Requirement of IFT-B-BBSome complex interaction in export of GPR161 from cilia.

Shohei Nozaki,Roiner Francisco Castro Araya,Kazuhisa Nakayama,Yohei Katoh

doi:10.1242/bio.043786

Shohei Nozaki, Roiner Francisco Castro Araya + Show 2 more

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https://doi.org/10.1242/bio.043786

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Journal: Biology open	Publication Date: Jan 1, 2019
Citations: 43	License type: CC BY 4.0

Affiliation: Kyoto University

Abstract

ABSTRACTThe intraflagellar transport (IFT) machinery, which includes the IFT-A and IFT-B complexes, mediates bidirectional trafficking of ciliary proteins. In addition to these complexes, the BBSome, which is composed of eight subunits that are encoded by the causative genes of Bardet-Biedl syndrome (BBS), has been proposed to connect the IFT machinery to ciliary membrane proteins, such as G protein-coupled receptors, to mediate their export from cilia. However, little is known about the connection between the IFT machinery and the BBSome. Using the visible immunoprecipitation assay, we here identified the interaction between IFT38 from the IFT-B complex and BBS1, BBS2 and BBS9 from the BBSome. Furthermore, by analyzing phenotypes of IFT38-knockout cells exogenously expressing wild-type IFT38 or its mutant lacking the ability to interact with BBS1+BBS2+BBS9, we showed that knockout cells expressing the IFT38 mutant have restored ciliogenesis; however, similar to BBS1-knockout cells, they demonstrated significant accumulation of GPR161 within cilia upon stimulation of Hedgehog signaling. These results indicate that the IFT-B–BBSome interaction is required for the export of GPR161 across the ciliary gate.

Highlights

Cilia are organelles that project from the surfaces of various eukaryotic cells, and are supported by the axoneme, which is a microtubule-based scaffold
By analyzing phenotypes of IFT38-KO cell lines exogenously expressing an IFT38 deletion construct, we showed that the intraflagellar transport (IFT)-B–BBSome interaction is required for export from cilia of GPR161, a G protein–coupled receptors (GPCRs) involved in Hh signaling
IFT-B–BBSome interaction is mediated by IFT38 and BBS1–BBS2–BBS9 To find a potential interface between the IFT machinery and the BBSome, we used the visible immunoprecipitation (VIP) assay, which is a convenient and flexible strategy to visually detect one-to-one protein interactions and one-to-many and many-to-many protein interactions (Funabashi et al, 2017, 2018; Hamada et al, 2018; Katoh et al, 2018, 2015, 2016)

Summary

Introduction

Cilia are organelles that project from the surfaces of various eukaryotic cells, and are supported by the axoneme, which is a microtubule-based scaffold. Cilia function as cellular antennae by mechanosensing extracellular stimuli, such as light and fluid flow, and chemosensing morphogenetic signals, such as Hedgehog (Hh) (Briscoe and Thérond, 2013; Mukhopadhyay and Rohatgi, 2014). Owing to their crucial roles, defects in cilia lead to a variety of congenital disorders, such as Bardet-Biedl syndrome (BBS), Joubert syndrome, nephronophthisis, Meckel syndrome, and short-rib thoracic dysplasia, which are collectively referred to as the ciliopathies, which accompany a wide spectrum of clinical manifestations, including retinal degeneration, polycystic kidneys, morbid obesity, and skeletal and brain malformations (Braun and Hildebrandt, 2017; Madhivanan and Aguilar, 2014). Received 9 April 2019; Accepted 24 August 2019 protein and lipid composition of the ciliary membrane differs greatly from that of the plasma membrane, due to the presence of the transition zone, which serves as a permeability/diffusion barrier at the base of cilia (Verhey and Yang, 2016; Wei et al, 2015)

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