ASAP1 is a dimeric ARF GTPase activating protein (GAP) and scaffolding protein that is recruited to the trans-Golgi network (TGN) through interactions with activated ARF4, PI(4,5)P2 and acidic phospholipids (Brown et al, 1998; Che et al, 2005; Nie et al, 2006). Once at the TGN, ASAP1 forms a tripartite complex with ARF4 and ciliary cargo, possibly by interacting with a putative C-terminal FR targeting motif present in a number of membrane proteins destined for the cilium, although this remains to be conclusively demonstrated (Corbit et al, 2005; Wang et al, 2012; reviewed in Bhogaraju et al, 2013). In addition to its role as an ARF GAP, ASAP1 also scaffolds the recruitment of a number of other proteins required for ciliary targeting, including RAB11 and the RAB11 effector FIP3 (Mazelova et al, 2009; Inoue et al, 2008; reviewed in Deretic 2013).

Recruitment of RAB11FIP3 to the trans-Golgi network (TGN) simulates ASAP1 to activate the ARF4 GTPase activity, causing the hydrolysis of GTP and the release of ARF4:GDP from the Golgi membrane (Mazelova et al, 2009; Inoue et al, 2008; reviewed in Deretic, 2013).

Recruitment of ASAP1 to the TGN facilitates the subsequent recruitment of both RAB11A and the RAB11 effector protein FIP3 to the ciliary targeting complex. RAB11FIP3 functions as a homodimer and can bind simultaneously to RAB11 and ARF4 through its C-terminal region (Inoue et al, 2008; Mazelova et al, 2009; Wang et al, 2012; Shiba et al, 2006; Eathiraj et al, 2006; Schonteich et al, 2007). RAB11FIP3 also interacts with the BAR domain of ASAP1 and in this way may play a role in stimulating the ARF GAP activity of ASAP1, promoting the inactivation ARF4 and its subsequent dissociation from the TGN (Inoue et al, 2008; reviewed in Deretic, 2013).

Membrane budding at the trans-Golgi network is promoted at least in part by the BAR domain of ASAP1, which is involved in sensing and inducing membrane curvature as well as providing the recognition site for small GTPases (Nie et al, 2006; Jian et al, 2009; Inoue et al, 2008; reviewed in Masuda et al, 2010). Oligomerization between ASAP1 and RAB11FIP3 may contribute to coat formation on vesicles budding from the TGN and destined for the plasma or ciliary membrane (Inoue et al, 2008; Mazelova et al, 2009; reviewed in Deretic, 2013).

Exocyst-mediated fusion of the Golgi-derived vesicle delivers the VxPx-containing membrane proteins to the ciliary membrane, although the precise mechanisms remain to be worked out (Mazelova et al, 2009; Wang et al, 2012; reviewed in Sung and Leroux, 2013). Vesicles carrying membrane proteins destined for the cilum may fuse at the periciliary membrane at the base of the cilium and deliver cargo to the IFT system. Ciliary membrane proteins may also diffuse laterally into the periciliary membrane after fusion of vesicles with the plasma membrane (reviewed in Hsiao et al, 2012; Sung and Leroux, 2013). Although not depicted in this reaction, there is evidence that some of the protein-protein interactions of the ciliary-targeting complex may persist into the periciliary or ciliary membrane region (Wang et al, 2012).

RAB8A is another small GTPase that is required for ciliogenesis. RAB8A is recruited to the ciliary targeting complex at the trans-Golgi network (TGN) through interactions of the RAB8A guanine nucleotide exchange factor (GEF) RAB3IP (also known as RABIN8) with ASAP1 and RAB11 (Wang et al, 2012; Westlake et al, 2011; Feng et al, 2012; reviewed in Deretic, 2013). RAB8A is recruited in the inactive GDP bound form, and is activated at the TGN by RAB3IP in a RAB11A-dependent fashion (Hatulla et al, 2002; Knodler et al, 2010; Westlake et al, 2012; Wang et al, 2012; Feng et al, 2012).

Once recruited to the ciliary targeting complex, RAB3IP/RABIN8 stimulates nucleotide exchange on RAB8A. Activated RAB8A is required for ciliogenesis and plays a role in mediating vesicle docking at the basal body, providing both lipid and protein content to the emerging cilium (Hattula et al, 2002; Knodler et al, 2010; Nachury et al, 2007; Wang et al, 2012; Westlake et al, 2011; Yoshimura et al, 2007; reviewed in Deretic, 2013; Sung and Leroux, 2013).

Proteomic studies suggest that the cilium is home to approximately a thousand proteins, and has a unique protein and lipid make up relative to the bulk cytoplasm and plasma membrane (Pazour et al, 2005; Ishikawa et al, 2012; Ostrowoski et al, 2002; reviewed in Emmer et al, 2010; Rohatgi and Snell, 2010). In addition, the cilium is a dynamic structure, and the axoneme is continually being remodeled by addition and removal of tubulin at the distal tip (Marshall and Rosenbaum, 2001; Stephens, 1997; Song et al, 2001). As a result, the function and structure of this organelle relies on the directed trafficking of protein and vesicles to the cilium. Small GTPases of the RAS, RAB, ARF and ARL families are involved in cytoskeletal organization and membrane traffic and are required to regulate the traffic from the Golgi and the trans-Golgi network to the cilium (reviewed in Deretic, 2013; Li et al, 2012). ARF4 is a Golgi-resident GTPase that acts in conjunction with a ciliary-targeting complex consisting of the ARF-GAP ASAP1, RAB11A, the RAB11 effector FIP3 and the RAB8A guanine nucleotide exchange factor RAB3IP/RABIN8 to target cargo bearing a putative C-terminal VxPx targeting motif to the cilium. A well-studied example of this system involves the trafficking of rhodopsin to the retinal rod photoreceptors, a specialized form of the cilium (reviewed in Deretic, 2013). ARL3, ARL13B and ARL6 are all small ARF-like GTPases with assorted roles in ciliary trafficking and maintenance. Studies in C. elegans suggest that ARL3 and ARL13B have opposing roles in maintaining the stability of the anterograde IFT trains in the cilium (Li et al, 2010). In addition, both ARL3 and ARL13B have roles in facilitating the traffic of subsets of ciliary cargo to the cilium. Myristoylated cargo such as peripheral membrane protein Nephrocystin-3 (NPHP3) is targeted to the cilium in a UNC119- and ARL3-dependent manner, while ARL13B is required for the PDE6-dependent ciliary localization of INPP5E (Wright et al, 2011; Humbert et al, 2012; reviewed in Li et al, 2012). ARL6 was also identified as BBS3, a gene that when mutated gives rise to the ciliopathy Bardet-Biedl syndrome (BBS). ARL6 acts upstream of a complex of 8 other BBS-associated proteins known as the BBSome. ARL6 and the BBSome are required for the ciliary targeting of proteins including the melanin concentrating hormone receptor (MCHR) and the somatostatin receptor (SSTR3), among others (Nachury et al, 2007; Loktev et al, 2008; Jin et al, 2010; Zhang et al, 2011). Both the BBSome and ARL6 may continue to be associated with cargo inside the cilium, as they are observed to undergo typical IFT movements along the axoneme (Fan et al, 2004; Lechtreck et al, 2009; reviewed in Li et al, 2012).