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...

ganglioside-enriched microdomain ͉ tetraspanin ͉ integrin ͉ anchorage-independent

We have studied in vivo neo-galactosylation in Saccharomyces cerevisiae and analyzed the critical factors involved in this system. Two heterologous genes, gma12 + encoding α1,2-galactosyltransferase (α1,2 GalT) from Schizosaccharomyces pombe and UGT2 encoding UDP-galactose (UDP-Gal) transporter from human, were functionally expressed to examine the intracellular conditions required for galactosylation. Detection by fluorescence labeled α-galactose specific lectin revealed that 50% of the cells incorporated galactose to cell surface mannoproteins only when the gma12 + and hUGT2 genes were coexpressed in galactose media. Integration of both genes in the ∆mnn1 background cells increased galactosylation to 80% of the cells. Correlation between cell surface galactosylation and UDP-galactose transport activity indicated that an exogenous supply of UDP-Gal transporter rather than α1,2 GalT played a key role for efficient galactosylation in S.cerevisiae. In addition, this heterologous system enabled us to study the in vivo function of S.pombe α1,2 GalT to prove that it transfers galactose to both N-and O-linked oligosaccharides. Structural analysis indicated that this enzyme transfers galactose to O-mannosyl residue attached to polypeptides and produces Galα1,2-Man1-O-Ser/Thr structure. Thus, we have successfully generated a system for efficient galactose incorporation which is originally absent in S.cerevisiae, suggesting further possibilities for in vivo glycan remodeling toward therapeutically useful galactose containing heterologous proteins in S.cerevisiae.