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 ...

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 ciliary kinesin KIF17 is a cargo, but not a motor, of the IFT-B complex. The ciliary entry of KIF17 through the transition zone depends on its binding to IFT-B, as well as on its nuclear localization signal.