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

Abstract

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.

Rab35 controls cilium length, function and membrane composition

“…Previous studies have shown that Arl13b loss leads to aberrant SMO accumulation in cilia, suggesting that Arl13b limits the ciliary accumulation of SMO, possibly by promoting its ciliary export . We wondered, therefore, whether elevated ciliary ARL13B levels in RAB35‐depleted cells are required for the SMO localisation defect in these cells.…”

“…Arl13b consists of an N‐terminal guanine nucleotide‐binding (G) domain and an unusually long C‐terminal tail with coiled coil (CC) and proline‐rich (PR) domains (Fig B) . All these domains were previously shown to be important for protein–protein interactions; for example, IFT‐B proteins interact with Arl13b via its PR domain, while INPP5E binds to the G‐domain . Arl13b also contains a C‐terminal RVEP motif for ciliary targeting and N‐terminal palmitoylation for ciliary membrane anchoring .…”

RABL2 interacts with the intraflagellar transport-B complex and CEP19 and participates in ciliary assembly

“…Expression vectors for the IFT-B subunits were described previously (Katoh et al. , 2016; Nozaki et al. , 2017).…”

“…The cell lysates were immunoprecipitated with GST–anti-GFP Nb prebound to glutathione–Sepharose 4B beads, and bound proteins were subjected to SDS–PAGE and immunoblotting analysis using anti-HA, anti-mRFP, anti-tRFP, or anti-GFP antibodies as described previously (Katoh et al. , 2015, 2016; Nozaki et al. , 2017).…”

Intraflagellar transporter protein (IFT27), an IFT25 binding partner, is essential for male fertility and spermiogenesis in mice

“…Defects in the IFT components have been shown to cause a spectrum of diseases, ranging from developmental defects to obesity, diabetes, and cancer. Many of these diseases, or ciliopathies, manifest as genetic syndromes, such as Joubert syndrome, Bardet-Biedel (BBS), Meckel-Gruber (MKS), and Nephronophthisis (NPHP)(Barker et al, 2014; Nozaki et al, 2017; Srivastava and Sayer, 2014; Su et al, 2014; Suspitsin and Imyanitov, 2016). …”