Institute of Molecular Medicine, College of Future Technology, Peking University, Beijing 100871, China.
STAR Protoc. 2022 Apr 19;3(2):101333. doi: 10.1016/j.xpro.2022.101333. eCollection 2022 Jun 17.
Amphipathic phospholipids translocated by scramblases play a central role in facilitating lipid movement across the membrane bilayer, especially at the endoplasmic reticulum (ER) membranes. Here, we present a protocol for assessing the activity of the ER-localized lipid scramblase TMEM41B. We detail an fluorescent liposome-based phospholipid scrambling assay and metabolic labeling in living cells using alkyne-choline. The scramblase activity of other VTT (VMP1, TMEM41, and Tvp38) domain-containing proteins, such as TMEM41A and VMP1, can be assayed. For complete details on the use and execution of this protocol, please refer to Huang et al. (2021).
双性磷脂通过翻转酶易位在促进跨膜双层脂质运动中发挥核心作用，特别是在内质网（ER）膜中。在此，我们提供了一种评估 ER 定位的脂质翻转酶 TMEM41B 活性的方案。我们详细描述了基于荧光脂质体的磷脂翻转测定法，以及使用炔烃胆碱在活细胞中的代谢标记。其他含有 VTT（VMP1、TMEM41 和 Tvp38）结构域的蛋白，如 TMEM41A 和 VMP1 的翻转酶活性也可以进行测定。有关此方案使用和执行的完整详细信息，请参阅 Huang 等人（2021 年）。
