Van Der Linden Inja M, Herrera Sara Abad, Montigny Cédric, Lenoir Guillaume, Pomorski Thomas Günther, Uzun Huriye D
Department of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Bochum, Germany.
Centre for Genomic Regulation (CGR), The Barcelona Institute of Science and Technology, Barcelona, Spain.
Bio Protoc. 2025 Jul 20;15(14):e5393. doi: 10.21769/BioProtoc.5393.
Flippases, a functionally distinct group of transmembrane proteins that flip lipids from the extracellular or luminal side to the cytosolic side of biological membranes, are key players in many important physiological processes, such as membrane trafficking and cellular signaling. To study the function of these membrane proteins under chemically defined conditions, reconstituting them into artificial vesicles is a crucial and effective approach. There are various methods for protein reconstitution involving different detergents and detergent removal techniques to integrate membrane proteins into artificial vesicles. In this protocol, we describe the reconstitution of the yeast flippase complex Drs2-Cdc50, which translocates phosphatidylserine across membranes of the trans-Golgi network at the expense of ATP hydrolysis. The flippase complex is incorporated into liposomes using a zwitterionic detergent, followed by detergent removal via dialysis-a gentle and effective strategy that helps preserve protein function. To evaluate the activity of the reconstituted flippase complex, two complementary assays are employed: (1) a fluorescence-based quenching assay to measure lipid transport; and (2) an ATPase assay using an ATP-regenerating system to measure ATP hydrolysis. Together, these methods provide a robust platform for analyzing the functional reconstitution of Drs2-Cdc50 in a defined membrane environment. Key features • Provides a gentle reconstitution approach via detergent removal by dialysis. • Measures ATPase activity using an NADH-coupled assay with an ATP-regenerating system. • Assesses lipid flippase activity with a sodium dithionite-based assay with fluorescent lipid derivatives. • Provides a well-defined experimental setup for direct characterization of lipid flippases.
翻转酶是一类功能独特的跨膜蛋白，可将脂质从生物膜的细胞外侧或腔侧翻转至胞质侧，是许多重要生理过程（如膜运输和细胞信号传导）的关键参与者。为了在化学定义的条件下研究这些膜蛋白的功能，将它们重组到人工囊泡中是一种关键且有效的方法。有多种蛋白质重组方法，涉及不同的去污剂和去污剂去除技术，以将膜蛋白整合到人工囊泡中。在本方案中，我们描述了酵母翻转酶复合物Drs2-Cdc50的重组，该复合物以ATP水解为代价将磷脂酰丝氨酸转运穿过反式高尔基体网络的膜。使用两性离子去污剂将翻转酶复合物掺入脂质体中，但随后通过透析去除去污剂——这是一种温和且有效的策略，有助于保留蛋白质功能。为了评估重组翻转酶复合物的活性，采用了两种互补的测定方法：（1）基于荧光的淬灭测定法来测量脂质转运；（2）使用ATP再生系统的ATP酶测定法来测量ATP水解。总之，这些方法为在确定的膜环境中分析Drs2-Cdc50的功能重组提供了一个强大的平台。关键特性 • 通过透析去除去污剂提供了一种温和的重组方法。 • 使用带有ATP再生系统的NADH偶联测定法测量ATP酶活性。 • 使用基于连二亚硫酸钠的荧光脂质衍生物测定法评估脂质翻转酶活性。 • 为直接表征脂质翻转酶提供了明确的实验设置。
