https://ror.org/01aj84f44 Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
https://ror.org/03yrrjy16 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Life Sci Alliance. 2024 Oct 25;8(1). doi: 10.26508/lsa.202402990. Print 2025 Jan.
In eukaryotes, integration of sterols into the vacuolar/lysosomal membrane is critically dependent on the Niemann-Pick type C (NPC) system. The system consists of an integral membrane protein, called NCR1 in yeast, and NPC2, a luminal soluble protein that transfers sterols to the N-terminal domain (NTD) of NCR1 before membrane integration. Both proteins have been implicated in sterol homeostasis of yeast and humans. Here, we investigate sterol and lipid binding of the NCR1/NPC2 transport system and determine crystal structures of the sterol binding NTD. The NTD binds both ergosterol and cholesterol, with nearly identical conformations of the binding pocket. Apart from sterols, the NTD can also bind fluorescent analogs of phosphatidylinositol, phosphatidylcholine, and phosphatidylserine, as well as sphingosine and ceramide. We confirm the multi-lipid scope of the NCR1/NPC2 system using photo-crosslinkable and clickable lipid analogs, namely, pac-cholesterol, pac-sphingosine, and pac-ceramide. Finally, we reconstitute the transfer of pac-sphingosine from NPC2 to the NTD in vitro. Collectively, our results support that the yeast NPC system can work as versatile machinery for vacuolar homeostasis of structurally diverse lipids, besides ergosterol.
在真核生物中，固醇整合到液泡/溶酶体膜中严重依赖于尼曼-匹克 C 型（NPC）系统。该系统由一个整合膜蛋白组成，在酵母中称为 NCR1，以及 NPC2，一种腔可溶性蛋白，在膜整合之前将固醇转移到 NCR1 的 N 端结构域（NTD）。这两种蛋白都与酵母和人类的固醇稳态有关。在这里，我们研究了 NCR1/NPC2 运输系统的固醇和脂质结合，并确定了固醇结合 NTD 的晶体结构。NTD 结合麦角固醇和胆固醇，结合口袋的构象几乎相同。除了固醇之外，NTD 还可以结合磷脂酰肌醇、磷脂酰胆碱和磷脂酰丝氨酸的荧光类似物，以及神经酰胺和鞘氨醇。我们使用光交联和点击化学脂质类似物，即 pac-胆固醇、pac-神经酰胺和 pac-鞘氨醇，证实了 NCR1/NPC2 系统的多脂质范围。最后，我们在体外重新构建了 NPC2 到 NTD 的 pac-神经酰胺转移。总的来说，我们的结果支持酵母 NPC 系统可以作为结构多样的脂质，除了麦角固醇外，液泡稳态的多功能机制。
