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. Author manuscript; available in PMC: 2013 Dec 15.
Published in final edited form as: Vision Res. 2012 Aug 7;75:5–10. doi: 10.1016/j.visres.2012.07.015

Molecular assemblies that control rhodopsin transport to the cilia

Dusanka Deretic 1,2, Jing Wang 1
PMCID: PMC3514645  NIHMSID: NIHMS404036  PMID: 22892112

Abstract

This review will focus on the conserved molecular mechanisms for the specific targeting of rhodopsin and rhodopsin-like sensory receptors to the primary cilia. We will discuss the molecular assemblies that control the movement of rhodopsin from the central sorting station of the cell, the trans-Golgi network (TGN), into membrane-enclosed rhodopsin transport carriers (RTCs), and their delivery to the primary cilia and the cilia-derived sensory organelles, the rod outer segments (ROS). Recent studies reveal that these processes are initiated by the synergistic interaction of rhodopsin with the active form of the G-protein Arf4 and the Arf GTPase activating protein (GAP) ASAP1. During rhodopsin progression, ASAP1 serves as an activation platform that brings together the proteins necessary for transport to the cilia, including the Rab11a-Rabin8-Rab8 complex involved in ciliogenesis. These specialized molecular assemblies, through successive action of discrete modules, cooperatively determine how rhodopsin and other rhodopsin-like signaling receptors gain access to primary cilia.

Keywords: Arfs, Arf GAPs, Cilium, Rabs, Rhodopsin, Trafficking

ROS is a modified primary cilium

Primary cilia are specialized membrane projections that are found on most eukaryotic cell types. Although their architecture varies, from a simple membrane outgrowth to elaborate specialized organelles, such as the retinal photoreceptor rod outer segments (ROS), their common function is to capture extracellular signals. Together with other components of the signal transduction complexes, sensory receptors are highly concentrated in ciliary membranes allowing for exceptional sensitivity to external stimuli. In the case of vision, the architecture and the molecular composition of the ROS provide for the optimum performance underlying extraordinary light sensitivity, i.e. the light receptor rhodopsin and associated phototransduction machinery concentrated in the stacked disk membranes ensure the capture of a single photon of light (Burns & Arshavsky, 2005, Rieke & Baylor, 1998).

The challenge in maintaining the distinctive light sensitivity of the ROS is partially met by the tight control of the protein entrance across the base, the connecting cilium, which is equivalent to the transition zone of primary (non-motile) cilia. Rhodopsin constitutes the main ciliary-targeted cargo protein in rod photoreceptors. Its delivery to ROS is an outstanding case of ciliary receptor transport, with connecting cilia trafficking ~1000 rhodopsin molecules per second (Besharse, 1986). Light sensitivity is maintained through the continuous renewal of the light sensing rhodopsin-laden membranes that are shed and engulfed by the adjacent retinal pigment epithelium, combined with the light-dependent entry and exit of the cytosolic components of the signal transduction machinery and the selective delivery of the lipid-modified peripheral membrane proteins (Calvert, et al., 2006, Insinna & Besharse, 2008, Najafi, et al., 2011, Wright, et al., 2011, Zhang, et al., 2011).

The selective inclusion of ROS-specific molecular complexes and the exclusion of the macromolecules that function in other parts of the cell are ongoing processes throughout the life of the photoreceptor, which often go awry when the key proteins required for the function and maintenance of this specialized organelle are affected by disease-causing mutations. The transition zone of the photoreceptor cilia houses a network of highly conserved proteins affected by a wide range of human diseases and developmental disorders known as ciliopathies. These include Senior-Loken, Jeune and Bardet-Biedel Syndrome (BBS), which are characterized by both retinal degeneration and cystic kidneys, frequently combined with obesity, polydactyly and sensory impairments (Blacque & Leroux, 2006, Fliegauf, et al., 2007, Gerdes, et al., 2009).

Rhodopsin trafficking to the ciliary base

Like all membrane proteins, rhodopsin follows the intracellular path from the site of its synthesis, the endoplasmic reticulum (ER) to the Golgi complex responsible for N-linked oligosaccharide modifications, sorting and transport. The ER and the Golgi are localized in the myoid part of the rod inner segment (RIS)(Fig 1A). After exiting the Golgi complex, rhodopsin reaches the TGN where it is specifically incorporated into rhodopsin transport carriers (RTCs) (Deretic, 2010), which traverse the mitochondria-rich ellipsoid region on their way to the base of the cilia. All primary cilia are contiguous with, but separated from the plasma membrane by the periciliary diffusion barrier. The exact nature of the periciliary diffusion barrier in photoreceptor cells is unclear at present, but is likely to combine specific lipid ordering, Septin rings and transition zone protein complexes (Besharse, 1986, Chih, et al., 2012, Garcia-Gonzalo, et al., 2011, Hu, et al., 2010, Nachury, et al., 2010, Vieira, et al., 2006). The periciliary membrane and the specialized membrane domain called the periciliary ridge complex (PRC) (Papermaster, et al., 1985, Peters, et al., 1983), function as a site for docking and fusion of RTCs.

