title: Probing nuclear pore complex architecture with proximity-dependent biotinylation.
abstract: Proximity-dependent biotin identification (BioID) is a method for identifying protein associations that occur in vivo. By fusing a promiscuous biotin ligase to a protein of interest expressed in living cells, BioID permits the labeling of proximate proteins during a defined labeling period. In this study we used BioID to study the human nuclear pore complex (NPC), one of the largest macromolecular assemblies in eukaryotes. Anchored within the nuclear envelope, NPCs mediate the nucleocytoplasmic trafficking of numerous cellular components. We applied BioID to constituents of the Nup107-160 complex and the Nup93 complex, two conserved NPC subcomplexes. A strikingly different set of NPC constituents was detected depending on the position of these BioID-fusion proteins within the NPC. By applying BioID to several constituents located throughout the extremely stable Nup107-160 subcomplex, we refined our understanding of this highly conserved subcomplex, in part by demonstrating a direct interaction of Nup43 with Nup85. Furthermore, by using the extremely stable Nup107-160 structure as a molecular ruler, we defined the practical labeling radius of BioID. These studies further our understanding of human NPC organization and demonstrate that BioID is a valuable tool for exploring the constituency and organization of large protein assemblies in living cells.
authors.affiliation: Sanford Children's Health Research Center, Sanford Research, Sioux Falls, SD 57104;
authors.forename: Dae In
authors.initials: DI
authors.lastname: Kim
authors.affiliation: Sanford Children's Health Research Center, Sanford Research, Sioux Falls, SD 57104;
authors.forename: K C
authors.initials: KC
authors.lastname: Birendra
authors.affiliation: Sanford-Burnham Proteomics Facility, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037;
authors.forename: Wenhong
authors.initials: W
authors.lastname: Zhu
authors.affiliation: Sanford-Burnham Proteomics Facility, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037;
authors.forename: Khatereh
authors.initials: K
authors.lastname: Motamedchaboki
authors.affiliation: Institut Jacques Monod, Unité Mixte de Recherche 7592, Centre National de la Recherche Scientifique, Université Paris Diderot, Sorbonne Paris Cité, F-75205 Paris, France; and.
authors.forename: Valérie
authors.initials: V
authors.lastname: Doye
authors.affiliation: Sanford Children's Health Research Center, Sanford Research, Sioux Falls, SD 57104;Department of Pediatrics, Sanford School of Medicine, University of South Dakota, Sioux Falls, SD 57105 kyle.roux@sanfordhealth.org.
authors.forename: Kyle J
authors.initials: KJ
authors.lastname: Roux
mesh_terms: D000465:Algorithms; D001710:Biotin / Q000737:chemistry*; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry*; D002467:Cell Nucleus / Q000378:metabolism; D002853:Chromatography, Liquid; D003593:Cytoplasm / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008025:Ligases / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000737:chemistry; D009685:Nuclear Envelope / Q000378:metabolism; D022022:Nuclear Pore / Q000737:chemistry*; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry*; D011485:Protein Binding; D019809:Streptavidin / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D014162:Transfection
chemical_list: D008562:Membrane Glycoproteins; D028861:Nuclear Pore Complex Proteins; C508154:Nup93 protein, human; C049312:nuclear pore protein p62; D049452:Green Fluorescent Proteins; D001710:Biotin; D019809:Streptavidin; D008025:Ligases; D019731:Carbon-Nitrogen Ligases