Retinal rod outer segment membranes (ROS) have been isolated by a simplified procedure from fresh and frozen retinas. When cattle and frog ROS membrane proteins are can Rana pipiens, weighing 50-150 g (Connecticut Valley Corp.), were treated with tetracycline for 5 days by esophageal intubation in order to prevent sepsis (Gibbs et al., 1966).

Thirteen mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa (ADRP) have been produced by transfection of cloned cDNA into tissue culture cells. Three mutants [class I: Phe45 --Leu, termination (deletion of C-terminal positions 344-348), and Pro-347 --Leul resemble wild-type rhodopsin in yield, regenerability with 11-cis-retinal, and plasma membrane localization. Ten mutants Met, His, Thr-58 --Arg, Val-87 -* Asp, Gly-89 Asp, Gly-106 -Trp, Arg-135 --Leu, Arg-135 Trp, Tyr-178 -+ Cys, and Gly] accumulate to significantly lower levels, regenerate with 11-cis-retinal variably or not at all, and are transported inefficiently to the plasma membrane, remaining primarily in the endoplasmic reticulum. These data suggest that there are at least two distinct biochemical defects associated with different rhodopsin mutants in ADRP.Retinitis pigmentosa (RP) is a group of inherited disorders that cause a progressive loss of retinal function. The hallmarks ofRP are decreased rod sensitivity, progressive loss of visual fields, a diminished electroretinographic response referable to photoreceptors, and characteristic pigmentary deposits in the retina (1).Recently, some patients with autosomal dominant RP (ADRP) were found to carry mutations in the gene encoding rhodopsin, the visual pigment mediating rod vision (2-5). The mutations cosegregate with RP and are absent from control populations with normal vision. In a previous study of 161 unrelated families with ADRP, 39 were found to carry 1 of 13 different point mutations in the rhodopsin coding region (5). The goal of the present study is to define the biochemical differences between wild-type (wt) rhodopsin and the variants responsible for ADRP. For this purpose we have produced in tissue culture cells each of the 13 mutant human rhodopsins described above and determined their yield, regenerability with 11-cis-retinal, and subcellular localization. MATERIALS AND METHODSTissue Culture Expression. A rhodopsin cDNA clone was isolated from a human retina cDNA library (6, 7), and a fragment containing the entire coding region was inserted into the expression plasmid pCIS (8). In vitro mutagenesis and production of opsin after transient or stable transfection of 293S cells were performed as described (9, 10).Absorbance Spectra. Cells from 20 10-cm plates were collected 60 hr after transient transfection, and membranes were prepared as described (9) except that the final membrane pellet was solubilized in 0.3 ml of 0.1 M sodium phosphate, pH 6.5/1 mM EDTA/1% 3-[(3-cholamidopropyl)-dimethylammoniol-1-propanesulfonate (CHAPS; Sigma). The solubilized sample was regenerated with li-cis-retinal, incubated with 50 mM hydroxylamine for 30 min, and the photobleaching difference spectrum was determined (9).Immunoblotting. Membrane samples prepared from cells 60 hr after transfection were mixed with an equal volume of 2x Laemmli sample buffer (lx = 0.125 M TrisHCl, pH 6.8/4% SDS/20o glycerol/10%o 2-mercaptoethanol/0.012% bromophenol blue), resolved on a SDS/12.5%...

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...