Detecting Membrane Protein‐protein Interactions Using the Mammalian Membrane Two‐hybrid (MaMTH) Assay

Punit Saraon1, Ingrid Grozavu2, Sang Hyun Lim2, Jamie Snider1, Zhong Yao1, Igor Stagljar3

1 Donnelly Centre, University of Toronto, Toronto, Ontario, 2 Department of Biochemistry, University of Toronto, Toronto, Ontario, 3 Department of Molecular Genetics, University of Toronto, Toronto, Ontario
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.15
Online Posting Date:  March, 2017
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Abstract

Protein‐protein interactions (PPIs) play an integral role in numerous cellular processes. Membrane protein interactions, in particular, are critical in cellular responses to stresses and stimuli, with dysfunction of these PPIs (e.g., due to aberrant expression and/or mutation of interaction partners) leading to a diverse array of pathological states. Exploration of the interaction space and dynamics of membrane proteins is difficult due to the limitations of current techniques used to study proteins in the biochemically complex environment of biological membranes. In the protocols below, we describe a newly developed membrane protein interaction assay called the Mammalian‐Membrane Two‐Hybrid (MaMTH), designed specifically for the detection of integral membrane PPIs in the context of living mammalian cells. Prior to using MaMTH, cell lines of interest are genetically modified to encode a reporter of choice. MaMTH “bait” and “prey” constructs of interest are also generated using Gateway cloning technology. The assay is then performed by co‐transfection of baits and preys, with bait‐prey interaction quantifiably assessed by way of a reporter signal (e.g., light (luciferase), fluorescence (GFP). © 2017 by John Wiley & Sons, Inc.

Keywords: membrane proteomics; mammalian‐membrane two‐hybrid; MaMTH; protein‐protein interactions; interactome

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Cell Culture and MaMTH Firefly Luciferase Assays
  • Support Protocol 1: Generation of MaMTH‐Modified Reporter Cells
  • Support Protocol 2: Generation of MaMTH Bait and Prey Constructs
  • Support Protocol 3: Immunoblot Analysis for Bait and Prey Expression
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Cell Culture and MaMTH Firefly Luciferase Assays

  Materials
  • MaMTH‐modified HEK293T cells (see protocol 2)
  • Phosphate‐buffered saline (PBS; Gibco)
  • Cell culture trypsin or TrypLE (Gibco)
  • Dulbecco's modified Eagle medium (DMEM; Gibco)
  • Fetal bovine serum (FBS; Gibco)
  • 100× penicillin and streptomycin solution (Gibco)
  • 2× BES [N,N‐bis(2‐hydroxyethyl)‐2‐aminoethanesulfonic acid] buffer (see recipe)
  • 2.5 M calcium chloride (CaCl 2)
  • 5× Reporter Lysis buffer (Promega, cat. no. E397A; requires freezing)
  • 5× Cell Culture Lysis reagent (Promega, cat. no. E153A)
  • Complete protease inhibitor cocktail tablet (Roche), optional
  • Luciferase substrate (see recipe)
  • Cell culture plates (VWR)
  • 37°C, 5% CO 2 incubator
  • Vortex mixer
  • Luminometer
NOTE: The steps below are described for a 96‐well plate format. If using another plate size adjust volumes as required (see Table 15.0.1 for examples).

Support Protocol 1: Generation of MaMTH‐Modified Reporter Cells

  Materials
  • HEK293T cells, passage <10 (or any other cells to be modified)
  • Dulbecco's modified Eagle medium (DMEM; Gibco)
  • Fetal bovine serum (FBS; Invitrogen)
  • Lentiviral reporter constructs containing 8× lexAops‐reporter (luciferase or GFP) or 5 × GAL4UAS‐reporter (luciferase or GFP) in pLD‐Gateway‐Puro‐NVF vector (Available from the Stagljar lab)
  • psPAX2 and pMD2 lentiviral packaging plasmids (both available from Addgene)
  • X‐tremeGene 9 transfection reagent (Roche)
  • Opti‐MEM serum‐reduced medium (Gibco)
  • Bovine serum albumin (BSA; BioShop Canada)
  • Phosphate‐buffered saline (PBS; Invitrogen)
  • Hygromycin (Thermo Fisher Scientific)
  • Plasmid construct expressing GAL4 and LexA transcription factors
  • 6‐cm tissue culture plate
  • 37°C, 5% CO 2 incubator
  • 96‐, 24‐, and 6‐well plates

Support Protocol 2: Generation of MaMTH Bait and Prey Constructs

  Materials
  • 10× Tris‐EDTA (TE) buffer (100 mM Tris·Cl pH 8.0, 10 mM EDTA pH 8.0, sterile filtered; Teknova)
  • cDNA for gene of interest
  • BP Clonase enzyme (Thermo Fisher Scientific)
  • pDONR223 entry clone vector (Addgene)
  • Proteinase K (20 mg/ml, Thermo Fisher Scientific)
  • E. coli DH5alpha competent bacterial cells
  • Ice
  • Lennox broth (LB) medium (20 g/liter; BioShop Canada)
  • M13 forward primer (for entry clones)
  • LR clonase II enzyme (Thermo Fisher Scientific)
  • 50 ng destination vector (specially designed MaMTH bait or prey constructs available from the Stagljar lab)
  • Lennox broth (LB) medium (20 g/liter) mixed with 100 mg spectinomycin per 1 liter of LB (BioShop Canada)
  • Lennox broth (LB) medium (20 g/L) mixed with 100 mg of ampicillin per 1 liter of LB (BioShop Canada)
  • Lennox broth (LB) agar plates (20 g/L of each) with 100 mg of ampicillin per 1 liter of LB‐agar (BioShop Canada)
  • 200‐μl PCR tubes (BIOplastics BV)
  • Vortex mixer 1.5‐ml microcentrifuge tubes
  • 15‐ml culture tubes (VWR International)

Support Protocol 3: Immunoblot Analysis for Bait and Prey Expression

  Materials
  • Loading buffer (see recipe)
  • 4% to 12% Criterion XT Bis‐Tris Gel (15‐well; Bio‐Rad)
  • Running buffer (see recipe)
  • Tris/glycine transfer buffer (see recipe)
  • Ponceau stain (see recipe)
  • Bovine serum albumin (BSA)
  • Tween‐20 (Sigma‐Aldrich)
  • Phosphate‐buffered saline (PBS; Gibco)
  • Anti‐V5 antibody for bait expression (Cell Signaling Technology)
  • Anti‐flag antibody for prey expression (Cell Signaling Technology)
  • HRP‐labeled Rabbit Secondary antibody (ThermoFisher Scientific)
  • Super Signal West Pico Chemiluminescence Substrate (Thermo Fisher Scientific)
  • 1.5‐ml polypropylene tube
  • Electrophoresis chamber
  • Nitrocellulose membrane (Thermo Fisher Scientific)
  • Immunoblot transfer chamber
  • Immunoblot cassette
  • X‐ray film for immunoblot detection (Thermo Fisher Scientific)
  • X‐ray developer
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Figures

Videos

Literature Cited

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Key Reference
  Petschnigg et al., 2014. See above.
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