Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
STAR Protoc. 2022 Apr 4;3(2):101274. doi: 10.1016/j.xpro.2022.101274. eCollection 2022 Jun 17.
Membrane proteins (MPs) are essential in many cellular functions. To maintain proteostasis, MPs are downregulated via ubiquitination and degradation. Here, we describe an optimized protocol to analyze MP degradation using quantitative western blot and flow cytometry-based approaches. We use the degradation of Ypq1, a vacuole membrane lysine transporter, to demonstrate the protocol, which can be adapted for other organelle MPs and thus provide useful tools to study MP regulation in yeast and other model organisms. For complete details on the use and execution of this protocol, please refer to Arines et al. (2021) and Yang et al. (2020).
膜蛋白（MPs）在许多细胞功能中至关重要。为了维持蛋白质的平衡，MPs 通过泛素化和降解进行下调。在这里，我们描述了一种使用定量 Western blot 和基于流式细胞术的方法分析 MP 降解的优化方案。我们使用液泡膜赖氨酸转运蛋白 Ypq1 的降解来演示该方案，该方案可以适应其他细胞器的 MPs，从而为研究酵母和其他模式生物中的 MP 调控提供有用的工具。有关该方案使用和执行的完整详细信息，请参阅 Arines 等人（2021 年）和 Yang 等人（2020 年）。
