Active Transport and Diffusion Barriers Restrict Joubert Syndrome-Associated ARL13B/ARL-13 to an Inv-like Ciliary Membrane Subdomain

Abstract

Cilia are microtubule-based cell appendages, serving motility, chemo-/mechano-/photo- sensation, and developmental signaling functions. Cilia are comprised of distinct structural and functional subregions including the basal body, transition zone (TZ) and inversin (Inv) compartments, and defects in this organelle are associated with an expanding spectrum of inherited disorders including Bardet-Biedl syndrome (BBS), Meckel-Gruber Syndrome (MKS), Joubert Syndrome (JS) and Nephronophthisis (NPHP). Despite major advances in understanding ciliary trafficking pathways such as intraflagellar transport (IFT), how proteins are transported to subciliary membranes remains poorly understood. Using Caenorhabditis elegans and mammalian cells, we investigated the transport mechanisms underlying compartmentalization of JS-associated ARL13B/ARL-13, which we previously found is restricted at proximal ciliary membranes. We now show evolutionary conservation of ARL13B/ARL-13 localisation to an Inv-like subciliary membrane compartment, excluding the TZ, in many C. elegans ciliated neurons and in a subset of mammalian ciliary subtypes. Compartmentalisation of C. elegans ARL-13 requires a C-terminal RVVP motif and membrane anchoring to prevent distal cilium and nuclear targeting, respectively. Quantitative imaging in more than 20 mutants revealed differential contributions for IFT and ciliopathy modules in defining the ARL-13 compartment; IFT-A/B, IFT-dynein and BBS genes prevent ARL-13 accumulation at periciliary membranes, whereas MKS/NPHP modules additionally inhibit ARL-13 association with TZ membranes. Furthermore, in vivo FRAP analyses revealed distinct roles for IFT and MKS/NPHP genes in regulating a TZ barrier to ARL-13 diffusion, and intraciliary ARL-13 diffusion. Finally, C. elegans ARL-13 undergoes IFT-like motility and quantitative protein complex analysis of human ARL13B identified functional associations with IFT-B complexes, mapped to IFT46 and IFT74 interactions. Together, these findings reveal distinct requirements for sequence motifs, IFT and ciliopathy modules in defining an ARL-13 subciliary membrane compartment. We conclude that MKS/NPHP modules comprise a TZ barrier to ARL-13 diffusion, whereas IFT genes predominantly facilitate ARL-13 ciliary entry and/or retention via active transport mechanisms.

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

“…A previous yeast two-hybrid study suggested that IFT46 and IFT74 interacted with ARL13B (Cevik et al, 2013). We therefore addressed whether IFT74 can also interact directly with ARL13B.…”

“…Although a previous study using proteomic analysis of affinity purified proteins and yeast two-hybrid analysis suggested that ARL13B interacts with some subunit(s) of the IFT-B complex (Cevik et al, 2013), it remained uncertain as to which subunit(s) directly interact with ARL13B. 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.…”

“…3H). In addition, the C-terminal RVVP sequence in Caenorhabditis elegans ARL-13 was suggested to determine its correct localization within cilia (Cevik et al, 2013). These sequences match the consensus ciliary-targeting motif (RVxP) found in several ciliary proteins (Geng et al, 2006).…”

“…PDE6D is known to bind to C-terminally prenylated proteins, including INPP5E, and ARL3 stimulates release of PDE6D from prenylated proteins (Fansa et al, 2016;Ismail et al, 2011). On the other hand, Blacque and colleagues have revealed that the IFT-B complex interacts with ARL13B and proposed that IFT-B facilitates the ciliary localization of ARL13B (namely, ARL13B might be a cargo of the IFT machinery), although it was uncertain which subunit(s) of the IFT-B complex are responsible for its interaction with ARL13B (Cevik et al, 2013).…”

Arfs at a Glance

“…Arl6 in its GTP-bound form is required to recruit the BBSome to the plasma membrane to drive cargo-sorting into cilia (Jin et al, 2010). Arl13 is mutated in patients with Joubert Syndrome, a cerebral disorder causing mental retardation, and functions in intraflagellar transport (Cevik et al, 2013). The retinitis pigmentosa 2 protein (RP2) is a GAP for Arl3 (Veltel et al, 2008), which functions in primary cilia to promote normal kidney and photoreceptor development (Schrick et al, 2006).…”