Determining the Topology of an Integral Membrane Protein

Neil Green1, Hong Fang1, Kai‐Uwe Kalies2, Victor Canfield3

1 Vanderbilt University School of Medicine, Nashville, Tennessee, 2 Max Delbruck Center for Molecular Medicine, Berlin, 3 Pennsylvania State University College of Medicine, Hershey, Pennsylvania
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 5.2
DOI:  10.1002/0471143030.cb0502s00
Online Posting Date:  May, 2001
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A variety of methods have been developed for assigning the aqueous domains of integral membrane proteins to either side of a biological membrane. Once the sequence of a protein is known from its DNA sequence it is possible to study the topology of the protein. This unit provides protocols in which the water‐soluble domains can be tested for their accessibility to reagents added to membranes with a defined orientation. Tagging of hydrophilic regions of the protein with different epitopes and probing of their orientation with respect to the membrane is also described. Finally, a procedure for fusion of a reporter enzyme to truncated fragments of the protein is provided. The fusion protein is used as a sensor of sequence disposition relative to the membrane.

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Protease Digestion
  • Basic Protocol 2: Immunofluorescence Staining
  • Support Protocol 1: Epitope Tagging
  • Basic Protocol 3: Reporter Gene Fusions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Protease Digestion

  • Canine pancreatic microsomal membranes (see unit 11.4)
  • Magnesium/sucrose/BSA (MSB) buffer (see recipe)
  • 20% (w/v) Triton X‐100
  • 10 mg/ml proteinase K (see recipe)
  • 100% (w/v) trichloroacetic acid (TCA)(see recipe)
  • recipeSDS sample buffer( appendix 2A)
  • Antibodies directed against a series of peptides corresponding to specific hydrophilic regions of the target protein
  • Control antibodies directed against a luminal protein or a luminal domain of a membrane protein in the membrane system to be analyzed
  • Additional reagents and equipment for preparing canine pancreatic microsomes (unit 11.4), separating proteins by SDS‐PAGE (unit 6.1), and detecting proteins by immunoblotting (unit 6.2)

Basic Protocol 2: Immunofluorescence Staining

  • HEK 293 cells (ATCC #CRL 1573)
  • 4% (w/v) paraformaldehyde (see recipe)
  • Nonidet P‐40/goat serum/BSA (NGB) solution (see recipe)
  • Anti‐HA mouse monoclonal antibody 12CA5 (Boehringer Mannheim)
  • Rhodamine‐conjugated rabbit anti‐mouse immunoglobulin G (IgG)
  • DMEM/FBS/HEPES (DFH) solution (see recipe)
  • Fluoromount G mounting medium (Fisher)
  • 6‐well tissue culture plates
  • Glass coverslips, 22‐mm diameter
  • Additional reagents and equipment for immunofluorescence staining of fixed mammalian cells (unit 4.3), epifluorescence (unit 4.2) or confocal laser microscopy, and growing cultured mammalian cells (unit 1.1)

Support Protocol 1: Epitope Tagging

  • TE buffer ( appendix 2A)
  • Plasmid DNA encoding the protein of interest
  • E. coli cells to be transformed
  • Additional reagents and equipment for synthesizing oligonucleotides ( appendix 3A), ligating DNA ( appendix 3A), transforming E. coli( appendix 3A), isolating plasmid DNA from E. coli ( appendix 3A), identifying plasmids by restriction endonuclease digestion ( appendix 3A), and sequencing oligonucleotides ( appendix 3A)

Basic Protocol 3: Reporter Gene Fusions

  • Reporter plasmid (Fig. ): pA189invHD (available from Neil Green, Vanderbilt University)
  • S. cerevisia estrain FC2‐12B (MATα trp1‐1 leu2‐1 ura3‐52 his4‐401 HOL1‐1 can1‐1; available from Neil Green, Vanderbilt University)
  • SD +HIS agar plates (see recipe)
  • SD +HOL agar plates (see recipe)
  • Thermocycler
  • Additional reagents and equipment for the polymerase chain reaction (PCR; appendix 3A), agarose gel electrophoresis ( appendix 3A), restriction endonuclease digestion ( appendix 3A), and transformation of E. coli and S. cerevisiae ( appendix 3A).
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Literature Cited

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