Intraflagellar transport (IFT) is essential for assembly and maintenance of cilia and flagella as well as ciliary motility and signaling. IFT is mediated by multisubunit complexes, including IFT-A, IFT-B, and the BBSome, in concert with kinesin and dynein motors. Under high salt conditions, purified IFT-B complex dissociates into a core subcomplex composed of at least nine subunits and at least five peripherally associated proteins. Using the visible immunoprecipitation assay, which we recently developed as a convenient protein-protein interaction assay, we determined the overall architecture of the IFT-B complex, which can be divided into core and peripheral subcomplexes composed of 10 and 6 subunits, respectively. In particular, we identified TTC26/IFT56 and Cluap1/IFT38, neither of which was included with certainty in previous models of the IFT-B complex, as integral components of the core and peripheral subcomplexes, respectively. Consistent with this, a ciliogenesis defect of Cluap1-deficient mouse embryonic fibroblasts was rescued by exogenous expression of wild-type Cluap1 but not by mutant Cluap1 lacking the binding ability to other IFT-B components. The detailed interaction map as well as comparison of subcellular localization of IFT-B components between wildtype and Cluap1-deficient cells provides insights into the functional relevance of the architecture of the IFT-B complex.Cilia and flagella are microtubule-based appendages on the surfaces of a wide variety of eukaryotic cells. Their assembly and maintenance by intraflagellar transport (IFT) 3 were revealed in Chlamydomonas reinhardtii by the pioneering studies of Rosenbaum and colleagues (1). Subsequently, due to the critical roles for cilia and flagella in various physiological and developmental processes, including cell motility, signaling, and sensory reception, these structures have been studied intensively in metazoans (2-4). IFT, which moves various proteins bidirectionally between the base and tip of cilia/flagella along a microtubule-based structure called the axoneme, is mediated by the large IFT particles with the aid of the anterograde molecular motor kinesin and the retrograde motor dynein. Under high salt conditions, the IFT particle purified from Chlamydomonas flagella can be divided into two complexes, IFT-A and IFT-B. These complexes are composed of ϳ6 and ϳ14 subunits, respectively, and are thought to connect cargo proteins with molecular motors (4, 5). Mutational analyses in Chlamydomonas and other ciliated organisms suggested that the IFT-A and IFT-B complexes are primarily involved in retrograde and anterograde ciliary trafficking, respectively. Biochemical studies revealed the approximate architecture of the Chlamydomonas IFT-A and IFT-B complexes (6 -12), and subsequent studies by Lorentzen and colleagues (13-15) revealed the structural basis of the interactions among several IFT-B subunits. The Chlamydomonas IFT-B complex consists of the core subcomplex, including at least nine subunits (IFT88, and at least five peripherally ...
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.
ARL13B (a small GTPase) and INPP5E (a phosphoinositide 5-phosphatase) are ciliary proteins encoded by causative genes of Joubert syndrome. We here showed, by taking advantage of a visible immunoprecipitation assay, that ARL13B interacts with the IFT46-IFT56 (IFT56 is also known as TTC26) dimer of the intraflagellar transport (IFT)-B complex, which mediates anterograde ciliary protein trafficking. However, the ciliary localization of ARL13B was found to be independent of its interaction with IFT-B, but dependent on the ciliary-targeting sequence RVEP in its C-terminal region. ARL13B-knockout cells had shorter cilia than control cells and exhibited aberrant localization of ciliary proteins, including INPP5E. In particular, in ARL13B-knockout cells, the IFT-A and IFT-B complexes accumulated at ciliary tips, and GPR161 (a negative regulator of Hedgehog signaling) could not exit cilia in response to stimulation with Smoothened agonist. This abnormal phenotype was rescued by the exogenous expression of wild-type ARL13B, as well as by its mutant defective in the interaction with IFT-B, but not by its mutants defective in INPP5E binding or in ciliary localization. Thus, ARL13B regulates IFT-A-mediated retrograde protein trafficking within cilia through its interaction with INPP5E.
