Protocol to analyze lipid asymmetry in the plasma membrane

Yugo Miyata; Katsumori Segawa

doi:10.1016/j.xpro.2022.101870

Protocol to analyze lipid asymmetry in the plasma membrane

STAR Protoc. 2022 Dec 16;3(4):101870. doi: 10.1016/j.xpro.2022.101870. Epub 2022 Nov 21.

Authors

Yugo Miyata¹, Katsumori Segawa²

Affiliations

¹ Department of Medical Chemistry, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
² Department of Medical Chemistry, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Electronic address: segawa.mche@tmd.ac.jp.

Abstract

The plasma membrane containing cholesterol exhibits phospholipid asymmetry, with phosphatidylcholine and sphingomyelin enriched in its outer leaflet and phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) on the cytoplasmic side. We herein describe steps for bacterial expression of recombinant proteins that bind to membrane lipids, followed by affinity purification. Using fluorescence-labeled phospholipid analogs, we further detail the assay to detect flippase activity, which maintains the single-sided distribution of PtdSer and PtdEtn, in mammalian cells. For complete details on the use and execution of this protocol, please refer to Segawa et al. (2021).¹.

Keywords: Cell Biology; Cell Membrane; Flow Cytometry/Mass Cytometry; Molecular Biology; Protein expression and purification.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Biological Transport
Cell Membrane / metabolism
Mammals / metabolism
Phosphatidylcholines* / metabolism
Phospholipids* / metabolism

Substances

Phospholipids
Phosphatidylcholines