title: New Gateway-compatible vectors for a high-throughput protein-protein interaction analysis by a bimolecular fluorescence complementation (BiFC) assay in plants and their application to a plant clathrin structure analysis.
abstract: Protein-protein interactions (PPI) play key roles in various biological processes. The bimolecular fluorescence complementation (BiFC) assay is an excellent tool for routine PPI analyses in living cells. We developed new Gateway vectors for a high-throughput BiFC analysis of plants, adopting a monomeric Venus split just after the tenth β-strand, and analyzed the interaction between Arabidopsis thaliana coated vesicle coatmers, the clathrin heavy chain (CHC), and the clathrin light chain (CLC). In competitive BiFC tests, CLC interacted with CHC through a coiled-coil motif in the middle section of CLC. R1340, R1448, and K1512 in CHC and W94 in CLC are potentially key amino acids underlying the inter-chain interaction, consistent with analyses based on homology modeling. Our Gateway BiFC system, the V10-BiFC system, provides a useful tool for a PPI analysis in living plant cells. The CLC-CHC interaction identified may facilitate clathrin triskelion assembly needed for cage formation.
authors.affiliation: a Department of Molecular and Functional Genomics , Interdisciplinary Center for Science Research, Organization of Research, Shimane University , Matsue , Japan.
authors.forename: Kohji
authors.initials: K
authors.lastname: Nishimura
authors.affiliation: b Department of Life Science and Biotechnology , Shimane University , Matsue , Japan.
authors.forename: Syouta
authors.initials: S
authors.lastname: Ishikawa
authors.affiliation: b Department of Life Science and Biotechnology , Shimane University , Matsue , Japan.
authors.forename: Erika
authors.initials: E
authors.lastname: Matsunami
authors.affiliation: c Department of Pharmacology , National Research Institute for Child Health and Development , Setagaya , Japan.|d Graduate School of Medical and Dental Sciences , Tokyo Medical and Dental University , Bunkyo , Japan.
authors.forename: Junji
authors.initials: J
authors.lastname: Yamauchi
authors.affiliation: e Department of Life Science and Informatics , Maebashi Institute of Technology , Maebashi , Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Homma
authors.affiliation: f Institute of Molecular Plant Sciences , University of Edinburgh , Edinburgh , UK.
authors.forename: Christine
authors.initials: C
authors.lastname: Faulkner
authors.affiliation: f Institute of Molecular Plant Sciences , University of Edinburgh , Edinburgh , UK.
authors.forename: Karl
authors.initials: K
authors.lastname: Oparka
authors.affiliation: b Department of Life Science and Biotechnology , Shimane University , Matsue , Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Jisaka
authors.affiliation: b Department of Life Science and Biotechnology , Shimane University , Matsue , Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Nagaya
authors.affiliation: b Department of Life Science and Biotechnology , Shimane University , Matsue , Japan.
authors.forename: Kazushige
authors.initials: K
authors.lastname: Yokota
authors.affiliation: a Department of Molecular and Functional Genomics , Interdisciplinary Center for Science Research, Organization of Research, Shimane University , Matsue , Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Nakagawa
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002966:Clathrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
chemical_list: D029681:Arabidopsis Proteins; D002966:Clathrin
keywords: Arabidopsis thaliana; BiFC; Venus; clathrin; protein–protein interaction