title: A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.
abstract: We have developed a new technique for proximity-dependent labeling of proteins in eukaryotic cells. Named BioID for proximity-dependent biotin identification, this approach is based on fusion of a promiscuous Escherichia coli biotin protein ligase to a targeting protein. BioID features proximity-dependent biotinylation of proteins that are near-neighbors of the fusion protein. Biotinylated proteins may be isolated by affinity capture and identified by mass spectrometry. We apply BioID to lamin-A (LaA), a well-characterized intermediate filament protein that is a constituent of the nuclear lamina, an important structural element of the nuclear envelope (NE). We identify multiple proteins that associate with and/or are proximate to LaA in vivo. The most abundant of these include known interactors of LaA that are localized to the NE, as well as a new NE-associated protein named SLAP75. Our results suggest BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment.
authors.affiliation: Sanford Children's Health Research Center, Sanford Research/University of South Dakota, Sioux Falls, SD 57104, USA. Kyle.Roux@sanfordhealth.org
authors.forename: Kyle J
authors.initials: KJ
authors.lastname: Roux
authors.forename: Dae In
authors.initials: DI
authors.lastname: Kim
authors.forename: Manfred
authors.initials: M
authors.lastname: Raida
authors.forename: Brian
authors.initials: B
authors.lastname: Burke
mesh_terms: D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007797:Laminin / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D007797:Laminin; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C084098:laminin A; D001710:Biotin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli