A Fluorescence-Based Flippase Assay to Monitor Lipid Transport by Drs2-Cdc50
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 funct...
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Bio-protocol LLC
2025-07-01
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| author | Inja Van Der Linden Sara Herrera Cédric Montigny Guillaume Lenoir Thomas Pomorski Huriye Uzun |
| author_facet | Inja Van Der Linden Sara Herrera Cédric Montigny Guillaume Lenoir Thomas Pomorski Huriye Uzun |
| author_sort | Inja Van Der Linden |
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| description | 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. |
| format | Article |
| id | doaj-art-04b5e3d6f5e9402e9cd219835b95ea41 |
| institution | Kabale University |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Bio-protocol LLC |
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| spelling | doaj-art-04b5e3d6f5e9402e9cd219835b95ea412025-08-20T03:34:36ZengBio-protocol LLCBio-Protocol2331-83252025-07-01151410.21769/BioProtoc.5393A Fluorescence-Based Flippase Assay to Monitor Lipid Transport by Drs2-Cdc50Inja Van Der Linden0Sara Herrera1Cédric Montigny2Guillaume Lenoir3Thomas Pomorski4Huriye Uzun5Department of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Bochum, GermanyDepartment of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Bochum, GermanyCentre for Genomic Regulation (CGR), The Barcelona Institute of Science and Technology, Barcelona, SpainUniversité Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell, Gif-sur-Yvette, FranceUniversité Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell, Gif-sur-Yvette, FranceDepartment of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Bochum, GermanyDepartment of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, DenmarkDepartment of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Bochum, GermanyDepartment of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, DenmarkFlippases, 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.https://bio-protocol.org/en/bpdetail?id=5393&type=0 |
| spellingShingle | Inja Van Der Linden Sara Herrera Cédric Montigny Guillaume Lenoir Thomas Pomorski Huriye Uzun A Fluorescence-Based Flippase Assay to Monitor Lipid Transport by Drs2-Cdc50 Bio-Protocol |
| title | A Fluorescence-Based Flippase Assay to Monitor Lipid Transport by Drs2-Cdc50 |
| title_full | A Fluorescence-Based Flippase Assay to Monitor Lipid Transport by Drs2-Cdc50 |
| title_fullStr | A Fluorescence-Based Flippase Assay to Monitor Lipid Transport by Drs2-Cdc50 |
| title_full_unstemmed | A Fluorescence-Based Flippase Assay to Monitor Lipid Transport by Drs2-Cdc50 |
| title_short | A Fluorescence-Based Flippase Assay to Monitor Lipid Transport by Drs2-Cdc50 |
| title_sort | fluorescence based flippase assay to monitor lipid transport by drs2 cdc50 |
| url | https://bio-protocol.org/en/bpdetail?id=5393&type=0 |
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