Specific transbilayer translocation of dolichol-linked oligosaccharides by an endoplasmic reticulum flippase
Specific transbilayer translocation of dolichol-linked oligosaccharides by an endoplasmic reticulum flippase
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DOI：
10.1073/pnas.0810225106
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发表时间：
2009-01-20
影响因子：
11.1
通讯作者：
Menon, Anant K.
中科院分区：
文献类型：
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作者：
Sanyal, Sumana;Menon, Anant K.
蛋白质 N-糖基化的寡糖供体 Glc(3)Man(9)GlcNAc(2)-PP-dolichol 通过多步途径合成，该途径起始于内质网 (ER) 的细胞质面，终止于发生糖基化反应的管腔。这需要脂质中间体 Man(5)GlcNAc(2)-PP-dolichol (M5-DLO) 跨内质网膜进行跨双层易位或翻转。除了需要特定的转运蛋白或翻转酶之外，M5-DLO 或任何其他脂质在 ER 上翻转的机制尚不清楚。我们最近证明了由去污剂溶解的内质网膜蛋白重构的蛋白脂质体中的 M5-DLO 翻转活性，并表明它不依赖于 ATP，并且需要在大约 4S 时沉淀的胰蛋白酶敏感蛋白。通过使用缺乏甘油磷脂翻转酶活性的活性富集部分，我们现在报告 M5-DLO 在重构系统中以 tau 时间常数 200 分钟快速翻转。大于 M5-DLO 的 DLO 易位也很差，tau 的长度范围从大约 10 分钟到 >200 分钟。我们得出的结论是：(i) DLO 聚糖中甘露糖的数量和排列对 DLO 被翻转酶易位的能力具有深远的影响，(ii) 聚糖大小本身并不决定 DLO 是否会被翻转，(iii) 翻转酶对 M5-DLO 具有高度特异性。我们的结果提出了 DLO 头基和翻转酶之间相互作用的简单结构模型。
The oligosaccharide donor for protein N-glycosylation, Glc(3)Man(9)GlcNAc(2)-PP-dolichol, is synthesized via a multistep pathway that starts on the cytoplasmic face of the endoplasmic reticulum (ER) and ends in the lumen where the glycosylation reaction occurs. This necessitates transbilayer translocation or flipping of the lipid intermediate Man(5)GlcNAc(2)-PP-dolichol (M5-DLO) across the ER membrane. The mechanism by which M5-DLO-or any other lipid-is flipped across the ER is unknown, except that specific transport proteins or flippases are required. We recently demonstrated M5-DLO flipping activity in proteoliposomes reconstituted from detergent-solubilized ER membrane proteins and showed that it was ATP-independent and required a trypsin-sensitive protein that sedimented at approximately 4S. By using an activity-enriched fraction devoid of glycerophospholipid flippase activity, we now report that M5-DLO is rapidly flipped in the reconstituted system with a time constant tau 200 min. DLOs larger than M5-DLO are also poorly translocated, with tau ranging from approximately 10 min to >200 min. We conclude that (i) the number and arrangement of mannoses in the DLO glycan has a profound effect on the ability of the DLO to be translocated by the flippase, (ii) glycan size per se does not dictate whether a DLO will be flipped, and (iii) the flippase is highly specific for M5-DLO. Our results suggest a simple structural model for the interaction between the DLO head group and the flippase.