Figure 1.

Figure 1

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(A) Diagram of a photoreceptor cell. Primary cilium originates from the basal body (BB) in the rod inner segment (RIS) and the axoneme elaborates the rod outer segment (ROS). The transition zone is a “connecting cilium”, the gateway to the ROS. Golgi and the TGN are localized in the myoid region (M) of the RIS. RTCs that bud from the TGN are targeted to the base of the cilium (arrow), through the ellipsoid region (E) filled with mitochondria (m). N, nucleus; Sy, synapse. (B) Molecular interactions taking place during rhodopsin progression to the ciliary base. At the TGN, activated Arf4 interacts with rhodopsin and they recruit ASAP1 into the ternary complex. ASAP1 likely initiates membrane deformation through its BAR domain while mediating GTP-hydrolysis on Arf4, which then dissociates from the TGN. Next, ASAP1 selectively binds Rab11a, which also associates with rhodopsin. ASAP1 and Rab11a recruit Rabin8 and Rab8. On RTCs, ASAP1 serves as a scaffold for the Rab11a/Rabin8/Rab8 complex, which controls the activation of Rab8. Activated Rab8 regulates RTCs fusion and the delivery of rhodopsin across the diffusion barrier surrounding the cilium. Rhodopsin then proceeds through the transition zone (or the connecting cilium) into the ciliary axoneme that forms the ROS.

ASAP1 as a component of a putative protein coat that regulates rhodopsin trafficking

Precious little is known about how membrane cargo moves from the TGN to the specialized organelles like primary cilia. Earlier studies have identified ubiquitous protein complexes that coat membranes and direct movement of cargo between intracellular organelles. For example, membrane transport between the ER and the Golgi, as well as the intra-Golgi transport, are mediated by the COPI and COPII families of coat proteins (Lee, et al., 2004). Our studies suggest that rhodopsin post-TGN trafficking to the cilia is regulated by a new, specialized system that differs from the well-studied assemblies that control the distribution of proteins within a cell. This molecular assembly functions as an effector of Arf4 at the TGN, where it forms a ciliary targeting module comprised of (i) the Arf GTPase activating protein (GAP) ASAP1, (ii) the small GTPase Rab11 and (iii) the Rab11/Arf effector FIP3 (Mazelova, et al., 2009a) (Fig 1B). Arf4 is a member of the Arf family of G-proteins that regulate membrane transport (Donaldson & Jackson, 2011), whereas Rab11 belongs to the Rab family of GTPases that together with protein coats, membrane tethers and SNAREs regulate intracellular membrane targeting (Cai, et al., 2007). FIP3 is a dual Arf/Rab11 effector that may serve to coordinate the activities of Arfs and Rabs (Hales, et al., 2001, Hickson, et al., 2003). FIP3 also interacts with ASAP1 (Inoue, et al., 2008), thus linking all known components of the ciliary targeting module.

ASAP1 is a multifunctional scaffold protein that acts as a regulator of rhodopsin trafficking through GTP hydrolysis on Arf4 and functions as an Arf4 effector that regulates RTC budding (Mazelova et al., 2009a). Arf4 mutant I46D that selectively abolishes ASAP1-mediated GTP hydrolysis disrupts RTC budding, causing rhodopsin mislocalization and rapid retinal degeneration in transgenic animals (Mazelova et al., 2009a). The functional domains of ASAP1 include the Arf GAP domain, pleckstrin homology (PH), SH3, proline-rich, and N-terminal BAR domain (Nie & Randazzo, 2006, Randazzo & Hirsch, 2004). The phosphorylation of the C-terminal SH3 and proline-rich domains, involved in protein-protein interactions, affects the conformation and the activity of ASAP1 (Nie & Randazzo, 2006, Randazzo & Hirsch, 2004). The N-terminal BAR (Bin/amphiphysin/Rvs) domain (Peter, et al., 2004), mediates membrane tubulation and homodimerization of ASAP1, and acts as an autoinhibitor of its GAP activity (Jian, et al., 2009, Nie, et al., 2006).

