The transbilayer redistribution of spin-labeled phospholipid analogues (SL-PL) with choline, serine, and ethanolamine head groups (PC, PS, and PE, respectively) was studied on intact disc vesicles of bovine rod outer segment membranes in the dark and after illumination. Redistribution was measured by the extraction of spin-labeled lipid analogues from the outer leaflet of membrane using the bovine serum albumin back-exchange assay. In the dark, PS was distributed asymmetrically, favoring the outer leaflet, whereas PC and PE showed small if any asymmetry. Green illumination for 1 min caused lipid head group-specific reorganization of SL-PL. Extraction of SL-PS by bovine serum albumin showed a fast transient (<10 min) enhancement, which was further augmented by a peptide stabilizing the active metarhodopsin II conformation. The data suggest a direct release of 1 molecule of bound PS per rhodopsin into the outer leaflet and subsequent redistribution between the two leaflets. SL-PE and SL-PC showed more complex kinetics, in both cases consistent with a prolonged period of reduced extraction (2 phospholipids per rhodopsin in each case). The different phases of SL-PL reorganization after illumination may be related to the formation and decay of the active rhodopsin species and to their subsequent regeneration process.Rhodopsin, the photoreceptor of the retinal rod, and its Gprotein transducin (G t ) 1 are archetypes of the G-protein-coupled receptor and heterotrimeric G-protein families, respectively. Rhodopsin is embedded in the membranes of the rod outer segment, which are arranged in a long, closely spaced stack of disc-like saccules. G t is bound to the cytoplasmic surface of the disc membranes. To provide an effective target for the light, rhodopsin is very densely packed, so that the receptor accounts for half of the dry weight of the disc membranes. It is composed of the apoprotein opsin, comprising seven transmembrane helices and the chromophore 11-cis-retinal, which is covalently bound to Lys 296 in the seventh helix via a protonated Schiff base and acts as a highly effective inverse agonist. Following the absorption of a photon, the retinal isomerizes to a strained all-trans conformation, which induces a series of conformational rearrangements of the opsin moiety. The final product of this reaction sequence is the active metarhodopsin II (Meta II) conformation, which contains all-trans-retinal as a covalently bound agonist, and is capable of catalyzing the activation of the retinal G-protein transducin (for reviews see Refs. 1-3). Identified physicochemical events that accompany the transition into the active state include proton transfer reactions and helix movements. Deprotonation of the Schiff base bond, protonation of its counterion Glu 113 , and the uptake of a proton from the aqueous solution (4) mediated by the highly conserved opsin residue Glu 134 are determinants of the active state. Based on computer simulations and mutagenesis studies, similar results have been obtained for the ␣ 1B -adrenergic...

We studied the transbilayer redistribution of phospholipids in bovine rod outer segment membranes on thoroughly washed, Ficoll-floated osmotically intact disc vesicles; freshly prepared membranes separated from the disc stack by osmotic shock; and intact disc stacks with a permeabilized plasma membrane (A-discs, B-discs C-discs, respectively). In all cases, spin-labelled phospholipid analogues (SL-PL) with choline, serine and ethanolamine head groups (PtdCho, PtdSer and PtdEtn, respectively) were taken up into the outer leaflet of the membranes by . 90% and within less than 30 s after SL-PL addition, as deduced from the disappearance of spin-label from the suspension medium and from the specific ESR spectrum of membrane-associated spinlabel. Using BSA extraction, the amount of SL-PL in the outer leaflet of the bilayer was determined. It decreased with a mean half-time of , 5 min at 25 8C, indicating rapid redistribution of all spin-labelled phospholipids into the inner leaflet of the disc membranes. After 1 h, PtdCho and PtdEtn were distributed almost symmetrically, whereas PtdSer was 35 : 65% (in/out). Using subsequent incubation with BSA, the outward movement (flop) of the analogues was observed directly, demonstrating that inward and outward movements proceed in thermodynamic equilibrium. No effect of N-ethylmaleimide or ATP on the redistribution could be measured, which makes it unlikely that energy-consuming translocase or flippase processes are involved in the redistribution in the dark. We reason that the solubilization zone around