title: Proximity-Dependent Biotin Identification (BioID) in Dictyostelium Amoebae.
abstract: The identification of a bona fide lamin-like protein in Dictyostelium made this lower eukaryote an attractive model organism to study evolutionarily conserved nuclear envelope (NE) proteins important for nuclear organization and human laminopathies. Proximity-dependent biotin identification (BioID), reported by Roux and colleagues, is a powerful discovery tool for lamin-associated proteins. In this method, living cells express a bait protein (e.g., lamin) fused to an R118G-mutated version of BirA, an Escherichia coli biotinylase. In the presence of biotin, BirA-R118G biotinylates target proteins in close proximity in vivo, which are purified using streptavidin and identified by immunoblotting or mass spectrometry. We adapted the BioID method for use in Dictyostelium amoebae. The protocols described here successfully revealed Dictyostelium lamin-like protein NE81 proximity to Sun1, a conserved inner nuclear membrane protein.
authors.affiliation: Institute for Biochemistry and Biology, Department of Cell Biology, University of Potsdam, Potsdam, Germany.
authors.forename: Petros
authors.initials: P
authors.lastname: Batsios
authors.affiliation: Institute for Biochemistry and Biology, Department of Cell Biology, University of Potsdam, Potsdam, Germany.
authors.forename: Irene
authors.initials: I
authors.lastname: Meyer
authors.affiliation: Institute for Biochemistry and Biology, Department of Cell Biology, University of Potsdam, Potsdam, Germany. Electronic address: rgraef@uni-potsdam.de.
authors.forename: Ralph
authors.initials: R
authors.lastname: Gräf
mesh_terms: D061245:Axenic Culture; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D002467:Cell Nucleus / Q000378:metabolism; D004023:Dictyostelium / Q000166:cytology / Q000378:metabolism*; D034882:Lamins / Q000502:physiology*; D009685:Nuclear Envelope / Q000378:metabolism; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000502:physiology*; D025941:Protein Interaction Mapping; D015800:Protozoan Proteins / Q000302:isolation & purification / Q000502:physiology*; D013194:Staining and Labeling
chemical_list: D034882:Lamins; D009687:Nuclear Proteins; D015800:Protozoan Proteins; D001710:Biotin
keywords: BioID; Dictyostelium; Lamin; Nuclear envelope; Nuclear lamina; Nucleus; Protein–protein interaction; Sun1