Karaoglu D, Kelleher D J, Gilmore R
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0103, USA.
J Biol Chem. 1997 Dec 19;272(51):32513-20. doi: 10.1074/jbc.272.51.32513.
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-kDa 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.
寡糖基转移酶已从酿酒酵母中纯化出来，是一种由四个或六个亚基组成的异源寡聚复合物。在此，通过将一个表位编码序列连接到先前已鉴定的亚基基因OST3上，研究了寡糖基转移酶在体内的亚基组成和化学计量。与表位标记的Ost3p共免疫沉淀的七种多肽中的五种（Ost1p、Wbp1p、Swp1p、Ost2p和Ost5p）与通过传统纯化方法获得的多肽相同。另外两种共沉淀多肽，表观分子量分别为60 kDa和3.6 kDa，分别被鉴定为78 kDa的Stt3蛋白和36个残基的Ost4蛋白。Stt3p和Ost4p先前在参与N-连接糖基化的基因产物筛选中被鉴定出来。对体内放射性标记的亚基和放射性碘化的纯化酶进行定量分析表明，酵母寡糖基转移酶由等摩尔量的八个亚基组成。将免疫沉淀的寡糖基转移酶暴露于温和的蛋白质变性剂中，产生了一个由Stt3p、Ost3p和Ost4p组成的亚复合物。这些实验，连同亚基相互作用的遗传和生化证据，表明该酶由以下三个亚复合物组成：(a) Stt3p-Ost4p-Ost3p，(b) Swp1p-Wbp1p-Ost2p，和(c) Ost1p-Ost5p。