the photoreceptor rhodopsin may be the locus of rapid redistribution of the highly unsaturated disc phospholipid.Keywords: disc membrane; flip-flop; phospholipid asymmetry; photoreceptor.The outer segments of retinal rod photoreceptor cells comprise an extensive area of folded membranes, which are arranged mainly in a long, closely spaced stack of disc-like saccules. Disc membranes are organized as lipid bilayers with rhodopsin as the major protein. Lipids account for approximately half of the dry weight of the membranes, and the discs consist of 45 mol% phosphatidylcholine (PtdCho), < 42 mol% phosphatidylethanolamine (PtdEtn) and 13.7 mol% phosphatidylserine (PtdSer) [1]. The most striking feature of the disc lipid composition is the unusually high content of long-chain polyunsaturated acids, particularly in PtdEtn and PtdSer, while the fatty acid residues of PtdCho are more saturated [2]. Several lines of evidence suggest that the fluid phospholipid environment of the disc membrane is important for the rod outer segment (ROS) photoreceptor functions, including the stability and functional regenerability of rhodopsin [3] and the influence of the lipid headgroup and acyl chain composition of phospholipids on the equilibrium between the functionally active photointermediate metarhodopsin II and metarhodopsin I [4]. Depletion of phospholipids, arising from essential v3 fatty acid deficiency, leads to loss of visual function with slowly progressing retinal dystrophy [5,6].Recent i...

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In retinal rod photoreceptor cells, transducin (G t ) and cyclic GMP phosphodiesterase (PDE) are peripherally anchored to the cytoplasmic surface of the disk saccules. We have examined the role of specific phospholipids in the interaction of these proteins with native osmotically intact disk vesicles, employing spin-labeled phospholipid analogues (2% of total phospholipids) and bovine serum albumin back-exchange assay. Inactive GDPbound transducin exclusively reduced the extraction of negatively charged phosphatidylserine. The effect disappeared upon activation of the G-protein with guanosine 5-O-(3-thiotriphosphate) (GTP␥S). PDE affected the extraction of the zwitterionic phosphatidylcholine and, to a smaller extent, of phosphatidylethanolamine. When active G t GTP␥S interacted with the PDE to form the active effector, the interaction with phosphatidylcholine was specifically enhanced. Each copy of the G-protein bound 3 ؎ 1 molecules of phosphatidylserine, whereas the PDE bound a much larger amount (70 ؎ 10) of a mixture of phosphatidylcholine and ethanolamine. The results are interpreted as a head group-specific and state-dependent interaction of the signaling proteins with the phospholipids of the photoreceptor membrane.The transduction of the light signal in the retinal rod photoreceptor is a well studied model system for G-protein-coupled signal transduction. It involves the sequential activation of rhodopsin, transducin (G t ), 1 and cGMP phosphodiesterase (PDE) on the cytoplasmic surface of the disk saccules that fill the rod outer segment (ROS). Absorption of light transforms rhodopsin into the activated metarhodopsin II state, which then interacts with G t to rapidly catalyze the exchange of GDP for GTP in the nucleotide-binding site of the G t ␣-subunit. G t ␣-GTP dissociates and couples to the effector PDE (1, 2). Activation of PDE results in a decrease in the cytosolic cGMP concentration which in turn leads to closure of cGMP-regulated channels in the plasma membrane, hyperpolarization, and neuronal signaling (see Ref. 3).Heterotrimeric G t is peripherally attached to the disk membrane by weak hydrophobic and ionic interactions. Both Nterminal acylation of the G t ␣-subunit and C-terminal farnesylation of the G t␥ -subunit are required for membrane association of G t (4). Electrostatic interactions, especially of G t ␤␥, further enhance the membrane binding to negatively charged surfaces or to vesicles containing the acidic lipid phosphatidylserine (5-7). Inactive transducin in its GDP-bound form is attached as a G t ␣␤␥ heterotrimer to the membrane. G t couples to activated rhodopsin, triggering GDP release as a first step of catalytic nucleotide exchange. The interaction with the receptor occurs directly from the membrane-bound state of the holoprotein. After activation of transducin, G t ␣ dissociates from G t ␤␥ and couples stoichiometrically to the effector PDE.The PDE is a heterotetrameric protein composed of the undissociable ␣␤ complex (8) and two identical ␥-subunits (9). Full activation of ...