Department of Molecular Biology and Genetics, Aarhus University, Aarhus C 8000, Denmark.
Department of Molecular Biology and Genetics, Aarhus Institute of Advanced Studies, Aarhus University, Aarhus C 8000, Denmark.
Proc Natl Acad Sci U S A. 2024 Apr 9;121(15):e2315575121. doi: 10.1073/pnas.2315575121. Epub 2024 Apr 3.
The membrane protein Niemann-Pick type C1 (NPC1, named NCR1 in yeast) is central to sterol homeostasis in eukaryotes. NCR1 is localized to the vacuolar membrane, where it is suggested to carry sterols across the protective glycocalyx and deposit them into the vacuolar membrane. However, documentation of a vacuolar glycocalyx in fungi is lacking, and the mechanism for sterol translocation has remained unclear. Here, we provide evidence supporting the presence of a glycocalyx in isolated vacuoles and report four cryo-EM structures of NCR1 in two distinct conformations, named tense and relaxed. These two conformations illustrate the movement of sterols through a tunnel formed by the luminal domains, thus bypassing the barrier presented by the glycocalyx. Based on these structures and on comparison with other members of the Resistance-Nodulation-Division (RND) superfamily, we propose a transport model that links changes in the luminal domains with a cycle of protonation and deprotonation within the transmembrane region of the protein. Our model suggests that NPC proteins work by a generalized RND mechanism where the proton motive force drives conformational changes in the transmembrane domains that are allosterically coupled to luminal/extracellular domains to promote sterol transport.
膜蛋白尼曼-匹克 C1 型（NPC1，在酵母中称为 NCR1）是真核生物甾醇稳态的核心。NCR1 定位于液泡膜，据推测它在那里携带甾醇穿过保护性糖萼并将其沉积到液泡膜中。然而，真菌液泡糖萼的存在尚未得到证实，甾醇转运的机制仍不清楚。在这里，我们提供了支持在分离的液泡中存在糖萼的证据，并报告了 NPC1 的四个 cryo-EM 结构，处于两种不同的构象，分别称为紧张和放松。这两种构象说明了甾醇通过由腔域形成的隧道的运动，从而绕过了糖萼的屏障。基于这些结构以及与其他 Resistance-Nodulation-Division（RND）超家族成员的比较，我们提出了一个运输模型，该模型将腔域的变化与蛋白质跨膜区域内质子化和去质子化循环联系起来。我们的模型表明，NPC 蛋白通过广义的 RND 机制发挥作用，其中质子动力驱动跨膜结构域的构象变化，这些变化与腔域/细胞外域的变构偶联，以促进甾醇的运输。
