Membrane topology of the Alg14 endoplasmic reticulum UDP-GlcNAc transferase subunit
Membrane topology of the Alg14 endoplasmic reticulum UDP-GlcNAc transferase subunit
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DOI：
10.1074/jbc.m704410200
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发表时间：
2007-10-05
影响因子：
4.8
通讯作者：
Dean, Neta
中科院分区：
文献类型：
--
作者：
Averbeck, Nicole;Keppler-Ross, Sabine;Dean, Neta
N-连接糖基化在内质网中开始，合成高度保守的多醇连接寡糖前体。催化该前体第二次糖添加的 UDP-GlcNAc 糖基转移酶存在于至少两个亚基 Alg14 和 Alg13 的大多数真核生物中。 Alg14 是一种膜蛋白，它将可溶性 Alg13 催化亚基从胞质溶胶募集到内质网 (ER) 膜的表面，并在此发生反应。在这里，我们研究了酿酒酵母 Alg14 的膜拓扑结构及其对 ER 膜缔合的要求。大多数算法预测 Alg14 包含一个或多个跨膜螺旋（跨膜域 (TMD)）。我们提供的证据表明 Alg14 包含 C 端胞质尾和位于 ER 腔内的 N 端。然而，我们还证明缺乏这种 TMD 的 Alg14 具有功能性，并且仍然与 ER 膜外周相关，这表明其他结构域可以介导 ER 关联。这些结论基于对 Alg13/Alg14 嵌合体的功能分析，该嵌合体包含在 Alg14 两端融合的 Alg13 或缺乏预测 TMD 的截短 Alg14 变体；完整 ER 膜中 Alg14 的蛋白酶保护测定；以及在高 pH 条件下提取含 Alg14 的 ER 膜。这些酵母 Alg13-Alg14 嵌合体概括了在某些原生动物中进化的 Alg13-Alg14 结构域排列的系统发育多样性。它们编码含有以任一方向与 Alg14 结构域融合的 Alg13 结构域的单个多肽，包括那些缺乏 Alg14 TMD 的多肽。因此，这种 Alg13-Alg14 UDP-GlcNAc 转移酶代表了通过裂变和融合进化的二分糖基转移酶的前所未有的例子。
N-linked glycosylation begins in the endoplasmic reticulum with the synthesis of a highly conserved dolichol-linked oligosaccharide precursor. The UDP-GlcNAc glycosyltransferase catalyzing the second sugar addition of this precursor consists in most eukaryotes of at least two subunits, Alg14 and Alg13. Alg14 is a membrane protein that recruits the soluble Alg13 catalytic subunit from the cytosol to the face of the endoplasmic reticulum ( ER) membrane where this reaction occurs. Here, we investigated the membrane topology of Saccharomyces cerevisiae Alg14 and its requirements for ER membrane association. Alg14 is predicted by most algorithms to contain one or more transmembrane spanning helices (transmembrane domains (TMDs)). We provide evidence that Alg14 contains a C-terminal cytosolic tail and an N terminus that resides within the ER lumen. However, we also demonstrate that Alg14 lacking this TMD is functional and remains peripherally associated with ER membranes, suggesting that additional domains can mediate ER association. These conclusions are based on the functional analysis of Alg13/Alg14 chimeras containing Alg13 fused at either end of Alg14 or truncated Alg14 variants lacking the predicted TMD; protease protection assays of Alg14 in intact ER membranes; and extraction of Alg14-containing ER membranes with high pH. These yeast Alg13-Alg14 chimeras recapitulate the phylogenetic diversity of Alg13-Alg14 domain arrangements that evolved in some protozoa. They encode single polypeptides containing an Alg13 domain fused to Alg14 domain in either orientation, including those lacking the Alg14 TMD. Thus, this Alg13-Alg14 UDP-GlcNAc transferase represents an unprecedented example of a bipartite glycosyltransferase that evolved by both fission and fusion.