosophila, and the nematode Caenorhabditis elegans with significant identity to the deduced amino acid sequence of human eIF6, suggesting the presence of homologues of human eIF6 in these organisms.It is now well established that the initiation of protein synthesis in eukaryotic cells begins with the recognition of the initiation AUG codon of mRNA by the small ribosomal subunit (40S) containing bound initiatior methionyl-tRNA (Met-tRNA f ) to form the 40S initiation complex (40S⅐mRNA⅐Met-tRNA f ). Subsequently, a 60S ribosomal subunit joins the 40S initiation complex to form the 80S initiation complex (80S⅐mRNA⅐Met-tRNA f ) that is competent to undergo peptide bond synthesis during the elongation phase of protein synthesis (for a review, see refs. 1-5). After termination of translation, ribosomes are released from the polysomal complex as 80S particles that are in equilibrium with 40S and 60S ribosomal subunits (6, 7). Because a new round of initiation requires separated subunits, a mechanism must exist for maintaining a pool of both ribosomal subunits or for generating them from 80S ribosomes. Two protein factors, eukaryotic translation initiation factor 3 (eIF3) and eIF6, have been implicated in this process (1-5). It has been reported that eIF3, a multisubunit protein complex of M r Ͼ 500,000, binds to the 40S ribosomal subunit and prevents its association with the 60S ribosomal subunit to form 80S ribosomes (8-11). In contrast to eIF3, eIF6, a monomeric protein of about 25 kDa, has been shown to bind to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit (12)(13)(14)(15). This ribosomal subunit anti-association property was used as an assay to identify and purify eIF6 from both wheat germ (12, 13) and mammalian cell extracts (14, 15). However, unlike eIF3 whose requirement for the binding of Met-tRNA f and mRNA to the 40S ribosomal subunit to form the 40S initiation complex is firmly established, the requirement of eIF6 in the initiation of protein synthesis both in vivo and in vitro has not yet been defined. This is in part because of the relatively low abundance of eIF6 in eukaryotic cells (14,15) and the inability to obtain sufficient quantities of the pure protein in stable form to carry out structural and detaile...

Distinct Functions of Eukaryotic Translation Initiation Factors eIF1A and eIF3 in the Formation of the 40 S Ribosomal Preinitiation Complex

“…Under similar assay conditions, eIF6, which binds to 60 S ribosomal subunits, prevents the association of 40 S and 60 S ribosomal subunits (data not shown here; see Refs. [21][22][23]). These results demonstrate that in contrast to other reports (18 -20), eIF1A and eIF3 by themselves do not have ribosomal subunit antiassociation activity in the absence of Met-tRNA f and other initiation components.…”

Overexpression of p27BBP in head and neck carcinomas and their lymph node metastases

“…One potential marker is an intriguing protein, p27 BBP , that has already been reported to be highly increased in colorectal carcinoma and related to its progression. 1 Previous research has established that this protein, originally identified as an interactor of the large cytodomain of integrin h4 2 and as a putative eukaryotic initiation factor (eIF6), 3 is an essential regulator of the 60S ribosome assembly. 4,5 Its evolutionary conservation (more than 85% identity between human and yeast) suggests that p27 BBP controls a crucial event within the protein synthesis pathway that has been maintained throughout eukaryotic evolution.…”

“…However, p27 BBP has also been found in a cytoplasmic soluble form and in a cytoskeletonassociated insoluble form. 2,3 These multiple locations suggest that p27 BBP may play different roles in different cellular structures but also suggest that a potential unifying role as a local controller of ribosome assembly may be served by p27 BBP wherever protein synthesis occurs.…”

Eukaryotic Translation Initiation Factor-6 Enhances Histamine and IL-2 Production in Mast Cells

“…eIF-6 has been purified from a variety of eukaryotic cells, including wheat germ, calf liver, and rabbit reticulocytes (12)(13)(14)(15). In addition, we and others have cloned the murine and human genes that encode this protein (10,16,17). eIF-6 is highly conserved among eukaryotic cells, and human and murine eIF-6 share 97.5% amino acid sequence homology.…”

“…In all species, eIF-6 is a monomer with a molecular mass of appoximately 26 kDa (12)(13)(14)(15). eIF-6 has been reported to bind to the 60S ribosomal subunit and prevent its association with the 40S ribosomal subunit in several different eukaryotic organisms (13)(14)(15)(16)18). Several lines of evidence indicate that eIF-6 is important for ribosome biogenesis by regulating cellular levels of free 60S subunit (11,19,20).…”