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Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)
Published on: April 20, 2015
Determining membrane protein topologies in single cells and high-throughput screening applications
Christian Wunder1, Jennifer Lippincott-Schwartz, Holger Lorenz
1National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA.
Current Protocols in Cell Biology
|December 15, 2010
Summary
The fluorescence protease protection (FPP) assay determines membrane protein topology, a key factor in protein function. This study provides two detailed protocols for FPP application in single cells and high-throughput screening.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Protein localization and topology are critical for cellular function.
- Determining membrane protein topology is essential for understanding protein function.
- Existing methods for topology determination can be limited in scope.
Purpose of the Study:
- To introduce and detail the fluorescence protease protection (FPP) assay for determining membrane protein topology.
- To provide practical, step-by-step protocols for implementing the FPP assay.
- To adapt the FPP assay for both single-cell analysis and high-throughput screening.
Main Methods:
- The FPP assay relies on expressing fluorescent-protein fusion proteins.
- Selective permeabilization of the plasma membrane is a key requirement.
- The assay can be applied to a wide range of cell types and organelles.
Main Results:
- The FPP assay has been successfully used to determine protein topologies in various organelles, including the endoplasmic reticulum, Golgi apparatus, mitochondria, peroxisomes, and autophagosomes.
- Two distinct protocols for the FPP assay are presented: one for fluorescence microscopy of single adherent cells and another for high-throughput screening.
- The assay is versatile and applicable across diverse cellular contexts.
Conclusions:
- The FPP assay is a powerful and versatile technique for determining membrane protein topology.
- The provided protocols facilitate the application of FPP in both detailed single-cell studies and large-scale screening efforts.
- Accurate protein topology determination using FPP aids in understanding protein function and cellular processes.
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