Unit

UNIT 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening Applications

  1. Christian Wunder1,2,
  2. Jennifer Lippincott-Schwartz1,
  3. Holger Lorenz3

Published Online: 1 DEC 2010

DOI: 10.1002/0471143030.cb0507s49

Current Protocols in Cell Biology

Current Protocols in Cell Biology

How to Cite

Wunder, C., Lippincott-Schwartz, J. and Lorenz, H. 2010. Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening Applications. Current Protocols in Cell Biology. 49:5.7:5.7.1–5.7.12.

Author Information

  1. 1

    National Institute of Child Health and Human Development, National Institutes of Health, Bethesda Maryland

  2. 2

    Institut Curie, Centre de Recherche, Traffic, Signaling and Delivery Group; CNRS UMR144, Paris, France

  3. 3

    Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Heidelberg, Germany

Publication History

  1. Published Online: 1 DEC 2010
  2. Published Print: DEC 2010

Abstract

Correct localization and topology are crucial for a protein's cellular function. To determine topologies of membrane proteins, a new technique, called fluorescence protease protection (FPP) assay, has recently been established. The sole requirements for FPP are the expression of fluorescent-protein fusion proteins and the selective permeabilization of the plasma membrane, permitting a wide range of cell types and organelles to be investigated. Proteins topologies in organelles like endoplasmic reticulum, Golgi apparatus, mitochondria, peroxisomes, and autophagosomes have already been determined by FPP. Here, two different step-by-step protocols of the FPP assay are provided. First, we describe the FPP assay using fluorescence microscopy for single adherent cells, and second, we outline the FPP assay for high-throughput screening applications. Curr. Protoc. Cell Biol. 49:5.7.1-5.7.12. © 2010 by John Wiley & Sons, Inc.

Keywords:

  • protein topology;
  • fluorescence microscopy;
  • high-throughput screening