Dysfunctions of primary cilia and cilia-derived sensory organelles underlie a multitude of human disorders, including retinal degeneration, yet membrane targeting to the cilium remains poorly understood. Here, we show that the newly identified ciliary targeting VxPx motif present in rhodopsin binds the small GTPase Arf4 and regulates its association with the trans-Golgi network (TGN), which is the site of assembly and function of a ciliary targeting complex. This complex is comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf effector FIP3, and the Arf GTPase-activating protein ASAP1. ASAP1 mediates GTP hydrolysis on Arf4 and functions as an Arf4 effector that regulates budding of post-TGN carriers, along with FIP3 and Rab11. The Arf4 mutant I46D, impaired in ASAP1-mediated GTP hydrolysis, causes aberrant rhodopsin trafficking and cytoskeletal and morphological defects resulting in retinal degeneration in transgenic animals. As the VxPx motif is present in other ciliary membrane proteins, the Arf4-based targeting complex is most likely a part of conserved machinery involved in the selection and packaging of the cargo destined for delivery to the cilium.

Mislocalization of the photopigment rhodopsin may be involved in the pathology of certain inherited retinal degenerative diseases. Here, we have elucidated rhodopsin's targeting signal which is responsible for its polarized distribution to the rod outer segment (ROS). Various green fluorescent protein (GFP)/rhodopsin COOH-terminal fusion proteins were expressed specifically in the major red rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The fusion proteins were targeted to membranes via lipid modifications (palmitoylation and myristoylation) as opposed to membrane spanning domains. Membrane association was found to be necessary but not sufficient for efficient ROS localization. A GFP fusion protein containing only the cytoplasmic COOH-terminal 44 amino acids of Xenopus rhodopsin localized exclusively to ROS membranes. Chimeras between rhodopsin and α adrenergic receptor COOH-terminal sequences further refined rhodopsin's ROS localization signal to its distal eight amino acids. Mutations/deletions of this region resulted in partial delocalization of the fusion proteins to rod inner segment (RIS) membranes. The targeting and transport of endogenous wild-type rhodopsin was unaffected by the presence of mislocalized GFP fusion proteins.

Rab8 is a GTPase involved in membrane trafficking. In photoreceptor cells, rab8 is proposed to participate in the late stages of delivery of rhodopsin-containing post-Golgi membranes to the plasma membrane near the base of the connecting cilium. To test the function of rab8 in vivo, we generated transgenic Xenopus laevis expressing wild-type, constitutively active (Q67L), and dominant negative (T22N) forms of canine rab8 in their rod photoreceptors as green fluorescent protein (GFP) fusion proteins. Wild-type and constitutively active GFP-rab8 proteins were primarily associated with Golgi and post-Golgi membranes, whereas the dominant negative protein was primarily cytoplasmic. Expression of wild-type GFP-rab8 had minimal effects on cell survival and intracellular structures. In contrast, GFP-rab8T22N caused rapid retinal degeneration. In surviving peripheral rods, tubulo-vesicular structures accumulated at the base of the connecting cilium. Expression of GFP-rab8Q67L induced a slower retinal degeneration in some tadpoles. Transgene effects were transmitted to F1 offspring. Expression of the GFP-rab8 fusion proteins appears to decrease the levels of endogenous rab8 protein. Our results demonstrate a role for rab8 in docking of post-Golgi membranes in rods, and constitute the first report of a transgenic X. laevis model of retinal degenerative disease. INTRODUCTIONVertebrate rod photoreceptors are highly polarized neurons. They possess a light-detecting organelle, the rod outer segment (OS), which is separated from the cell body (the inner segment [IS]) by a connecting cilium (CC) and is composed of a stack of rhodopsin-containing membranous disks. OS membranes are continuously renewed (Young and Droz, 1968). Amphibian rods synthesize photosensitive membranes at an extremely high rate, estimated at 3.2 m 2 /min/ cell for Xenopus laevis (Besharse et al., 1977;Besharse, 1986). Amphibian rods are an excellent system for studying neuronal membrane transport, because the components involved are likely to be hypertrophied to accommodate high synthetic rates. Furthermore, rods are sensitive to trafficking disruptions; mutations in the rhodopsin gene that disrupt a sorting signal result in IS accumulation of rhodopsin and cause retinitis pigmentosa (Berson 1993;Sung et al., 1994;Deretic et al., 1998;Tam et al., 2000).Eukaryotic cells possess complex mechanisms to control membrane trafficking between intracellular compartments. Rab proteins are highly conserved GTPases involved in trafficking, although their role is obscure. The human genome contains genes encoding at least 60 rab family members (Bock et al., 2001), each of which may participate in a different trafficking pathway (Novick and Zerial, 1997). Rab8 and rab6, as well as other GTP-binding proteins, are associated with rhodopsin-containing post-Golgi membranes isolated from frog photoreceptors (Deretic et al., 1995;Deretic and Papermaster, 1993). Peptides derived from the effector region of rab6 (Deretic, 1998) and the C terminus of rhodopsin inhibit the format...

