SUMMARY UNC119 is widely expressed among vertebrates and invertebrates. Here we report that UNC119 recognized the acylated N-terminus of the rod photoreceptor transducin α-subunit (Tα) as well as C. elegans G proteins Odr-3 and Gpa-13. The crystal structure of human UNC119 at 1.95 Å resolution revealed an immunoglobulin-like β-sandwich fold. Pulldowns and isothermal titration calorimetry revealed a tight interaction between UNC119 and acylated Gα peptides. Co-crystallization of UNC119 with an acylated Tα N-terminal peptide at 2.0 Å revealed that the lipid chain is buried deeply into UNC119's hydrophobic cavity. UNC119 bound TαGTP inhibiting its GTPase activity, thereby providing a stable UNC119-TαGTP complex that is capable of diffusing from the inner segment back to the outer segment following light-induced translocation. UNC119 deletion in both mouse and C. elegans lead to G protein mislocalization. These results establish UNC119 as a novel Gα-subunit cofactor that is essential for G-protein trafficking in sensory cilia.

The retinoid cycle is a recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. Photoreceptor-specific retinol dehydrogenase (prRDH) catalyzes reduction of all-trans-retinal to all-trans-retinol and is thought to be a key enzyme in the retinoid cycle. We disrupted mouse prRDH (human gene symbol RDH8) gene expression by targeted recombination and generated a homozygous prRDH knock-out (prRDH؊/؊) mouse. Histological analysis and electron microscopy of retinas from 6-to 8-week-old prRDH؊/؊ mice revealed no structural differences of the photoreceptors or inner retina. For brief light exposure, absence of prRDH did not affect the rate of 11-cis-retinal regeneration or the decay of Meta II, the activated form of rhodopsin. Absence of prRDH, however, caused significant accumulation of all-trans-retinal following exposure to bright lights and delayed recovery of rod function as measured by electroretinograms and single cell recordings. Retention of all-trans-retinal resulted in slight overproduction of A2E, a condensation product of all-trans-retinal and phosphatidylethanolamine. We conclude that prRDH is an enzyme that catalyzes reduction of all-trans-retinal in the rod outer segment, most noticeably at higher light intensities and prolonged illumination, but is not an essential enzyme of the retinoid cycle.Reduction and oxidation of retinoids are key reactions of the retinoid cycle (visual cycle), which is critical for the production of the chromophore of rhodopsin, 11-cis-retinal (1, 2). When light strikes the visual pigments (rhodopsin and cone opsins) in photoreceptors, it causes the 11-cis-retinylidene chromophore to isomerize to its all-trans configuration, before all-trans-retinal is released from the binding site of the pigments (3) (see Scheme 1). The NADPH-dependent reduction of all-trans-retinal in photoreceptor outer segments is the first step in the regeneration of bleached visual pigment. The reduction occurs directly on the cytoplasmic surface of outer segment disk membranes. Once all-trans-retinal escapes into the internal disk space, it is pumped out to the cytosol by a photoreceptorspecific ATP-binding transporter (4 -8). Several all-trans-retinol dehydrogenases (RDHs) 1 from the photoreceptor cells have been identified. First, Haeseleer et al. (9) cloned a cone-specific enzyme from the short-chain dehydrogenase/reductase (SDR) family with properties that suggest participation in the retinoid cycle. Next, Rattner and colleagues (10) reported the identification of a novel member of the SDR family, photoreceptor RDH (prRDH or RDH8), that localized to photoreceptors and possessed enzymatic properties closely matching those previously reported for RDH activity in ROS. The authors suggested that prRDH is the enzyme responsible for the reduction of all-trans-retinal to all-trans-retinol within the photoreceptor outer segment. The sequence homology among SDRs is typically low (20 -40%), but the structural homology is high and most protein folds are conserved (11). prR...

