Functional characterization of Ost3p. Loss of the 34-kD subunit of the Saccharomyces cerevisiae oligosaccharyltransferase results in biased underglycosylation of acceptor substrates.
Functional characterization of Ost3p. Loss of the 34-kD subunit of the Saccharomyces cerevisiae oligosaccharyltransferase results in biased underglycosylation of acceptor substrates.
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DOI：
10.1083/jcb.130.3.567
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发表时间：
1995-08
期刊：
影响因子：
--
通讯作者：
Gilmore R
中科院分区：
文献类型：
--
作者：
Karaoglu D;Kelleher DJ;Gilmore R
在粗面内质网腔内，寡糖基转移酶催化高甘露糖寡糖部分从脂质连接的寡糖供体整体转移到新生多肽中的天冬酰胺受体位点。将酿酒酵母寡糖基转移酶纯化为由表观分子量为64 kD（Ost 1 p）、45 kD（Wbp 1 p）、34 kD、30 kD（Swp 1 p）、16 kD和9 kD的六个亚基（α-ζ）组成的异低聚体复合物。在这里，我们报告的Ost 3 p的结构和功能特性，它对应于34 kD的γ-亚基的寡糖基转移酶。与Ost 1 p、Wbp 1 p和Swp 1 p不同，Ost 3 p的表达对于酵母的生存力不是必需的。相反，ost 3无效突变酵母在固体或液体培养基中以野生型速率生长，而与培养温度无关。尽管如此，在体外寡糖基转移酶测定中，从ost 3无效突变体膜制备的去污剂提取物的活性比从野生型膜制备的提取物低两倍。此外，Ost 3 p的丧失伴随着体内可溶性和膜结合糖蛋白的显著糖基化不足。与先前表征的寡糖基转移酶中的ost 1 -1突变体和寡糖组装途径中的alg 5突变体相比，ost 3无效突变酵母似乎在几种膜糖蛋白的糖基化中选择性受损。后一种观察结果表明，Ost 3 p可以增强体内寡糖转移到受体底物的子集。
Within the lumen of the rough endoplasmic reticulum, oligosaccharyltransferase catalyzes the en bloc transfer of a high mannose oligosaccharide moiety from the lipid-linked oligosaccharide donor to asparagine acceptor sites in nascent polypeptides. The Saccharomyces cerevisiae oligosaccharyltransferase was purified as a heteroligomeric complex consisting of six subunits (alpha-zeta) having apparent molecular masses of 64 kD (Ost1p), 45 kD (Wbp1p), 34 kD, 30 kD (Swp1p), 16 kD, and 9 kD. Here we report a structural and functional characterization of Ost3p which corresponds to the 34-kD gamma-subunit of the oligosaccharyltransferase. Unlike Ost1p, Wbp1p, and Swp1p, expression of Ost3p is not essential for viability of yeast. Instead, ost3 null mutant yeast grow at wild-type rates on solid or in liquid media irrespective of culture temperature. Nonetheless, detergent extracts prepared from ost3 null mutant membranes are twofold less active than extracts prepared from wild-type membranes in an in vitro oligosaccharyltransferase assay. Furthermore, loss of Ost3p is accompanied by significant underglycosylation of soluble and membrane- bound glycoproteins in vivo. Compared to the previously characterized ost1-1 mutant in the oligosaccharyltransferase, and the alg5 mutant in the oligosaccharide assembly pathway, ost3 null mutant yeast appear to be selectively impaired in the glycosylation of several membrane glycoproteins. The latter observation suggests that Ost3p may enhance oligosaccharide transfer in vivo to a subset of acceptor substrates.