Our studies indicate that targeting of cilliary cargo presented in the context of Arf4 involves a stepwise assembly of ASAP1-associated complexes. This mechanism of action is distinctly different from that of the BBSome, a coat protein complex that also acts as an effector of Arf family proteins (Arl6), which functions in ciliary sorting of the GPCR Sstr3, although it lacks the capacity to deform the membrane (Jin, et al., 2010). These differences parallel the differences in the assembly of the COPI and COPII coat protein complexes involved in bi-directional ER-Golgi trafficking (Lee et al., 2004). ASAP1 resembles the Sec23 component of the COPII coat, which acts as a GAP for the Arf-family GTPase Sar1, and controls the forward direction of vesicle traffic (Lord, et al., 2011). The en-block assembly of the BBSome resembles the assembly of the COPI coat involved in retrograde trafficking. The release of the COPII coat is regulated by the phosphorylation of the Sec23/Sec24 complex, so it is possible that the phosphorylation of the C-terminal protein-protein interaction domains of ASAP1 also regulates its release from RTCs.

Recently, a combination of proteomic, structural and comparative genomic studies has revealed that membrane-deforming machineries, which control intracellular trafficking by generating vesicles and tubules carrying cargo proteins, belong to three families: i) protocoatomer-derived, which function in endo- and exocytosis (clathrin, COPI and COPII), ii) ESCRT, which function in the generation of multivesicular bodies and iii) BAR domain-associated complexes, which function in the Golgi-to-endosome trafficking as well as in endo- and phagocytosis (amphiphysin, FBP-17) (Field, et al., 2011). In this context, ASAP1 could be viewed as a component of a BAR domain-associated complex that regulates ciliary targeting of sensory receptors such as rhodopsin. ASAP1 could also be a component of a membrane-deforming protein coat. Its coat-like properties include the regulation by a member of the Arf family (Arf4), oligomerization and membrane curvature-inducing activity, and formation of a heterotrimeric complex with Rab11 and FIP3 (Inoue et al., 2008), which also acts as an oligomer. FIP3 may form a coat with ASAP1, or may link the ASAP1-based coat to the dynein motor (Horgan, et al., 2010) for transport of RTCs to the base of the cilium.

Rhodopsin has two functional ciliary targeting signals, the VxPx and the FR, which are recognized by Arf4 and ASAP1, respectively

One of the main functions of the canonical coat complexes is the recognition of intracellular targeting signals in cargo proteins (Springer, et al., 1999). Rhodopsin possesses the C-terminal VxPx targeting motif that regulates Arf4 binding and rhodopsin targeting in vitro and in vivo (Concepcion, et al., 2002, Deretic, et al., 1998, Deretic, et al., 2005, Green, et al., 2000, Li, et al., 1996, Tam, et al., 2000). The highly conserved VxPx motif is also functional in polycystins 1 and 2, and the cyclic nucleotide-gated channel CNGB1b subunit (Geng, et al., 2006, Jenkins, et al., 2006, Ward, et al., 2011). The second ciliary targeting signal, the FR motif, which is present in several other GPCRs (Corbit, et al., 2005), lies within the cytoplasmic helix H8 of rhodopsin (Fig 2). Our recent studies indicate that the FR motif binds to ASAP1, implicating Arf4 and ASAP1 as the recognition module for the VxPx- and FR-containing cargo. We find that, unlike the WT, the rhodopsin FR-AA mutant defective in ASAP1 binding does not localize to cilia in the heterologous expression system, in epithelial IMCD3 cells. In this aspect, rhodopsin FR-AA mutant is comparable to smoothened Ciliary Localization Deficient (CLDSmo) mutant (Corbit et al., 2005). Interestingly, the GPCR Sstr3 that is sorted by the BBSome also contains a homologous motif that is apparently not employed for its ciliary targeting (Berbari, et al., 2008). This difference may reflect the cell specificity, or the existence of parallel pathways to cilia characterized by their dependency on the BBSome or ASAP1.

Figure 2.

Figure 2

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(A) Schematic of bovine rhodopsin with protein backbone shown with a transparent surface, chromophore in purple, palmitic acid residues in green and oligosaccharides in blue (after (Palczewski, et al., 2000)). The cytoplasmic C-terminal VxPx motif, which is the Arf4 binding site, is circled. The FR motif, which binds ASAP1 is within α helix H-8 (circled). (B) GPCRs containing the VxPx (top) and FR targeting sequences (bottom) are aligned. The VxPx C-terminal ciliary targeting signal (CTS) of rhodopsin is a hot spot for ADRP mutations, which are indicated in the figure (asterisk denotes a single amino acid substitution, arrow an insertion and bent arrow a truncation). Truncation mutation and insertion disrupting the rhodopsin CTS FR also result in ADRP. The sequence alignment of GPCRs containing the FR motif is according to (Corbit et al., 2005). However, we changed the alignment of Sstr3 to reflect the presence of the FR signal, which lies three amino acids downstream of the FK signal previously identified by Corbit et al. Interestingly, the FK signal has been mutagenized and found non-functional by Mykytyn and colleagues (Berbari et al., 2008), which leaves the role of the FR signal in the ciliary targeting of Sstr3 undetermined.

