Monitoring Non-vesicular Transport of Phosphatidylserine and Phosphatidylinositol 4-Phosphate in Intact Cells by BRET Analysis
| Autor: | Daniel J Toth, Mira Sohn, Tamas Balla |
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| Rok vydání: | 2019 |
| Předmět: |
0303 health sciences
education.field_of_study Phosphatidylinositol 4-phosphate 030302 biochemistry & molecular biology Population Biological membrane Pleckstrin homology domain Vesicular transport protein 03 medical and health sciences chemistry.chemical_compound chemistry Biophysics lipids (amino acids peptides and proteins) Phosphatidylinositol education Plant lipid transfer proteins Lipid Transport 030304 developmental biology |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493991358 |
| DOI: | 10.1007/978-1-4939-9136-5_2 |
| Popis: | Non-vesicular lipid transport via lipid transfer proteins (LTPs) at membrane contact sites (MCSs) is critical for the maintenance of the lipid composition of biological membranes. The ability to measure lipid transfer activity of diverse LTPs in live cells without interrupting the fine structural organization is essential to better understand the contribution of non-vesicular lipid transport to membrane organization. Here, we describe a semiquantitative method to analyze phosphatidylinositol 4-phosphate (PI4P) and phosphatidylserine (PS) changes at the plasma membrane (PM) as they relate to LTP functions. This live cell method is based on bioluminescence resonance energy transfer (BRET) measurements between a luciferase-tagged lipid-recognizing module and a PM-targeted fluorescent acceptor. Oxysterol-binding protein-related protein (ORP) 5 is a PI4P/PS lipid transfer protein which is stably tethered to the ER and also dynamically interacts with PM PI4P/PI(4,5)P2 via its pleckstrin homology (PH) domain. We show that this method is able to detect PI4P and PS changes in the PM upon acute recruitment of an ORP5 construct to the PM. This method is convenient and robust, utilizing population of cells in 96-well plates analyzed in a plate reader. We will also highlight potential further applications extending the method for other LTPs and other lipid cargoes. |
| Databáze: | OpenAIRE |
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