Bardet-Biedl syndrome (BBS)4
is a genetically heterogeneous human disorder characterized by a wide array of phenotypes, including obesity, polycystic kidney disease, retinal degeneration, polydactyly, and sensory impairments (1
). It represents a paradigm for ciliopathies, a class of genetic disorders sharing the common etiology of basal body and/or cilia dysfunction (5
). The physiological relevance of cilia, the motile and/or sensory organelles present on most vertebrate cell types, is emphasized by the growing number of pleiotropic diseases belonging to this class, which in addition to BBS includes the closely related Alström syndrome, as well Meckel syndrome, Joubert syndrome, and Senior-Løken syndrome.
Once thought to be a vestigial organelle, the nonmotile (primary) cilium is now well established as being essential for the transduction of chemical, visual, and mechanical sensory stimuli, and its receptor-studded membrane orchestrates a number of important signaling cascades necessary for vertebrate development, including Hedgehog, Wnt, and PDGFRαα signaling (6
The microtubule-based ciliary axoneme is assembled at the distal end of a basal body, a structure derived from the mother centriole that docks at the plasma membrane prior to ciliogenesis (13
). Nearly all cilia are built and maintained by a multiprotein intraflagellar transport (IFT) machinery that mobilizes ciliary cargo, for example structural components, receptors, and signaling molecules, into the organelle via an anterograde kinesin motor and transports components back to the base by way of a retrograde dynein motor (15
). The IFT machinery is thought to dock near the tips of the transitional fibers, which are structures of unknown composition that form a nine-membered pinwheel-like radial array that joins the distal end of the basal body to the ciliary membrane (19
). Near this site, effectively a “ciliary gate,” post-Golgi trafficking of vesicular cargoes destined for the cilium terminates by fusion with the membrane; the transport of the released cargoes into the organelle may then be facilitated by the IFT machinery (21
Few cellular factors are known to participate in the functional transition from vesicular trafficking to ciliary trafficking (24
) or, potentially, in the reverse process where ciliary components are trafficked out of the organelle. Although ciliary trafficking is nonvesicular, it relies on IFT protein subunits that share β-propeller/solenoid topologies (and ostensibly functions) similar to those of coat vesicle-associated proteins (23
). Our group previously discovered a member of the ARF-like family of small GTPases, ARL6, that could conceivably possess such a trafficking function. ARL6 is associated with Bardet-Biedl syndrome type 3 (BBS3) (27
), presently making it 1 of 14 BBS-associated proteins (4
). In Caenorhabditis elegans
sensory cilia, ARL6 was shown to undergo IFT (28
). Moreover, ARF-like proteins, which are generally implicated in membrane trafficking and regulation of microtubule-associated processes (29
), have at least two members that possess established ciliary functions. ARL3 orthologues from Leishmania
, C. elegans
, and mice are associated with cilia/ciliary photoreceptor functions (33
); similarly, ARL13B specifically localizes to cilia in C. elegans
, and in the mouse it participates in cilium formation and Hedgehog signaling (34
). The exclusive presence of ARL6 in organisms that possess cilia and its association with IFT (28
) therefore suggests a potential role for this small GTPase in coordinating or facilitating the functional bridging of intraciliary trafficking with vesicular trafficking.
Although all BBS proteins studied to date appear to play important roles in either trafficking cargo to the basal body/centrosome or within cilia via IFT (2
), ARL6/BBS3 is suggested to have a potentially distinct function from that of a core group of BBS proteins that assemble into a complex, termed the BBSome (37
). This notion arises from the finding that the BBSome, which consists of a biochemically separable protein complex containing BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, and BBS9 proteins, does not co-fractionate with BBS3 in a sucrose gradient (37
). Interestingly, one component of the BBSome (BBS1) interacts with Rabin8, a GDP/GTP exchange factor for the small GTPase RAB8. Rabin8 co-localizes with the BBSome at the centrosome/basal body and affects the nucleotide cycling of RAB8, which enters the cilium in its GTP-bound state and is necessary for ciliogenesis (37
). RAB8 was also recently shown to interact with Rabaptin5, which localizes at the basal body, and associates with an IFT protein termed Elipsa/DYF-11 (38
). Given the well established role of Rabaptin5 as a RAB5 effector in endocytosis, it was suggested that this Rabaptin5-RAB8-Elipsa/DYF-11 trio may provide critical functional interactions between the ciliary membrane (or vesicles associated with cilia) and the IFT machinery (39
). In C. elegans
, RAB-5 itself was shown to concentrate at the base of cilia together with an early endosome-associated STAM-Hrs complex that regulates polycystin-1/polycystin-2 protein removal from cilia (40
). How BBSome proteins, which are themselves associated with both the basal body and IFT, participate in this basal body-ciliary sorting-trafficking process remains elusive, and the role of BBS3, potentially distinct from core BBSome components, is presently unknown.
To shed light on the molecular and cellular functions of ARL6/BBS3, we first determined the crystal structure of the GTP-bound form of the human protein. The ARL6/BBS3 structure represents the first to be solved for a BBS or IFT protein and displays unique characteristics not found in other small GTPases. In combination with GTP binding functional assays, the structure demonstrates how single-residue mutations uncovered in BBS patients abrogate nucleotide binding and thus protein function. Furthermore, we now demonstrate that ARL6 in ciliated mammalian cells localizes to the distal end basal bodies, near or at transition fibers, and thus in close proximity to the site of vesicle docking/fusion at the base of cilia. Consistent with a role for ARL6 at this subcellular location, we show that disrupting its cellular function, via overproduction of GDP- or GTP-locked forms, leads to ciliary anomalies in vivo. Furthermore, excess cellular wild-type ARL6 causes an up-regulation of Wnt signaling. Importantly, this effect is not observed with pathogenic variants of ARL6/BBS3. Together, our findings reveal that a BBS protein not directly (or tightly) associated with the BBSome localizes to a novel site within the basal body region, where it may regulate cilia (dis)assembly and, perhaps as a result, subsequent ciliary signaling events. We propose that ARL6/BBS3 may perform this role by modulating membrane trafficking at the base of the ciliary organelle.