Regulation of the putative protein coat

In addition to signal recognition, coat complexes also deform membranes. Our experiments suggest that activated Arf4 and ASAP1 are necessary and sufficient for ciliary cargo recognition in vitro, however membrane lipids probably affect complex formation under physiological conditions. This is highly likely because ASAP1 acts as a coincidence detector for acidic phospholipids and activated Arfs, and functions as an Arf effector by increasing membrane curvature (Nie et al., 2006). The formation of the rhodopsin-Arf4-ASAP1 complex in the presence of acidic phospholipids at the TGN likely results in membrane deformation and packaging of rhodopsin into budding RTCs, a conclusion supported by our finding that ASAP1 stimulates RTC budding in vitro (Mazelova et al., 2009a).

ASAP1 functions as a temporal regulator of ciliary traffic through GTP hydrolysis on Arf4. A conformational change involving the Arf-ASAP1 complex is required to disable the autoinhibition of GAP activity blocked by the BAR domain (Jian et al., 2009, Luo, et al., 2007). This autoinhibition can be alleviated by phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) and/or the binding of FIP3 to the BAR domain of ASAP1 (Inoue et al., 2008, Jian et al., 2009). Since FIP3 is a component of the ciliary targeting module that regulates rhodopsin trafficking, it could serve to activate the GAP activity of ASAP1 in photoreceptor cells. An appealing possibility is that PI(4,5)P2 determines the binding partner for ASAP1, since it regulates both its GAP activity and interaction with FIP3. Our studies implicate PI(4,5)P2 as a crucial component necessary for RTC fusion (Deretic, et al., 2004) and this function could involve the regulation of the assembly/disassembly of the putative ASAP1/FIP3 coat.

The role of rhodopsin as a cargo protein may be to stabilize the inhibitory conformation of the BAR domain of ASAP1, precluding premature GTP hydrolysis on Arf4. A cargo-induced pause could stall the complex and prolong the effector function of ASAP1, facilitating the collection of cargo into ciliary-targeted RTCs. This implies that in the absence of the ciliary cargo at the TGN, ASAP1-mediated GTP hydrolysis leads to the release of Arf4 from the membrane. This concept is supported by our study showing that Arf4 is absent from the photoreceptor TGN when its access to rhodopsin is blocked (Mazelova et al., 2009a).

ASAP1 acts as a scaffold for the Rab11a-Rabin8-Rab8 ciliogenesis cascade

The Rab11a-Rabin8-Rab8 cilia-generating module is a regulatory complex that governs ciliary targeting and ciliogenesis (Bryant, et al., 2010, Knodler, et al., 2010, Westlake, et al., 2011). The guanine nucleotide exchange factor (GEF) for Rab8, Rabin8, colocalizes with ASAP1 and Rab11 at the photoreceptor TGN. Rabin8 specifically associates with ASAP1 and the inactive form of Rab8, indicating that in photoreceptor cells ASAP1 acts as a scaffold for the Rab11a-Rabin8-Rab8 ciliogenesis cascade (Fig 1B). Interestingly, retinal photoreceptors possess an additional Rab8 GEF named Retinitis Pigmentosa GTPase Regulator (RPGR). Mutations in RPGR that reduce its GEF activity cause photoreceptor degeneration in X-linked retinitis pigmentosa (XLRP)(Murga-Zamalloa, et al., 2010). The presence of two Rab8 GEFs suggests that the additional level of Rab8 control may be needed for the extraordinary ciliary traffic that supports continuous ciliary membrane expansion in photoreceptor cells.

