Determining Membrane Protein Topologies in Single Cells and High‐Throughput Screening Applications

Christian Wunder1, Jennifer Lippincott‐Schwartz2, Holger Lorenz3

1 Institut Curie, Centre de Recherche, Traffic, Signaling and Delivery Group; CNRS UMR144, Paris, France, 2 National Institute of Child Health and Human Development, National Institutes of Health, Bethesda Maryland, 3 Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Heidelberg, Germany
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 5.7
DOI:  10.1002/0471143030.cb0507s49
Online Posting Date:  December, 2010
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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

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: FPP Assay for Fluorescence Microscopes
  • Support Protocol 1: Establish Conditions for Optimal Plasma Membrane Permeabilization and Destruction of FPs
  • Basic Protocol 2: FPP Assay for High‐Throughput Screening (HTS)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: FPP Assay for Fluorescence Microscopes

  Materials
  • Adherent cells (e.g., HeLa, COS‐7, NRK, HEK293) to be transfected with a soluble FP and/or a FP fusion plasmid
  • Cell culture medium for cells of interest (e.g., RPMI 1640 or DMEM, supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin)
  • FP expression plasmid (e.g., Clontech) encoding the fluorescent protein‐of‐interest
  • Transfection reagent (e.g., FuGENE 6; Roche)
  • FP (soluble) expression plasmid, e.g., mCFP, EGFP (Clontech)
  • KHM buffer (see recipe) or cell culture medium without FBS and without phenol red
  • Protease solution (see recipe)
  • Digitonin (see recipe)
  • Sterile tissue culture hood
  • 8‐well Lab‐Tek chamber slide (Nalge Nunc, cat. no. 155411)
  • Inverted fluorescence microscope (either widefield or confocal), capable of time lapse recording
  • Additional reagents and equipment for transfection of cells (unit 20.6)

Support Protocol 1: Establish Conditions for Optimal Plasma Membrane Permeabilization and Destruction of FPs

  • FP (soluble) expression plasmid, e.g., mCFP, EGFP (Clontech)
  • Membrane‐anchored, but cytosol‐facing FP, e.g., Caveolin 1‐YFP (Tagawa et al., ) or VSVG‐YFP (Presley et al., )
NOTE: Soluble‐ and membrane‐anchored FPs must have non‐overlapping spectral properties.

Basic Protocol 2: FPP Assay for High‐Throughput Screening (HTS)

  Materials
  • Adherent cells to be transfected with a soluble FP and/or a FP fusion plasmid
  • Transfection reagent (e.g., FuGENE 6; Roche)
  • Cell culture medium for cells of interest (e.g., RPMI 1640 or DMEM, supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin)
  • FP (soluble) expression plasmid, e.g., mCherry (Clontech)
  • FP expression plasmid containing DNA encoding the protein‐of‐interest
  • 0.05% (w/v) trypsin/0.53 mM EDTA (Invitrogen)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS)
  • KHM buffer (see recipe) or cell culture medium without FBS and without phenol red
  • Digitonin (see recipe)
  • Protease solution (see recipe)
  • Sterile tissue culture hood
  • 10‐cm cell culture dish
  • Hemacytometer
  • 96‐well tissue culture plates (Special Optics Low Fluorescence Assay Plates; Sigma‐Aldrich: Corning CLS3720)
  • Microplate fluorescence reader [e.g., from Varian (Cary Eclipse), BioTek, Tecan)]
  • Microplate washer and dispenser (e.g., EL406, BioTek; multichannel pipets may be used for initial trial experiments)
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Figures

Videos

Literature Cited

Literature Cited
   Janski, A.M. and Cornell, N.W. 1980. Subcellular distribution of enzymes determined by rapid digitonin fractionation of isolated hepatocytes. Biochem. J. 186:423‐429.
   Kuznetsov, A.V., Veksler, V., Gellerich, F.N., Saks, V., Margreiter, R., and Kunz, W.S. 2008. Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat. Protoc. 3:965‐976.
   Liscum, L. and Munn, N.J. 1999. Intracellular cholesterol transport. Biochim. Biophys. Acta 1438:19‐37.
   Lorenz, H., Hailey, D.W., and Lippincott‐Schwartz, J. 2006. Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization. Nat. Methods 3:205‐210.
   Plutner, H., Davidson, H.W., Saraste, J., and Balch, W.E. 1992. Morphological analysis of protein transport from the ER to Golgi membranes in digitonin‐permeabilized cells: Role of the P58 containing compartment. J. Cell Biol. 119:1097‐1116.
   Presley, J.F., Cole, N.B., Schroer, T.A., Hirschberg, K., Zaal, K.J., and Lippincott‐Schwartz, J. 1997. ER‐to‐Golgi transport visualized in living cells. Nature 389:81‐85.
   Tagawa, A., Mezzacasa, A., Hayer, A., Longatti, A., Pelkmans, L., and Helenius, A. 2005. Assembly and trafficking of caveolar domains in the cell: Caveolae as stable, cargo‐triggered, vesicular transporters. J. Cell Biol. 170:769‐779.
   Takagi, S., Otsuka, H., Akiyama, T., and Sankawa, U. 1982. Digitonin‐cholesterol complex formation—effects of varying the length of the side chain. Chem. Pharm. Bull. 30:3485‐3492.
   Wilson, R., Allen, A.J., Oliver, J., Brookman, J.L., High, S., and Bulleid, N.J. 1995. The translocation, folding, assembly and redox‐dependent degradation of secretory and membrane proteins in semi‐permeabilized mammalian cells. Biochem. J. 307:679‐687.
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