The dynein-2 complex mediates trafficking of ciliary proteins by powering the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes. Although 11 subunits are known to constitute the dynein-2 complex, with several light-chain subunits shared by the dynein-1 complex, the overall architecture of the dynein-2 complex has not been fully clarified. Utilizing the visible immunoprecipitation assay, we demonstrated the interaction modes among the dynein-2 subunits, including previously undefined interactions, such as that between WDR60 and the TCTEX1D2–DYNLT1/DYNLT3 dimer. The dynein-2 complex can be divided into three subcomplexes, namely DYNC2H1–DYNC2LI1, WDR34–DYNLL1/DYNLL2–DYNLRB1/DYNLRB2, and WDR60–TCTEX1D2–DYNLT1/DYNLT3. We established cell lines lacking WDR60 or TCTEX1D2, both of which are dynein-2–specific subunits encoded by ciliopathy-causing genes, and found that both WDR60-knockout (KO) and TCTEX1D2-KO cells show defects in retrograde ciliary protein trafficking, with WDR60-KO cells demonstrating more severe defects probably due to failed assembly of the dynein-2 complex. The exogenous expression of a WDR60 mutant lacking TCTEX1D2 binding partially restored retrograde trafficking to a level comparable to that of TCTEX1D2-KO cells. Thus, our results demonstrated that WDR60 plays a major role and TCTEX1D2 plays an auxiliary role in the dynein-2 complex to mediate retrograde ciliary protein trafficking.
A donor knock-in vector optimized for the CRISPR/Cas9 system is constructed and a practical system developed that enables efficient disruption of cilia-related genes by exploiting homology-independent repair. A second version of the system can be used to reduce off-target cleavage frequency and increase versatility.
Protein trafficking within cilia is mediated by the intraflagellar transport (IFT) machinery composed of large protein complexes. The BBSome consists of eight BBS proteins encoded by causative genes of Bardet-Biedl syndrome (BBS), and has been implicated in the trafficking of ciliary membrane proteins, including G protein-coupled receptors (GPCRs), by connecting the IFT machinery to cargo GPCRs. Membrane recruitment of the BBSome to promote cargo trafficking has been proposed to be regulated by the Arf-like small GTPase ARL6/BBS3, through its interaction with the BBS1 subunit of the BBSome. We here investigated how the BBSome core subcomplex composed of BBS1, BBS2, BBS7, and BBS9 assembles and interacts with ARL6, and found that the ARL6–BBS1 interaction is reinforced by BBS9. BBS1-knockout (KO) cells showed defects in the ciliary entry of other BBSome subunits and ARL6, and in ciliary retrograde trafficking and the export of the GPCRs, Smoothened and GPR161. The trafficking defect of these GPCRs was rescued by the exogenous expression of wild-type BBS1, but not by its mutant lacking BBS9-binding ability. Our data thus indicate that the intact BBSome is required for retrograde trafficking of GPCRs out of cilia.
The 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.
Compositions of proteins and lipids within cilia and on the ciliary membrane are maintained to be distinct from those of the cytoplasm and plasma membrane, respectively, by the presence of the ciliary gate. INPP5E is a phosphoinositide 5-phosphatase that is localized on the ciliary membrane by anchorage via its C-terminal prenyl moiety. In addition, the ciliary membrane localization of INPP5E is determined by the small GTPase ARL13B. However, it remained unclear as to how ARL13B participates in the localization of INPP5E. We here show that wild-type INPP5E, INPP5E(WT), in ARL13B-knockout cells and an INPP5E mutant defective in ARL13B binding, INPP5E(ΔCTS), in control cells were unable to show steady-state localization on the ciliary membrane. However, not only INPP5E(WT) but also INPP5E(ΔCTS) was able to rescue the abnormal localization of ciliary proteins in INPP5E-knockout cells. Analysis using the chemically induced dimerization system demonstrated that INPP5E(WT) in ARL13B-knockout cells and INPP5E(ΔCTS) in control cells were able to enter cilia, but neither was retained on the ciliary membrane due to the lack of the INPP5E–ARL13B interaction. Thus, our data demonstrate that binding of INPP5E to ARL13B is essential for its steady-state localization on the ciliary membrane but is dispensable for its entry into cilia.
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