title: Detecting Membrane Protein-protein Interactions Using the Mammalian Membrane Two-hybrid (MaMTH) Assay.
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.
authors.affiliation: Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Punit
authors.initials: P
authors.lastname: Saraon
authors.affiliation: Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.|Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Grozavu
authors.affiliation: Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.|Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Sang Hyun
authors.initials: SH
authors.lastname: Lim
authors.affiliation: Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Jamie
authors.initials: J
authors.lastname: Snider
authors.affiliation: Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Yao
authors.affiliation: Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.|Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.|Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Igor
authors.initials: I
authors.lastname: Stagljar
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000737:chemistry*; D006801:Humans; D025941:Protein Interaction Mapping / Q000379:methods*; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques / Q000295:instrumentation*
keywords: MaMTH; interactome; mammalian-membrane two-hybrid; membrane proteomics; protein-protein interactions