Spirig Urs, Bodmer Daniel, Wacker Michael, Burda Patricie, Aebi Markus
Institute of Microbiology, ETH Zurich, CH-8093 Zurich, Switzerland.
Glycobiology. 2005 Dec;15(12):1396-406. doi: 10.1093/glycob/cwj025. Epub 2005 Aug 11.
In the central reaction of N-linked glycosylation, the oligosaccharyltransferase (OTase) complex catalyzes the transfer of a lipid-linked core oligosaccharide onto asparagine residues of nascent polypeptide chains in the lumen of the endoplasmic reticulum (ER). The Saccharomyces cerevisiae OTase has been shown to consist of at least eight subunits. We analyzed this enzyme complex, applying the technique of blue native gel electrophoresis. Using available antibodies, six different subunits were detected in the wild-type (wt) complex, including Stt3p, Ost1p, Wbp1p, Swp1p, Ost3p, and Ost6p. We demonstrate that the small 3.4-kDa subunit Ost4p is required for the incorporation of either Ost3p or Ost6p into the complex, resulting in two, functionally distinct OTase complexes in vivo. Ost3p and Ost6p are not absolutely required for OTase activity, but modulate the affinity of the enzyme toward different protein substrates.
在N-连接糖基化的核心反应中，寡糖基转移酶（OTase）复合物催化内质网（ER）腔中脂质连接的核心寡糖转移到新生多肽链的天冬酰胺残基上。酿酒酵母OTase已被证明至少由八个亚基组成。我们应用蓝色非变性凝胶电泳技术分析了这种酶复合物。使用现有的抗体，在野生型（wt）复合物中检测到六个不同的亚基，包括Stt3p、Ost1p、Wbp1p、Swp1p、Ost3p和Ost6p。我们证明，3.4 kDa的小亚基Ost4p是将Ost3p或Ost6p整合到复合物中所必需的，这导致体内存在两种功能不同的OTase复合物。Ost3p和Ost6p对于OTase活性不是绝对必需的，但它们调节该酶对不同蛋白质底物的亲和力。
