The oligosaccharyltransferase complex from Saccharomyces cerevisiae -: Isolation of the OST6 gene, its synthetic interaction with OST3, and analysis of the native complex
The oligosaccharyltransferase complex from Saccharomyces cerevisiae -: Isolation of the OST6 gene, its synthetic interaction with OST3, and analysis of the native complex
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DOI：
10.1074/jbc.274.24.17249
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发表时间：
1999-06-11
影响因子：
4.8
通讯作者：
Lehle, L
中科院分区：
文献类型：
--
作者：
Knauer, R;Lehle, L
蛋白质的N-糖基化是一个重要的、高度保守的蛋白质修饰过程，它是由异源寡聚蛋白复合物寡糖基转移酶（OST）催化的。来自酵母的具有酶活性的OST制剂显示由四个亚基（Ost 1 p、Wbp 1 p、Ost 3 p、Swp 1 p）或六个亚基（除了所列出的四个亚基之外的Ost 2 p和Ost 5 p）组成。遗传学研究已经公开了Stt 3 p和Ost 4p作为N-糖基化所需的额外蛋白。在这项研究中，我们报告了一个新的OST基因的鉴定和功能表征，命名为OST 6，该基因与OST 3具有同源性，特别是具有惊人相似的膜拓扑结构。这两种基因都不是酵母生长所必需的。OST 6或OST 3的破坏仅引起N-糖基化的微小缺陷，但Delta ost 3 Delta ost 6双突变体显示合成表型，导致体内可溶性和膜结合糖蛋白的严重糖基化不足，并导致体外OST活性降低。此外，这两个基因中的每一个也具有特定的功能，因为影响细胞壁生物发生的试剂在相应的无效突变体中显示不同的生长表型。通过蓝色天然电泳和免疫检测，鉴定了一个类似于240-kDa的复合物，由Ost 1 p、Stt 3 p、Wbp 1 p、Ost 3 p、Ost 6p、Swp 1 p、Ost 2 p和Ost 5 p组成，表明可能迄今为止鉴定的所有OST蛋白都是OST复合物的组分。因此，这些基因的功能似乎对于募集有效N-糖基化所必需的完全活性复合物是必不可少的，
The key step of N-glycosylation of proteins, an essential and highly conserved protein modification, is catalyzed by the hetero-oligomeric protein complex oligosaccharyltransferase (OST), So far, eight genes have been identified in Saccharomyces cerevisiae that are involved in this process. Enzymatically active OST preparations from yeast were shown to be composed of four (Ost1p, Wbp1p, Ost3p, Swp1p) or six subunits (Ost2p and Ost5p in addition to the four listed). Genetic studies have disclosed Stt3p and Ost4p as additional proteins needed for N-glycosylation, In this study we report the identification and functional characterization of a new OST gene, designated OST6, that has homology to OST3 and in particular a strikingly similar membrane topology. Neither gene is essential for growth of yeast. Disruption of OST6 or OST3 causes only a minor defect in N-glycosylation, but an Delta ost3 Delta ost6 double mutant displays a synthetic phenotype, leading to a severe underglycosylation of soluble and membrane-bound glycoproteins in vivo and to a reduced OST activity in vitro. Moreover, each of the two genes has also a specific func tion, since agents affecting cell wall biogenesis reveal different growth phenotypes in the respective null mutants. By blue native electrophoresis and immunodetection, a similar to 240-kDa complex was identified consisting of Ost1p, Stt3p, Wbp1p, Ost3p, Ost6p, Swp1p, Ost2p, and Ost5p, indicating that probably all so far identified OST proteins are constituents of the OST complex, It is also shown that disruption of OST3 and OST6 leads to a defect in the assembly of the complex. Hence, the function of these genes seems to be essential for recruiting a fully active complex necessary for efficient N-glycosylation,