The unique structural feature of the dilysine (Lys206-Lys296) pair in the transferrin N-lobe (hTF/2N) has been postulated to serve a special function in the release of iron from the protein. These two lysines, which are located in opposite domains, hydrogen bond to each other in the iron-containing hTF/2N at neutral pH but are far apart in the apo-form of the protein. It has been proposed that charge repulsion resulting from the protonation of the dilysines at lower pH may be the trigger to open the cleft and facilitate iron release. The fact that the dilysine pair is positively charged and resides in a location close to the metal-binding center has also led to the suggestion that the dilysine pair is an anion-binding site for chelators. The present report provides comprehensive evidence to confirm that the dilysine pair plays this dual role in modulating release of iron. When either of the lysines is mutated to glutamate or glutamine or when both are mutated to glutamate, release of iron is much slower compared to the wild-type protein. This is due to the fact that the driving force for cleft opening is absent in the mutants or is converted to a lock-like interaction (in the case of the K206E and K296E mutants). Direct titration of the apo-proteins with anions as well as anion-dependent iron release studies show that the dilysine pair is part of an active anion-binding site which exists with the Lys296-Tyr188 interaction as a core. At this site, Lys296 serves as the primary anion-binding residue and Tyr188 is the main reporter for electronic spectral change, with smaller contributions from Lys206, Tyr85, and Tyr95. In iron-loaded hTF/2N, anion binding becomes invisible as monitored by UV-vis difference spectra since the spectral reporters Tyr188 and Tyr95 are bound to iron. Our data strongly support the hypothesis that the apo-hTF/2N exists in equilibrium between the open and closed conformations, because only in the closed form is Lys296 in direct contact with Tyr188. The current findings bring together observations, ideas, and experimental data from a large number of previous studies and shed further light on the detailed mechanism of iron release from the transferrin N-lobe. In iron-containing hTF/2N, Lys296 may still function as a target to introduce an anion (or a chelator) near to the iron-binding center. When the pH is lowered, the protonation of carbonate (synergistic anion for metal binding) and then the dilysine pair form the driving force to loosen the cleft, exposing iron; the nearby anion (or chelator) then binds to the iron and releases it from the protein.

Protein targeting is essential for domain specialization in polarized cells. In photoreceptors, three distinct membrane domains exist in the outer segment: plasma membrane, disk lamella, and disk rim. Peripherin/retinal degeneration slow (rds) and rom-1 are photoreceptor-specific members of the transmembrane 4 superfamily of transmembrane proteins, which participate in disk morphogenesis and localize to rod outer segment (ROS) disk rims. We examined the role of their C termini in targeting by generating transgenic Xenopus laevis expressing green fluorescent protein (GFP) fusion proteins. A GFP fusion containing residues 317-336 of peripherin/rds localized uniformly to disk membranes. A longer fusion (residues 307-346) also localized to the ROS but exhibited higher affinity for disk rims than disk lamella. In contrast, the rom-1 C terminus did not promote ROS localization. The GFP-peripherin/rds fusion proteins did not immunoprecipitate with peripherin/rds or rom-1, suggesting this region does not form intermolecular interactions and is not involved in subunit assembly. Presence of GFP-peripherin/rds fusions correlated with disrupted incisures, disordered ROS tips, and membrane whorls. These abnormalities may reflect competition of the fusion proteins for other proteins that interact with peripherin/rds. This work describes novel roles for the C terminus of peripherin/rds in targeting and maintaining ROS structure and its potential involvement in inherited retinal degenerations. INTRODUCTIONAlthough numerous membrane proteins have sequences that target them to intracellular compartments (e.g., endoplasmic reticulum, lysosomes, and nuclei), the molecular interactions governing the distribution of proteins to the plasma membrane of polarized cells are less clearly defined because of the diversity of unique proteins and membrane domains. Our group is particularly interested in the mechanisms governing the targeting of proteins to the specialized functional domains of rod photoreceptors. Rods are highly polarized and intricately organized neuronal cells, which reside in the outer retina. Perturbations of their complex cellular architecture lead to retinal degeneration (RD) by triggering apoptosis (Chang et al., 1993;Papermaster and Nir, 1994). It is therefore important to study how these cells create and maintain their unique morphology. Like other neurons, rods possess a synaptic terminal and cell body (named the inner segment or RIS). They also possess a unique organelle, called the outer segment (ROS), which is attached to the RIS by a nonmotile (9 ϩ 0) connecting cilium. The ROS consists of a large stack of flattened membranous disks ensheathed within a plasma membrane ( Figure 1A) and is the site of light capture and signal transduction. The distal ROS tips are shed daily, requiring concomitant synthesis and addition of new disks to the base of the ROS (Young and Droz, 1968).Nascent disks begin as evaginations of the plasma membrane but eventually seal and separate from it. Mature disks have two distinct domains...

