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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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