Our data suggest that Rab11a-Rabin8-Rab8 complex in addition to ciliogenesis also regulates ciliary access and acts as a ciliary destination module. This module allows the cargo that is vetted by the Arf4/ASAP1 cargo recognition module to pass across the periciliary diffusion barrier. Interestingly, our studies show that rhodopsin binds Rab11 and Rab8 directly, similar to other ciliary sensory receptors (Follit, et al., 2010, Ward et al., 2011). This binding might strengthen cargo interaction with the ciliary destination module. Both Rab11a and Rab8a interact with the Sec6/8 membrane tethering complex (Bryant et al., 2010). The Sec6/8 complex is localized at the base of the photoreceptor cilium (Mazelova, et al., 2009b), consistent with its role as an effector for the ciliary cargo destination module. The final steps in RTC fusion at the base of the cilium involve SNAREs Syntaxin 3 and SNAP-25. Surprisingly, docosahexaenoic acid (DHA), an essential polyunsaturated fatty acid of the ROS, increases Syntaxin 3 and SNAP-25 pairing, thus regulating ciliary membrane expansion (Mazelova et al., 2009b). Once in the ciliary membrane, rhodopsin likely moves towards the ROS through different molecular assemblies that include kinesin microtubule motors and the ubiquitous IFT machinery (Bhowmick, et al., 2009, Insinna, et al., 2009, Keady, et al., 2011, Lopes, et al., 2010, Pazour, et al., 2002).

Retinopathy-associated proteins and degenerative diseases

Mutations in the rhodopsin gene that cause autosomal dominant retinitis pigmentosa (ADRP) (http://www.retina-international.com/sci-news/rhomut.htm) affect both the VxPx and the FR targeting signals that are essential for the correct delivery to the ciliary membrane (Fig 2). Disruptions of the VxPx motif lead to some of the most severe forms of ADRP (Berson, et al., 2002, Bessant, et al., 1999). Our studies suggest that the FR targeting signal that interacts with ASAP1 critically affects ciliary access of rhodopsin in the heterologous expression system, in epithelial cells. The interaction of the rhodopsin FR ciliary targeting signal with ASAP1 in retinal photoreceptors remains to be confirmed, however ADRP mutations affecting the FR signal suggest that it also plays a role in ROS biogenesis.

One of the regulators of RTC trafficking to the ciliary base, Rab8 has recently emerged as a crucial regulator of ciliogenesis and ciliary targeting (Bryant et al., 2010, Deretic, et al., 1995, Knodler et al., 2010, Moritz, et al., 2001, Omori, et al., 2008, Westlake et al., 2011, Yoshimura, et al., 2007). Rab8 directly interacts with: (i) the ciliary cargo such as rhodopsin (our studies), Polycystin 1 and fibrocystin (Follit et al., 2010, Ward et al., 2011), (ii) the BBSome complex affected by mutations causing the Bardet-Biedl syndrome (Nachury, et al., 2007), (iii) RPGR, which is mutated in XLRP (Murga-Zamalloa et al., 2010) and (iv) CEP-290 (Kim, et al., 2008, Tsang, et al., 2008) that organizes NPHP, MKS and JBTS complexes at the transition zone, where they restrict the diffusion of membrane proteins across the ciliary base (Craige, et al., 2010, Garcia-Gonzalo et al., 2011, Sang, et al., 2011, van Reeuwijk, et al., 2011). Mutations in NPHP, MKS and JBTS proteins cause nephronophthisis, Meckel syndrome and Joubert syndrome. NPHP and MKS proteins interact with IFT complexes and support the ciliary transport of rhodopsin, but not peripherin (Zhao & Malicki, 2011). Finally, in rhodopsin trafficking Rab8 appears to be functionally linked to Tulp1, and mutations in TULP1 gene cause autosomal recessive RP and LCA (Hagstrom, et al., 2012). Thus, the molecular assemblies that control rhodopsin transport to the cilia that are described here exert the principal organizing function for the proteins involved in cilia structure and function, which account for the largest group among the genes linked to photoreceptor degeneration. Future studies of molecular assemblies that regulate the biogenesis of cilia and cilia-derived sensory organelles are likely to reveal the additional involvement of the specific members of the regulatory machinery described here in ciliopathies and other degenerative diseases.

  • Molecular assemblies involved in cilia structure and function are discussed.

  • Photoreceptor rod outer segment (ROS) is a modified primary cilium

  • Rhodopsin regulates ROS assembly through the G-protein Arf4 and its GAP ASAP1.

  • Arf4 and ASAP1 recognize rhodopsin VxPx and the FR targeting signals, respectively.

  • ASAP1 coordinates the proteins necessary for the ciliary transport of rhodopsin.

Acknowledgments

We thank lab members and collaborators for their valuable contributions and many colleagues for their generous gifts of reagents and fruitful discussions. We also thank Kris Palczewski for the image of bovine rhodopsin. Supported by the NIH grant EY-12421.

The abbreviations used are

RIS

Rod Inner Segment(s)

ROS

Rod Outer Segment(s)

RTC(s)

Rhodopsin Transport Carrier(s)

RP

Retinitis Pigmentosa

SNARE

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor

TGN

Trans-Golgi Network

Footnotes

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