To elucidate the molecular mechanisms underlying the light-sensitive retinal degeneration caused by the rhodopsin mutation P23H, which causes retinitis pigmentosa (RP) in humans, we expressed Xenopus laevis, bovine, human, and murine forms of P23H rhodopsin in transgenic X. laevis rod photoreceptors. All P23H rhodopsins caused aggressive retinal degeneration associated with low expression levels and retention of P23H rhodopsin in the endoplasmic reticulum (ER), suggesting involvement of protein misfolding and ER stress. However, light sensitivity varied dramatically between these RP models, with complete or partial rescue by dark rearing in the case of bovine and human P23H rhodopsin, and no rescue for X. laevis P23H rhodopsin. Rescue by dark rearing required an intact 11-cis-retinal chromophore binding site within the mutant protein and was associated with truncation of the P23H rhodopsin N terminus. This yielded an abundant nontoxic ϳ27 kDa form that escaped the ER and was transported to the rod outer segment. The truncated protein was produced in the greatest quantities in dark-reared retinas expressing bovine P23H rhodopsin and was not observed with X. laevis P23H rhodopsin. These results are consistent with a mechanism involving enhanced protein folding in the presence of 11-cis-retinal chromophore, with ER exit assisted by proteolytic truncation of the N terminus. This study provides a molecular mechanism for light sensitivity observed in other transgenic models of RP and for phenotypic variation among RP patients.

To study rhodopsin biosynthesis and transport in vivo, we engineered a fusion protein (rho-GFP) of bovine rhodopsin (rho) and green fluorescent protein (GFP). rho-GFP expressed in COS-1 cells bound 11-cis retinal, generating a pigment with spectral properties of rhodopsin (A max at 500 nm) and GFP (A max at 488 nm). rho-GFP activated transducin at 50% of the wild-type activity, whereas phosphorylation of rho-GFP by rhodopsin kinase was 10% of wild-type levels. We expressed rho-GFP in the rod photoreceptors of Xenopus laevis using the X. laevis principal opsin promoter. Like rhodopsin, rho-GFP localized to rod outer segments, indicating that rho-GFP was recognized by membrane transport mechanisms. In contrast, a rho-GFP variant lacking the Cterminal outer segment localization signal distributed to both outer and inner segment membranes. Confocal microscopy of transgenic retinas revealed that transgene expression levels varied between cells, an effect that is probably analogous to position-effect variegation. Furthermore, rho-GFP concentrations varied along the length of individual rods, indicating that expression levels varied within single cells on a daily or hourly basis. These results have implications for transgenic models of retinal degeneration and mechanisms of position-effect variegation and demonstrate the utility of rho-GFP as a probe for rhodopsin transport and temporal regulation of promoter function.Studies of protein targeting and renewal have largely been carried out in cultured cell systems. However, cultured cells often provide only an approximation of the in vivo situation, particularly when proteins are studied in heterologous cell systems or when interactions between cell types in highly organized tissues may be critical. Here we employ an in vivo system for the study of rhodopsin targeting and renewal in an animal in which simple genetic manipulations are easily accomplished, transgenic Xenopus laevis that express a fluorescent form of rhodopsin. In developing this system, we encountered variable protein expression patterns resembling heterochromatin-associated position-effect variegation. Because of the unique mechanisms of photoreceptor outer segment growth and renewal, we obtained a novel perspective on this previously described artifact of transgenesis.Rhodopsin is the light-sensing visual pigment of vertebrate rod photoreceptor cells, which mediate vision under dim light conditions. It consists of an apoprotein, opsin, with seven membrane-spanning domains and a covalently attached 11-cis retinal chromophore. Upon light absorption, the retinal chromophore undergoes cis-trans isomerization, eliciting a series of conformational changes that initiate intracellular signaling through the G-protein transducin, leading to closure of cation channels in the plasma membrane and hyperpolarization of the cell (1).Rhodopsin is found almost exclusively in the rod outer segment (ROS) 1 compartment of vertebrate rod photoreceptors. The ROS consists of a stack of membranous discs surrounded by a plasma me...

Mutations of the aspartic acid residue at position 63 of the N-lobe of human serum transferrin substantially alter the metal ion- and anion-binding properties of the protein. Substitution of serine, asparagine, glutamic acid, or alanine results in the loss of a key component of the interface in the interdomain cleft and the metal-binding ligand, aspartic acid, leading in all cases to an increased preference for NTA rather than carbonate as the "synergistic" anion relative to the wild-type protein. Excess bicarbonate is required to eliminate the NTA and obtain the "correct" visible spectrum. Carbonate replaces NTA via an intermediate. Blue shifts for the characteristic absorption band of each mutant show a range of effects on the Fe-O (Tyr) interaction. Titration with Co(III) yielded the molecular absorption coefficient for each mutant except D63A, where Co(III) appeared to oxidize the tyrosine residues and damage the ability of the mutant to bind metal. The chelator, Tiron, removes iron from hTF/2N with a simple saturation kinetic mode with respect to the ligand concentration. Chloride inhibits the release in an interesting manner: the effect is initially sharp and then levels off with a minimum k(obs) at [KCl] = 0.5 M. However, the reaction of the D63 mutants with Tiron results in the formation of the ternary complexes Fe-hTF/2N-Tiron. Significant red shifts for the characteristic absorption bands of these complexes suggest a different ligation of Tiron in the mutants from that in wild-type hTF/2N.