Architectures of multisubunit complexes revealed by a visible immunoprecipitation assay using fluorescent fusion proteins

J Cell Sci. 2015 Jun 15;128(12):2351-62. doi: 10.1242/jcs.168740. Epub 2015 May 11.

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.

Keywords: Anti-GFP nanobody; BBSome; Exocyst; Fluorescent fusion protein; Membrane traffic; Protein–protein interaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Carrier Proteins / metabolism*
  • Cell Membrane / metabolism
  • Cells, Cultured
  • Cytoskeletal Proteins
  • Green Fluorescent Proteins / metabolism*
  • HEK293 Cells
  • Humans
  • Immunoenzyme Techniques
  • Immunoprecipitation
  • Luminescent Proteins / metabolism*
  • Microscopy, Fluorescence
  • Microtubule-Associated Proteins
  • Multiprotein Complexes / metabolism*
  • Protein Binding
  • Protein Interaction Maps
  • Protein Subunits
  • Protein Transport
  • Proteins / metabolism*
  • Single-Domain Antibodies / metabolism
  • Two-Hybrid System Techniques
  • Vesicular Transport Proteins / metabolism*

Substances

  • BBIP1 protein, human
  • BBS4 protein, human
  • Carrier Proteins
  • Cytoskeletal Proteins
  • Luminescent Proteins
  • Microtubule-Associated Proteins
  • Multiprotein Complexes
  • Protein Subunits
  • Proteins
  • Single-Domain Antibodies
  • TTC8 protein, human
  • Vesicular Transport Proteins
  • enhanced green fluorescent protein
  • red fluorescent protein
  • Green Fluorescent Proteins