A genetic reversion analysis at the HIS4 locus in Saccharomyces cerevisiae has identified SUI1 as a component of the translation initiation complex which plays an important role in ribosomal recognition of the initiator codon. SUI1 is an essential protein of 12.3 kDa that is required in vivo for the initiation of protein synthesis. Here we present evidence that SUI1 is identical to the smallest subunit, p16, of eukaryotic translation initiation factor 3 (eIF-3) in S. cerevisiae. SUI1 and eIF3-p16 comigrate upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and cross-react with anti-SUI1 and anti-eIF3 antisera. Anti-SUI1 antisera immunoprecipitate all of the subunits of eIF3, whereas antisera against the eIF3 complex and the individual PRT1 and GCD10 subunits of eIF3 immunoprecipitate SUI1. Finally, the N-terminal amino acid sequence of a truncated form of eIF3-p16 matches the sequence of SUI1. eIF3 isolated from a sui1(ts) strain at 37 degrees C lacks SUI1 and fails to exhibit eIF3 activity in the in vitro assay for methionyl-puromycin synthesis. A free form of SUI1 separate from the eIF3 complex is found in S. cerevisiae but lacks activity in the in vitro assay. The results, together with prior genetic experiments, indicate that SUI1 is essential for eIF3 activity and functions as part of eIF3 and in concert with eIF2 to promote eIF2-GTP-Met-tRNAi ternary complex recognition of the initiator codon.

Eukaryotic translation initiation factor 3 (eIF3) in the yeast Saccharomyces cerevisiae comprises about eight polypeptides and plays a central role in the binding of methionyl-tRNA i and mRNA to the 40S ribosomal subunit. The fourth largest subunit, eIF3-p39, was gel purified, and a 12-amino-acid tryptic peptide was sequenced, enabling the cloning of the TIF34 gene. TIF34 encodes a 38,753-Da protein that corresponds to eIF3-p39 in size and antigenicity. Disruption of TIF34 is lethal, and depletion of eIF3-p39 by glucose repression of TIF34 expressed from a GAL promoter results in cessation of cell growth. As eIF3-p39 levels fall, polysomes become smaller, indicating a role for eIF3-p39 in the initiation phase of protein synthesis. Unexpectedly, depletion results in degradation of all of the subunit proteins of eIF3 at a rate much faster than the normal turnover rates of these proteins. eIF3-p39 has 46% sequence identity with the p36 subunit of human eIF3. Both proteins are members of the WD-repeat family of proteins, possessing five to seven repeat elements. Taken together, the results indicate that eIF3-p39 plays an important, although not necessarily direct, role in the initiation phase of protein synthesis and suggest that it may be required for the assembly and maintenance of the eIF3 complex in eukaryotic cells.Initiation of protein synthesis is promoted by at least 10 proteins called initiation factors (reviewed in reference 14). The largest and most complex of these is eukaryotic translation initiation factor 3 (eIF3), a factor comprising at least eight subunits. eIF3 plays a central role in the initiation pathway in mammalian cells (1a, 26). It binds to 40S ribosomal subunits and is implicated in dissociating 80S ribosomes into 40S and 60S subunits (1a, 6). It prevents dissociation of the MettRNA i ⅐ eIF2 ⅐ GTP ternary complex caused by addition of RNA (7) and stabilizes ternary complex binding to 40S ribosomal subunits (1a). eIF3 is required for mRNA binding to 40S and 80S ribosomes (1a, 26), in part by binding the eIF4G subunit of the cap-binding complex, eIF4F (12, 13). Therefore, knowledge of the structure and function of eIF3 is essential for understanding the mechanism of the initiation phase of protein synthesis.To better elucidate the function of eIF3 by the application of both biochemical and genetic methods, we have been studying the factor in the yeast Saccharomyces cerevisiae. Yeast eIF3 was initially isolated and purified by a biochemical approach that used an eIF3-dependent mammalian assay for the synthesis of methionyl-puromycin (Met-PM) (17). The resulting yeast complex comprised eight subunits with apparent masses of 16,21, 29, 33, 39, 62, 90, and 135 kDa. The genes for three of the subunits had been identified previously, but it was not realized that they encode subunits of eIF3. The second-largest subunit, p90, is encoded by PRT1; the temperature-sensitive prt1-1 mutant causes destabilization of Met-tRNA i binding to 40S ribosomal subunits (8,15). GCD10, first characterized genetical...

