title: Architectures of multisubunit complexes revealed by a visible immunoprecipitation assay using fluorescent fusion proteins.
abstract: In this study, we elucidated the architectures of two multisubunit complexes, the BBSome and exocyst, through a novel application of fluorescent fusion proteins. By processing lysates from cells co-expressing GFP and RFP fusion proteins for immunoprecipitation with anti-GFP nanobody, protein-protein interactions could be reproducibly visualized by directly observing the immunoprecipitates under a microscope, and evaluated using a microplate reader, without requiring immunoblotting. Using this 'visible' immunoprecipitation (VIP) assay, we mapped binary subunit interactions of the BBSome complex, and determined the hierarchies of up to four subunit interactions. We also demonstrated the assembly sequence of the BBSome around the centrosome, and showed that BBS18 (also known as BBIP1 and BBIP10) serves as a linker between BBS4 and BBS8 (also known as TTC8). We also applied the VIP assay to mapping subunit interactions of the exocyst tethering complex. By individually subtracting the eight exocyst subunits from multisubunit interaction assays, we unequivocally demonstrated one-to-many subunit interactions (Exo70 with Sec10+Sec15, and Exo84 with Sec10+Sec15+Exo70). The simple, versatile VIP assay described here will pave the way to understanding the architectures and functions of multisubunit complexes involved in a variety of cellular processes.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Katoh
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Shohei
authors.initials: S
authors.lastname: Nozaki
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: David
authors.initials: D
authors.lastname: Hartanto
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Miyano
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan kazunaka@pharm.kyoto-u.ac.jp.
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Nakayama
mesh_terms: D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes / Q000378:metabolism*; D011485:Protein Binding; D060066:Protein Interaction Maps; D021122:Protein Subunits; D021381:Protein Transport; D011506:Proteins / Q000378:metabolism*; D061905:Single-Domain Antibodies / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; D021122:Protein Subunits; D011506:Proteins; D061905:Single-Domain Antibodies; D033921:Vesicular Transport Proteins; D000097573:Red Fluorescent Protein; C000591362:BBIP1 protein, human; C429655:BBS4 protein, human; C478138:TTC8 protein, human; C485184:enhanced green fluorescent protein
keywords: Anti-GFP nanobody; BBSome; Exocyst; Fluorescent fusion protein; Membrane traffic; Protein–protein interaction