Biosynthesis of lipid-linked oligosaccharides in Saccharomyces cerevisiae -: Alg13p AND Alg14p form a complex required for the formation of GlcNAc2-PP-dolichol
Biosynthesis of lipid-linked oligosaccharides in Saccharomyces cerevisiae -: Alg13p AND Alg14p form a complex required for the formation of GlcNAc2-PP-dolichol
复制标题
DOI：
10.1074/jbc.m506358200
复制
发表时间：
2005-10-14
影响因子：
4.8
通讯作者：
Jakob, CA
中科院分区：
文献类型：
--
作者：
Bickel, T;Lehle, L;Jakob, CA
内质网中的N-糖基化是一种重要的蛋白质修饰，在从酵母到人类的进化过程中高度保守。在这里，我们确定和表征了两个重要的酵母蛋白具有同源性的细菌糖基转移酶，指定Alg 13 p和Alg 14 p，作为所需的形成Glc-NAc 2-PP-dolichol（Dol），在生物合成的第二步独特的脂质连接的核心寡糖。每个基因的下调导致蛋白质N-糖基化缺陷和体内GlcNAc（1）-PP-Dol的积累，如用[H-3]葡糖胺代谢标记所揭示的。来自抑制ALG 13或ALG 14的细胞的微粒体膜及其去污剂溶解的提取物不能催化N-乙酰葡糖胺从UDP-GlcNAc转移至[C-14] GlcNAc（1）-PP-Dol，但不损害GlcNAc（1）-PP-Dol或GlcNAc-GPI的形成。从溶解的提取物中免疫沉淀Alg 13 p导致Glc-NAc 2-PP-Dol的形成，但需要Alg 14 p的活性，因为从ALG 14下调的细胞中获得的Alg 13 p免疫沉淀物缺乏这种活性。在Western印迹分析中，它表明，Alg 13 p，其中没有明确的跨膜段已被预测，定位于膜和胞质溶胶;后者的形式，然而，是酶失活。相反，Alg 14 p仅与膜结合。ALG 14基因的抑制导致膜上Alg 13 p的耗竭。通过使用Alg 14-ZZ作为诱饵的IgG-Sepharose亲和层析，我们证明Alg 13-myc与Alg 14-ZZ共分级。这些数据表明，Alg 13 p与Alg 14 p结合形成复合物，形成催化GlcNAc（2）-PP-Dol生物合成的活性转移酶。
N-Glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to man. Here we identify and characterize two essential yeast proteins having homology to bacterial glycosyltransferases, designated Alg13p and Alg14p, as being required for the formation of Glc-NAc2-PP-dolichol ( Dol), the second step in the biosynthesis of the unique lipid-linked core oligosaccharide. Down-regulation of each gene led to a defect in protein N-glycosylation and an accumulation of GlcNAc(1)-PP-Dol in vivo as revealed by metabolic labeling with [H-3] glucosamine. Microsomal membranes from cells repressed for ALG13 or ALG14, as well as detergent-solubilized extracts thereof, were unable to catalyze the transfer of N-acetylglucosamine from UDP-GlcNAc to [C-14] GlcNAc(1)-PP-Dol, but did not impair the formation of GlcNAc(1)-PP-Dol or GlcNAc-GPI. Immunoprecipitating Alg13p from solubilized extracts resulted in the formation of Glc-NAc2-PP-Dol but required Alg14p for activity, because an Alg13p immunoprecipitate obtained from cells in which ALG14 was downregulated lacked this activity. In Western blot analysis it was demonstrated that Alg13p, for which no well defined transmembrane segment has been predicted, localizes both to the membrane and cytosol; the latter form, however, is enzymatically inactive. In contrast, Alg14p is exclusively membrane-bound. Repression of the ALG14 gene causes a depletion of Alg13p from the membrane. By affinity chromatography on IgG-Sepharose using Alg14-ZZ as bait, we demonstrate that Alg13-myc co-fractionates with Alg14-ZZ. The data suggest that Alg13p associates with Alg14p to a complex forming the active transferase catalyzing the biosynthesis of GlcNAc(2)-PP-Dol.