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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


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

PDF or HTML at Wiley Online Library

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
PDF or HTML at Wiley Online Library


Basic Protocol 1: Cell Culture and MaMTH Firefly Luciferase Assays

  • 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

  • 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

  • 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

  • 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
PDF or HTML at Wiley Online Library



Literature Cited

  Bensimon, A., Heck, A.J.R., and Aebersold, R. 2012. Mass spectrometry‐based proteomics and network biology. Annu. Rev. Biochem. 81:379‐405. Available at:‐biochem‐072909‐100424?url_ver=Z39.88‐2003& doi: 10.1146/annurev‐biochem‐072909‐100424.
  Dunham, W.H., Mullin, M., and Gingras, A.C. 2012. Affinity‐purification coupled to mass spectrometry: Basic principles and strategies. Proteomics 12:1576‐1590. doi: 10.1002/pmic.201100523.
  Johnsson, N. and Varshavsky, A. 1994. Split ubiquitin as a sensor of protein interactions in vivo. Proc. Natl. Acad. Sci. U.S.A. 91:10340‐10344. Available at: doi: 10.1073/pnas.91.22.10340.
  Kerppola, T.K. 2006. Visualization of molecular interactions by fluorescence complementation. Nat. Rev. Mol. Cell Biol. 7:449‐456. doi: 10.1038/nrm1929.
  Kittanakom, S., Barrios‐Rodiles, M., Petschnigg, J., Arnoldo, A., Wong, V., Kotlyar, M., Heisler, L.E., Jurisica, I., Wrana, J.L., Nislow, C., and Stagljar, I. 2014. CHIP‐MYTH: A novel interactive proteomics method for the assessment of agonist‐dependent interactions of the human 2‐adrenergic receptor. Biochem. Biophys. Res. Commun. 445:746‐756. doi: 10.1016/j.bbrc.2014.02.033.
  Lam, M.H.Y., Snider, J., Rehal, M., Wong, V., Aboualizadeh, F., Drecun, L., Wong, O., Jubran, B., Li, M., Ali, M., Jessulat, M., Deineko, V., Miller, R., Lee, M.E., Park, H.O., Davidson, A., Babu, M., and Stagljar, I. 2015. A comprehensive membrane interactome mapping of sho1p reveals Fps1p as a novel key player in the regulation of the HOG pathway in S. cerevisiae. J. Mol. Biol. 427:2088‐2103. doi: 10.1016/j.jmb.2015.01.016.
  Lievens, S., Lemmens, I., and Tavernier, J. 2009. Mammalian two‐hybrids come of age. Trends Biochem. Sci. 34:579‐588. doi: 10.1016/j.tibs.2009.06.009.
  Mak, A.B., Nixon, A.M.L., Kittanakom, S., Stewart, J.M., Chen, G.I., Curak, J., Gingras, A.C., Mazitschek, R., Neel, B.G., Stagljar, I., and Moffat, J. 2012. Regulation of CD133 by HDAC6 promotes ‐catenin signaling to suppress cancer cell differentiation. Cell Rep. 2:951‐963. doi: 10.1016/j.celrep.2012.09.016.
  Michnick, S.W., Ear, P.H., Manderson, E.N., Remy, I., and Stefan, E. 2007. Universal strategies in research and drug discovery based on protein‐fragment complementation assays. Nat. Revi. Drug Discov. 6:569‐582. Available at: doi: 10.1038/nrd2311.
  Paumi, C.M., Menendez, J., Arnoldo, A., Engels, K., Iyer, K.R., Thaminy, S., Georgiev, O., Barral, Y., Michaelis, S., and Stagljar, I. 2007. Mapping protein‐protein interactions for the yeast ABC transporter Ycf1p by integrated split‐ubiquitin membrane yeast two‐hybrid analysis. Mol. Cell 26:15‐25. doi: 10.1016/j.molcel.2007.03.011.
