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We report here the high-level expression of a synthetic gene for bovine rhodopsin in transfected monkey kidney COS-1 cells. Rhodopsin is produced in these cells to a level of0.3% of the cell protein, and it binds exogenously added 11-cis-retinal to generate the characteristic rhodopsin absorption spectrum. We describe a one-step immunoaftmity procedure for purification of the rhodopsin essentially to homogeneity. The COS-1 cell rhodopsin activates the GTPase activity of bovine transducin in a light-dependent manner with the same specific activity as that of purified bovine rhodopsin. Electron microscopy of immunogold-stained cells indicates that rhodopsin is located in the plasma membrane of the transfected cells and is oriented with the amino terminus on the extracellular side of the membrane. This orientation is analogous to that of rhodopsin in the disk membranes of photoreceptor cells in the bovine retina.Rhodopsin is the photoreceptor protein of vertebrate retinal rod cells (1, 2). Upon absorption of light, rhodopsin undergoes a structural change that allows it to activate the GTP-binding protein, transducin, and thus initiate a sequence of events that results in the hyperpolarization of the rod cell. Light transduction and its regulation is evidently mediated by a number of proteins in the rod outer segment (ROS).Bovine rhodopsin consists of a polypeptide chain of 348 amino acids whose sequence is known by both protein and DNA sequencing (3-5). 11-cis-Retinal linked as a Schiff base to the e-amino group of Lys-296 serves as the chromophore. The primary event following the capture of a photon by rhodopsin is the isomerization of 11-cis-retinal to all-transretinal. However, little is known about the nature of the structural changes induced in rhodopsin by this isomerization, the consequent interaction with transducin, or the mechanism of light/dark adaptation. We wish to study these questions by carrying out specific amino acid substitutions in the rhodopsin molecule by using recombinant DNA techniques. For site-specific mutagenesis, we have previously synthesized a gene for bovine rhodopsin that contains a suitable number of conveniently placed unique restriction sites (6, 7). These allow the replacement of specific restriction fragments by synthetic counterparts that contain the desired altered codons. The next requirement is the satisfactory expression of rhodopsin in its fully functional form. In this paper, we report on the high-level expression of the synthetic rhodopsin gene in mammalian cells using the expression vector p91023(B) (8, 9). The apoprotein (opsin) produced in these cells can be reconstituted by the addition of exogenous ll-cis-retinal. It has been purified essentially to homogeneity by a one-step immunoaffinity procedure and has been characterized. MATERIALS AND METHODSMaterials. COS-1 monkey kidney cells (10) Buffers and Media. Medium A was Dulbecco's modified Eagle's medium containing D-glucose (4.5 g/liter), streptomycin (100 mg/ml), penicillin (100 mg/ml), a supplement of 2 ...

P4-ATPases comprise a family of P-type ATPases that actively transport or flip phospholipids across cell membranes. This generates and maintains membrane lipid asymmetry, a property essential for a wide variety of cellular processes such as vesicle budding and trafficking, cell signaling, blood coagulation, apoptosis, bile and cholesterol homeostasis, and neuronal cell survival. Some P4-ATPases transport phosphatidylserine and phosphatidylethanolamine across the plasma membrane or intracellular membranes whereas other P4-ATPases are specific for phosphatidylcholine. The importance of P4-ATPases is highlighted by the finding that genetic defects in two P4-ATPases ATP8A2 and ATP8B1 are associated with severe human disorders. Recent studies have provided insight into how P4-ATPases translocate phospholipids across membranes. P4-ATPases form a phosphorylated intermediate at the aspartate of the P-type ATPase signature sequence, and dephosphorylation is activated by the lipid substrate being flipped from the exoplasmic to the cytoplasmic leaflet similar to the activation of dephosphorylation of Na+/K+-ATPase by exoplasmic K+. How the phospholipid is translocated can be understood in terms of a peripheral hydrophobic gate pathway between transmembrane helices M1, M3, M4, and M6. This pathway, which partially overlaps with the suggested pathway for migration of Ca2+ in the opposite direction in the Ca2+-ATPase, is wider than the latter, thereby accommodating the phospholipid head group. The head group is propelled along against its concentration gradient with the hydrocarbon chains projecting out into the lipid phase by movement of an isoleucine located at the position corresponding to an ion binding glutamate in the Ca2+- and Na+/K+-ATPases. Hence, the P4-ATPase mechanism is quite similar to the mechanism of these ion pumps, where the glutamate translocates the ions by moving like a pump rod. The accessory subunit CDC50 may be located in close association with the exoplasmic entrance of the suggested pathway, and possibly promotes the binding of the lipid substrate. This review focuses on properties of mammalian and yeast P4-ATPases for which most mechanistic insight is available. However, the structure, function and enigmas associated with mammalian and yeast P4-ATPases most likely extend to P4-ATPases of plants and other organisms.

