Studies on the Function of Oligosaccharyl Transferase Subunits

Abstract

In the yeast, Saccharomyces cerevisiae, oligosaccharyl transferase (OT) is composed of nine different transmembrane proteins. Using a glycosylatable peptide containing a photoprobe, we previously found that only one essential subunit, Ost1p, was specifically labeled by the photoprobe and recently have shown that it does not contain the recognition domain for the glycosylatable sequence Asn-Xaa-Thr/Ser. In this study we utilized additional glycosylatable peptides containing two photoreactive groups and found that these were linked to Stt3p and Ost3p. Stt3p is the most conserved subunit in the OT complex, and therefore 21 block mutants in the lumenal region were prepared. Of the 14 lethal mutant proteins only two, as well as one temperature-sensitive mutant protein, were incorporated into the OT complex. However, using microsomes prepared from these three strains, the labeling of Ost1p was markedly decreased upon photoactivation with the Asn-Bpa-Thr photoprobe. Based on the block mutants single amino acid mutations were prepared and analyzed. From all of these results, we conclude that the sequence from residues 516 to 520, WWDYG in Stt3p, plays a central role in glycosylatable peptide recognition and/or the catalytic glycosylation process.

New Findings on Interactions among the Yeast Oligosaccharyl Transferase Subunits Using a Chemical Cross-linker

“…Construction of Plasmids and Strains-QYY102 (W303-1a OST2:: HA-his5 ؉ S. pombe), QYY104 (W303-1a SWP::HA-his5 ؉ S. pombe), QYY105 (W303-1a OST5::HA-his5 ؉ S. pombe) were prepared using ME-3 plasmid and the same method as mentioned earlier (5,13). AYY7 (W3031-a OST6::3HA-his5 ϩ S. pombe) and AYY10 (W303-1a OST4:: 13myc::Kan R ) were prepared as follows: A 1019-bp DNA fragment encoding the OST6 gene was obtained by PCR amplification using genomic DNA of yeast as the template (primers were: 5Ј-GCTAG-CAGCTGATGAAGTGGTGTAGCACATAC-3Ј and 5Ј-GCTAGGGATC-CCAAAAACAATTGGGTACCCTGG-3Ј).…”

“…To understand how these nine subunits interact with each other, a genetic approach using the yeast two-hybrid screen was utilized, but it failed to show interactions between any of the luminal domains of the yeast OT subunits (3). Studies on the mechanism of enzyme catalysis have proposed that Wbp1p may contain a site for the binding of the lipidlinked oligosaccharide substrate (4), and Stt3p was shown to be directly involved in peptide substrate recognition and/or the catalytic glycosylation process (5). However, there is little detailed information on how these two subunits interact to catalyze the glycosylation reaction.…”

Preferential transfer of the complete glycan is determined by the oligosaccharyltransferase complex and not by the catalytic subunit

“…The structures of the transferred glycans differed widely from those transferred in eukaryotic cells (10). (iii) Point mutations in the sequence WWDYG, a motif present in all members of the Stt3p family, eliminated or sharply reduced OST activity (11). (iv) Genomic analysis showed that Stt3p is the only protein of the entire OST complex encoded by the genomes of trypanosomatid protozoa (6).…”

Dimeric organization of the yeast oligosaccharyl transferase complex

“…Studies in the last two decades have provided clues to the possible functions of the OT subunits in the N-glycosylation reaction, but only recently has it been established that one of the OT subunits, Stt3p, bears the active site of the OT reaction (10)(11)(12). Ost3p and Ost6p are believed to perform redundant functions in the OT reaction because the two proteins exhibit sequence similarity and strikingly similar hydropathy plots (13).…”