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.

Regulation of ciliary retrograde protein trafficking by the Joubert syndrome proteins ARL13B and INPP5E

“…To unequivocally determine the subunits responsible for interaction of the IFT-B complex with ARL13B, we applied the VIP assay, which was established in our laboratory as a simple and versatile method to determine proteinprotein interactions without performing SDS-PAGE and immunoblotting (Katoh et al, 2015(Katoh et al, , 2016. Using our VIP assay, we previously determined the overall architectures of the exocyst, BBSome and IFT-B complexes.…”

“…We then exploited one of the advantages of the flexible VIP assay system to detect the ARL13B-IFT-B interaction; previously, we successfully identified one-to-many and many-to-many subunit interactions in the exocyst and IFT-B complexes through the VIP assay (Katoh et al, 2015(Katoh et al, , 2016. When ARL13B-EGFP was coexpressed with all the core or peripheral subunits fused to tRFP or mChe in HEK293T cells, and lysates prepared from the transfected cells were processed for immunoprecipitation with glutathione S-transferase (GST)-fused anti-GFP nanobody (Nb) prebound to glutathioneSepharose beads, red fluorescent signals were detected when the core, but not peripheral, subunits were co-expressed with ARL13B (Fig.…”

“…GST-tagged anti-GFP Nb prebound to glutathione-Sepharose 4B beads were prepared as described previously (Katoh et al, 2015). SAG was purchased from Enzo Life Sciences, and Polyethylenimine Max was purchased from Polysciences.…”

“…Recently, we developed a novel and flexible method for detecting protein-protein interactions, which we named the visible immunoprecipitation (VIP) assay (Katoh et al, 2015(Katoh et al, , 2016. By taking advantage of this flexible method, we previously determined the architecture of the IFT-B complex (Katoh et al, 2016), as well as those of the BBSome (a complex composed of eight subunits encoded by the causative genes of BBS) and exocyst complexes (Katoh et al, 2015).…”

“…By taking advantage of this flexible method, we previously determined the architecture of the IFT-B complex (Katoh et al, 2016), as well as those of the BBSome (a complex composed of eight subunits encoded by the causative genes of BBS) and exocyst complexes (Katoh et al, 2015). In the present study, we used the VIP assay to determine which subunit(s) of the IFT-B complex participate in its interaction with ARL13B, and found that the IFT46-IFT56 dimer (IFT56 is also known as TTC26) is responsible for the interaction of IFT-B with ARL13B [note that IFT56 is the most recently identified IFT-B component (Ishikawa et al, 2014), and IFT46 and IFT56 have been shown to form a heterodimer (Swiderski et al, 2014)].…”