  Petschnigg, J., Groisman, B., Kotlyar, M., Taipale, M., Zheng, Y., Kurat, C.F., Sayad, A., Sierra, J.R., Usaj, M.M., Snider, J., Nachman, A., Krykbaeva, I., Tsao, M.S., Moffat, J., Pawson, T., Lindquist, S., Jurisica, I., and Stagljar, I. 2014. The mammalian‐membrane two‐hybrid assay (MaMTH) for probing membrane‐protein interactions in human cells. Nat. Methods 11:585‐592. Available at: doi:10.1038/nmeth.2895.
  Petschnigg, J., Kotlyar, M., Blair, L., Jurisica, I., Stagljar, I., and Ketteler, R. 2016. Systematic identification of oncogenic EGFR interaction partners. J. Mol. Biol. S0022‐2836(16)30535‐30536.
  Rojo‐Niersbach, E., Morley, D., Heck, S., and Lehming, N. 2000. A new method for the selection of protein interactions in mammalian cells. Biochem. J. 348 Pt 3:585‐590. Available at: doi: 10.1042/bj3480585.
  Snider, J., Kittanakom, S., Damjanovic, D., Curak, J., Wong, V., and Stagljar, I. 2010. Detecting interactions with membrane proteins using a membrane two‐hybrid assay in yeast. Nat. Protoc. 5:1281‐1293. Available at: doi: 10.1038/nprot.2010.83.
  Snider, J., Kotlyar, M., Saraon, P., Yao, Z., Jurisica, I., and Stagljar, I. 2015. Fundamentals of protein interaction network mapping. Mol. Syst. Biol. 11:848‐848. Available at: doi: 10.15252/msb.20156351.
  Snider, J., Hanif, A., Lee, M.E., Jin, K., Yu, A.R., Graham, C., Chuk, M., Damjanovic, D., Wierzbicka, M., Tang, P., Balderes, D., Wong, V., Jessulat, M., Darowski, K.D., San Luis, B.J., Shevelev, I., Sturley, S.L., Boone, C., Greenblatt, J.F., Zhang, Z., Paumi, C.M., Babu, M., Park, H.O., Michaelis, S., and Stagljar, I. 2013. Mapping the functional yeast ABC transporter interactome. Nat. Chem. Biol. 9:565‐572. Available at: doi: 10.1038/nchembio.1293.
  Stagljar, I., Korostensky, C., Johnsson, N., and te Heesen, S. 1998. A genetic system based on split‐ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc. Natl. Acad. Sci. U.S.A. 95:5187‐5192. Available at: doi: 10.1073/pnas.95.9.5187.
  Stevens, T.J. and Arkin, I.T. 2000. Do more complex organisms have a greater proportion of membrane proteins in their genomes? Proteins 39:417‐420. doi: 10.1002/(SICI)1097‐0134(20000601)39:4%3c417::AID‐PROT140%3e3.0.CO;2‐Y.
  Suter, B., Kittanakom, S., and Stagljar, I. 2008. Two‐hybrid technologies in proteomics research. Curr. Opin. Biotechnol. 19:316‐323. doi: 10.1016/j.copbio.2008.06.005.
  Thaminy, S., Auerbach, D., Arnoldo, A., and Stagljar, I. 2003. Identification of novel ErbB3‐interacting factors using the split‐ubiquitin membrane yeast two‐hybrid system. Genome Res. 13:1744‐1753. doi: 10.1101/gr.1276503.
  Usenovic, M., Knight, A.L., Ray, A., Wong, V., Brown, K.R., Caldwell, G.A., Caldwell, K.A., Stagljar, I., and Krainc, D. 2012. Identification of novel ATP13A2 interactors and their role in ‐synuclein misfolding and toxicity. Hum. Mol. Genet. 21:3785‐3794. doi: 10.1093/hmg/dds206.
  Yao, Z., Petschnigg, J., Ketteler, R., and Stagljar, I. 2015. Application guide for omics approaches to cell signaling. Nat. Chem. Biol. 11:387‐397. Available at: doi: 10.1038/nchembio.1809.
  Yao, Z., Darowski, K., St‐Denis, N., Wong, V., Offensperger, F., Villedieu, A., Amin, S., Malty, R., Aoki, H., Guo, H., Xu, Y., Iorio, C., Kotlya, M., Emili, A., Jurisica, I., Neel, B.G., Babu, M., Gingras, A.C., and Stagljar, I. 2016. A global analysis of the receptor tyrosine kinase‐protein phosphatase interactome. Mol. Cell. S1097‐2765(16)30817‐6.
Key Reference
  Petschnigg et al., 2014. See above.
PDF or HTML at Wiley Online Library