title: Rapid mapping of interactions between Human SNX-BAR proteins measured in vitro by AlphaScreen and single-molecule spectroscopy.
abstract: Protein dimerization and oligomerization is commonly used by nature to increase the structural and functional complexity of proteins. Regulated protein assembly is essential to transfer information in signaling, transcriptional, and membrane trafficking events. Here we show that a combination of cell-free protein expression, a proximity based interaction assay (AlphaScreen), and single-molecule fluorescence allow rapid mapping of homo- and hetero-oligomerization of proteins. We have applied this approach to the family of BAR domain-containing sorting nexin (SNX-BAR) proteins, which are essential regulators of membrane trafficking and remodeling in all eukaryotes. Dimerization of BAR domains is essential for creating a concave structure capable of sensing and inducing membrane curvature. We have systematically mapped 144 pairwise interactions between the human SNX-BAR proteins and generated an interaction matrix of preferred dimerization partners for each family member. We find that while nine SNX-BAR proteins are able to form homo-dimers, several including the retromer-associated SNX1, SNX2, and SNX5 require heteromeric interactions for dimerization. SNX2, SNX4, SNX6, and SNX8 show a promiscuous ability to bind other SNX-BAR proteins and we also observe a novel interaction with the SNX3 protein which lacks the BAR domain structure.
authors.affiliation: From the ‡Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072 Australia;
authors.forename: Emma
authors.initials: E
authors.lastname: Sierecki
authors.affiliation: From the ‡Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072 Australia;
authors.forename: Loes M
authors.initials: LM
authors.lastname: Stevers
authors.affiliation: From the ‡Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072 Australia;
authors.forename: Nichole
authors.initials: N
authors.lastname: Giles
authors.affiliation: From the ‡Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072 Australia;
authors.forename: Mark E
authors.initials: ME
authors.lastname: Polinkovsky
authors.affiliation: From the ‡Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072 Australia;
authors.forename: Mehdi
authors.initials: M
authors.lastname: Moustaqil
authors.affiliation: From the ‡Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072 Australia;
authors.forename: Sergey
authors.initials: S
authors.lastname: Mureev
authors.affiliation: From the ‡Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072 Australia;
authors.forename: Wayne A
authors.initials: WA
authors.lastname: Johnston
authors.affiliation: §University of Queensland, Diamantina Institute, Translational Research Institute, Brisbane, Australia;
authors.forename: Mareike
authors.initials: M
authors.lastname: Dahmer-Heath
authors.affiliation: §University of Queensland, Diamantina Institute, Translational Research Institute, Brisbane, Australia;
authors.forename: Dubravka
authors.initials: D
authors.lastname: Skalamera
authors.affiliation: ¶School of Pharmacy, The University of Queensland, Brisbane, Australia.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Gonda
authors.affiliation: §University of Queensland, Diamantina Institute, Translational Research Institute, Brisbane, Australia;
authors.forename: Brian
authors.initials: B
authors.lastname: Gabrielli
authors.affiliation: From the ‡Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072 Australia;
authors.forename: Brett M
authors.initials: BM
authors.lastname: Collins
authors.affiliation: From the ‡Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072 Australia; y.gambin@uq.edu.au k.alexandrov@uq.edu.au.
authors.forename: Kirill
authors.initials: K
authors.lastname: Alexandrov
authors.affiliation: From the ‡Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072 Australia;
authors.forename: Yann
authors.initials: Y
authors.lastname: Gambin
mesh_terms: D019281:Dimerization; D006801:Humans; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D058305:Sorting Nexins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D058305:Sorting Nexins