The highly conserved Stt3 protein is a subunit of the yeast oligosaccharyltransferase and forms a subcomplex with Ost3p and Ost4p

被引:78
作者
Karaoglu, D [1 ]
Kelleher, DJ [1 ]
Gilmore, R [1 ]
机构
[1] Univ Massachusetts, Sch Med, Dept Biochem & Mol Biol, Worcester, MA 01655 USA
关键词
D O I
10.1074/jbc.272.51.32513
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The oligosaccharyltransferase has been purified from Saccharomyces cerevisiae as an hetero-oligomeric complex composed of four or six subunits. Here, the in vivo subunit composition and stoichiometry of the oligosaccharyltransferase were investigated by attaching an epitope coding sequence to a previously characterized subunit gene, OST3. Five (Ost1p, Wbp1p, Swp1p, Ost2p, and Ost5p) of the seven polypeptides that were coimmunoprecipitated with the epitope-tagged Ost3p were identical to those obtained by the conventional purification procedure. Two additional coprecipitating polypeptides with apparent molecular masses of 60 and 3.6 kDa were identified as the 78-bDa Stt3 protein and the 36-residue Ost4 protein, respectively. Stt3p and Ost4p were previously identified in screens for gene products involved in N-linked glycosylation, Quantification of the in vivo radiolabeled subunits and the radioiodinated purified enzyme shows that the yeast oligosaccharyltransferase is composed of equimolar amounts of eight subunits. Exposure of the immunoprecipitated oligosaccharyltransferase to mild protein denaturants yielded a subcomplex comprised of Stt3p, Ost3p, and Ost4p. These experiments, taken together with genetic and biochemical evidence for subunit interactions, suggest that the enzyme is composed of the following three subcomplexes: (a) Stt3p-Ost4p-Ost3p, (b) Swp1p-Wbp1p-Ost2p, and (c) Ost1p-Ost5p.
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页码:32513 / 32520
页数:8
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