The initiation of translation on eukaryotic mRNA is governed by the concerted action of polypeptides of the eIF-4F complex. One of these polypeptides, eIF-4G, is proteolytically inactivated upon infection with several members of the Picornaviridae family. This cleavage occurs by the action of virusencoded proteinases: 2Apro (entero-and rhinovirus) or L pro (aphthovirus). An indirect mode of eIF-4G cleavage through the activation of a second cellular proteinase has been proposed in the case of poliovirus. Although cleavage of eIF-4G by rhino-and coxsackievirus 2A pro has been achieved directly in vitro, a similar activity has not been documented to date for poliovirus 2Apro . We report here that a recombinant form of poliovirus 2A pro fused to maltose binding protein (MBP) directly cleaves human eIF-4G from a highly purified eIF-4F complex. Efficient cleavage of eIF-4G requires magnesium ions. The presence of other initiation factors such as eIF-3, eIF-4A or eIF-4B mimics in part the stimulatory effect of magnesium ions and probably stabilizes the cleavage products of eIF-4G generated by 2Apro . These results suggest that efficient cleavage of eIF-4G by MBP-2Apro requires a proper conformation of this factor. Finally, MBP-2Apro protein cleaves an eIF-4G-derived synthetic peptide at the same site as rhino-and coxsackievirus 2Apro (R485-G486).z 1998 Federation of European Biochemical Societies.

Eukaryotic initiation factor (eIF) 1A (formerly called eIF-4C) is a small protein that promotes dissociation of 80 S ribosomes into subunits, stabilizes methionyl-tRNA binding to 40 S ribosomal subunits, and is required for the binding of mRNA to ribosomes. The sequence of eIF-1A derived from its cloned cDNA possesses a high frequency of basic residues and acidic residues at its N and C termini, respectively. Northwestern blotting with a fragment of mRNA indicates that eIF-1A binds RNA. Overexpression of the human eIF-1A cDNA in Escherichia coli and subsequent purification enabled us to prepare large quantities of active factor. The level of eIF-1A in HeLa cells determined by Western immunoblotting is 0.01% of total protein, which corresponds to 0.2 molecules of eIF-1A/ribosome. The moderate abundance means that eIF-1A is equal to or in excess of native 40 S subunits and suggests that the factor may not be limiting for protein synthesis, a conclusion reinforced by the failure of overproduced eIF-1A to stimulate translation rates in transiently transfected COS-1 cells. S1 nuclease protection and primer extension analyses show that eIF-1A mRNA possesses an unusually long 5'-untranslated leader that is very G/C-rich (72%). Unexpectedly, the mRNA is efficiently translated in HeLa cells as judged by polysome profile analyses.

The subcellular localizations of the Dolichos biflorus seed lectin and the structurally related lectin (cross-reactive material [CRMI) from the stems and leaves of this plant were determined by immunofluorescence, immunocytochemistry, and cell fractionation procedures. Subcellular fractionation of the cotyledons using a nonaqueous procedure to minimize disruption of the protein bodies showed that the majority of the seed lectin was associated with the protein body fraction and some lectin was also present in the starch granules. Immunofluorescence and immunocytochemistry at the light microscopic level showed that the seed lectin was mainly localized at the peripheries of these organelles. Lectin was also found in the cytoplasm of the cells, although the amount appeared to be dependent upon the degree of protein body disruption.Immunofluorescence and immunocytochemistry studies of the stem and leaf lectin (CRM) indicated that a significant portion of this lectin may be associated with the cell walls, although lectin was also seen in the cytoplasm of plasmolyzed cells. Extraction and cell fractionation studies showed that a large portion of the CRM is readily solubilized and most of the remainder is pelleted at 1000g. The CRM can be extracted from these pellets by treatment with cellulase and pectinase; other reagents such as NaCI, detergents, and EDTA could also release significant amounts of CRM. These studies suggest that the CRM is noncovalently bound to the cell walls. A comparison of the distribution of exogenously supplied IlIIICRM with the endogenous CRM during extraction and cell fractionation indicates that soluble CRM is not adsorbed to the 1000g pellet during fractionation.The different subcellular distributions of these two structurally related lectins suggest that different tissues of the same plant may utilize lectins for different functions.The seeds of the Dolichos biflorus plant contain a lectin that is specific for terminal nonreducing a-N-acetylgalactosamine residues (13). This lectin is localized in the cotyledons where it accounts for about 10% of the nitrogen in the soluble mature seed extract. Upon germination of the seeds, the seed lectin disappears at about the same rate as the other cotyledon proteins (31). Peptide mapping studies and COOH-terminal amino acid analyses indicate that these subunits are identical to one another, except at their COOH-terminal ends where subunit I appears to be slightly longer than subunit 11 (6,7,29). Despite these similarities, the carbohydrate-binding activity of the lectin appears to reside in subunit 1 (12).Although the seed lectin has not been detected in other parts of the plant at any stage of its life cycle, the stems and leaves of the plant contain a glycoprotein that cross-reacts with antibodies to the seed lectin (31). This CRM' resembles the seed lectin in its amino acid and carbohydrate composition and is a 70,000 mol wt dimer consisting of one subunit with an electrophoretic mobility identical to subunit I of the seed lectin and anot...