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Many substrates for P-glycoprotein, an ABC transporter that mediates multidrug resistance in mammalian cells, have been shown to stimulate its ATPase activity in vitro. In the present study, we used this property as a criterion to search for natural and artificial substrates and/or allosteric regulators of ABCR, the rod photoreceptor-specific ABC transporter responsible for Stargardt disease, an early onset macular degeneration. ABCR was immunoaffinity purified to apparent homogeneity from bovine rod outer segments and reconstituted into liposomes. All-trans-retinal, a candidate ligand, stimulates the ATPase activity of ABCR 3-4-fold, with a half-maximal effect at 10 -15 M.

Photobleaching of rhodopsin in rod photoreceptors activates the visual cascade system leading to a decrease in cyclic GMP and the closure of cGMP-gated channels in the rod outer segment plasma membrane. Calcium plays an important role in the recovery of the rod outer segment to its dark state by regulating the resynthesis of cGMP by guanylate cyclase. Here we report that calmodulin, a Ca(2+)-binding protein present in the rod outer segment, increases the apparent Michaelis constant of the channel for cGMP. This results in a decrease in the rate of cation influx into the rod outer segment by two- to sixfold at low cGMP concentrations and has the effect of increasing the sensitivity of the channel to small changes in cGMP levels during phototransduction. Biochemical studies indicate that calcium-calmodulin binds to a protein of M(r) 240K which is tightly associated with the channel. On the basis of these studies, Ca2+ is suggested to play a central role in photorecovery and light adaptation, not only by regulating guanylate cyclase, possibly through recoverin, but also by modulating the cGMP-gated channel through calmodulin interaction with the 240K protein.

X-linked juvenile retinoschisis (XLRS, MIM 312700) is a common early onset macular degeneration in males characterized by mild to severe loss in visual acuity, splitting of retinal layers, and a reduction in the b-wave of the electroretinogram (ERG). The RS1 gene (MIM 300839) associated with the disease encodes retinoschisin, a 224 amino acid protein containing a discoidin domain as the major structural unit, an N-terminal cleavable signal sequence, and regions responsible for subunit oligomerization. Retinoschisin is secreted from retinal cells as a disulphide-linked homo-octameric complex which binds to the surface of photoreceptors and bipolar cells to help maintain the integrity of the retina. Over 190 disease-causing mutations in the RS1 gene are known with most mutations occurring as non-synonymous changes in the discoidin domain. Cell expression studies have shown that disease-associated missense mutations in the discoidin domain cause severe protein misfolding and retention in the endoplasmic reticulum, mutations in the signal sequence result in aberrant protein synthesis, and mutations in regions flanking the discoidin domain cause defective disulphide-linked subunit assembly, all of which produce a non-functional protein. Knockout mice deficient in retinoschisin have been generated and shown to display most of the characteristic features found in XLRS patients. Recombinant adeno-associated virus (rAAV) mediated delivery of the normal RS1 gene to the retina of young knockout mice result in long term retinoschisin expression and rescue of retinal structure and function providing a ‘proof of concept’ that gene therapy may be an effective treatment for XLRS.