The mouse Pde6d gene encodes a ubiquitous prenyl binding protein, termed PrBP/␦, of largely unknown physiological function. PrBP/␦ was originally identified as a putative rod cGMP phosphodiesterase (PDE6) subunit in the retina, where it is relatively abundant. To investigate the consequences of Pde6d deletion in retina, we generated a Pde6d ؊/؊ mouse by targeted recombination. Although manifesting reduced body weight, the Pde6d ؊/؊ mouse was viable and fertile and its retina developed normally. Immunocytochemistry showed that farnesylated rhodopsin kinase (GRK1) and prenylated rod PDE6 catalytic subunits partially mislocalized in Pde6d ؊/؊ rods, whereas rhodopsin was unaffected. In Pde6d ؊/؊ rod single-cell recordings, sensitivity to single photons was increased and saturating flash responses were prolonged. Pde6d ؊/؊ scotopic paired-flash electroretinograms indicated a delay in recovery of the dark state, likely due to reduced levels of GRK1 in rod outer segments. In Pde6d ؊/؊ cone outer segments, GRK1 and cone PDE6␣ were present at very low levels and the photopic b-wave amplitudes were reduced by 70%. Thus the absence of PrBP/␦ in retina impairs transport of prenylated proteins, particularly GRK1 and cone PDE, to rod and cone outer segments, resulting in altered photoreceptor physiology and a phenotype of a slowly progressing rod/cone dystrophy.prenyl binding protein ͉ rod/cone dystrophy ͉ vesicular transport ͉ isoprenylation ͉ membrane association

Bovine PDE␦ was originally copurified with rod cGMP phosphodiesterase (PDE) and shown to interact with prenylated, carboxymethylated C-terminal Cys residues. Other studies showed that PDE␦ can interact with several small GTPases including Rab13, Ras, Rap, and Rho6, all of which are prenylated, as well as the Nterminal portion of retinitis pigmentosa GTPase regulator and Arl2/Arl3, which are not prenylated. We show by immunocytochemistry with a PDE␦-specific antibody that PDE␦ is present in rods and cones. We find by yeast two-hybrid screening with a PDE␦ bait that it can interact with farnesylated rhodopsin kinase (GRK1) and that prenylation is essential for this interaction. In vitro binding assays indicate that both recombinant farnesylated GRK1 and geranylgeranylated GRK7 co-precipitate with a glutathione S-transferase-PDE␦ fusion protein. Using fluorescence resonance energy transfer techniques exploiting the intrinsic tryptophan fluorescence of PDE␦ and dansylated prenyl cysteines as fluorescent ligands, we show that PDE␦ specifically binds geranylgeranyl and farnesyl moieties with a K d of 19.06 and 0.70 M, respectively. Our experiments establish that PDE␦ functions as a prenyl-binding protein interacting with multiple prenylated proteins.

Lecithin retinol acyl transferase (LRAT) and retinal pigment epithelium protein 65 (RPE65) are key enzymes of the retinoid cycle. In Lrat Ϫ/Ϫ and Rpe65 Ϫ/Ϫ mice, models of human Leber congenital amaurosis, the retinoid cycle is disrupted and 11-cis-retinal, the chromophore of visual pigments, is not produced. The Lrat Ϫ/Ϫ and Rpe65 Ϫ/Ϫ retina phenotype presents with rapid sectorial cone degeneration, and the visual pigments, S-opsin and M/L-opsin, fail to traffic to cone outer segments appropriately. In contrast, rod opsin traffics normally in mutant rods. Concomitantly, guanylate cyclase 1, cone T␣-subunit, cone phosphodiesterase 6␣Ј (PDE6␣Ј), and GRK1 (G-protein-coupled receptor kinase 1; opsin kinase) are not transported to Lrat Ϫ/Ϫ and Rpe65 Ϫ/Ϫ cone outer segments. Aberrant localization of these membrane-associated proteins was evident at postnatal day 15, before the onset of ventral and central cone degeneration. Protein levels of cone T␣ and cone PDE6␣Ј were reduced, whereas their transcript levels were unchanged, suggesting posttranslational degradation. In an Rpe65 Ϫ/Ϫ Rho Ϫ/Ϫ double knock-out model, trafficking of cone pigments and membrane-associated cone phototransduction polypeptides to the outer segments proceeded normally after 11-cis-retinal administration. These results suggest that ventral and central cone opsins must be regenerated with 11-cis-retinal to permit transport to the outer segments. Furthermore, the presence of 11-cis-retinal is essential for proper transport of several membrane-associated cone phototransduction polypeptides in these cones.