In eukaryotes, the biosynthesis of a highly conserved dolichol-linked oligosaccharide (DLO) precursor Glc3Man9GlcNAc2-pyrophosphate-dolichol (PP-Dol) begins on the cytoplasmic face of the endoplasmic reticulum (ER) and ends within the lumen. Two functionally distinguished heteromeric glycosyltransferase (GTase) complexes are responsible for the cytosolic DLO assembly. Alg1, a β-1, 4 mannosyltransferase (MTase) physically interacts with Alg2 and Alg11 proteins to form the multienzyme complex which catalyzes the addition of all five mannose to generate the Man5GlcNAc2-PP-Dol intermediate. Despite the fact that Alg1 plays a central role in the formation of the multi-MTase has been confirmed, the topological information of Alg1 including the molecular mechanism of membrane association are still poorly understood. Using a combination of bioinformatics and biological approaches, we have undertaken a structural and functional study on Alg1 protein, in which the enzymatic activities of Alg1 and its variants were monitored by a complementation assay using the GALpr-ALG1 yeast strain, and further confirmed by a liquid chromatography–mass spectrometry-based in vitro quantitative assay. Computational and experimental evidence confirmed Alg1 shares structure similarity with Alg13/14 complex, which has been defined as a membrane-associated GT-B GTase. Particularly, we provide clear evidence that the N-terminal transmembrane domain including the following positively charged amino acids and an N-terminal amphiphilic-like α helix domain exposed on the protein surface strictly coordinate the Alg1 orientation on the ER membrane. This work provides detailed membrane topology of Alg1 and further reveals its biological importance at the spatial aspect in coordination of cytosolic DLO biosynthesis.

中文翻译：

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Alg1 beta-1,4甘露糖基转移酶的结构和功能分析揭示了其膜拓扑的生理重要性

在真核生物中，高度保守的与二元醇连接的寡糖（DLO）前体Glc3Man9GlcNAc2-焦磷酸二醇（PP-Dol）的生物合成始于内质网（ER）的细胞质表面，并在管腔内终止。两种功能上不同的异源糖基转移酶（GTase）复合物负责细胞溶质DLO组装。Alg1，一种β-1、4甘露糖基转移酶（MTase）与Alg2和Alg11蛋白发生物理相互作用，形成多酶复合物，该复合物催化所有五个甘露糖的添加，从而生成Man5GlcNAc2-PP-Dol中间体。尽管已经证实了Alg1在多MTase的形成中起着核心作用，但是仍然不清楚Alg1的拓扑信息，包括膜缔合的分子机制。GALpr - ALG1酵母菌株，并通过基于液相色谱-质谱的体外定量测定进一步证实。计算和实验证据证实，Alg1与Alg13 / 14复合物具有相似的结构，后者被定义为与膜相关的GT-B GTase。特别是，我们提供了明确的证据，包括以下带正电荷的氨基酸的N末端跨膜结构域和暴露于蛋白质表面的N末端两亲性α螺旋结构域严格协调了ER膜上的Alg1方向。这项工作提供了详细的Alg1膜拓扑结构，并进一步揭示了其在空间方面对协调胞质DLO生物合成的生物学重要性。