When human erythrocytes are incubated with spin-labeled analogues of sphingomyelin, phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine, with a short beta chain (C5) bearing a doxyl group at the fourth carbon position, the labeled lipids incorporate readily in the outer monolayer. The incorporation is followed in fresh erythrocytes by a selective inward diffusion of the amino derivatives. This observation led us to postulate the existence of a selective ATP-dependent system that would flip aminophospholipids from the outer to the inner monolayer [Seigneuret, M., & Devaux, P. F. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3751-3755]. This study further examines the nature of this selective transport and demonstrates that it is mediated by a specific membrane protein. By measurement of the initial rate of transverse diffusion of spin-labeled lipids incorporated at various concentrations in the membrane outer leaflet of packed erythrocytes, apparent Km values were determined for the phosphatidylserine and phosphatidylethanolamine analogues. A ratio of approximately equal to 1/9.4 [corrected] was obtained (KmPS/KmPE). Using spin-labels bearing either a 14N or a 15N isotope, we have carried out competition experiments allowing us to measure simultaneously the transport of two different phospholipids. By this procedure, we show that phosphatidylserine and phosphatidylethanolamine compete for the same transport site but that phosphatidylserine has a higher affinity, in agreement with a lower apparent Km. On the other hand, the slow diffusion of the phosphatidylcholine or sphingomyelin analogues has no influence on the transport of phosphatidylserine or phosphatidylethanolamine. Experiments carried out in ghosts loaded with ATP enabled us to determine the activation energies for phosphatidylserine and phosphatidylcholine transverse diffusion.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to fix spin-labeled acids at the boundary layer of membrane-bound proteins, spin-labeled long-chain derivatives (m,n)MSL (general formula, CH,(CH2),R-(CH2),COO(CH2)2-M, where R is an oxazolidine ring containing a nitroxide and M is a maleimide residue) were synthesized. The spin-labeled molecules bind covalently to at least two different classes of sulfhydryl groups on rhodopsin in disc membrane fragments from bovine retina. One class of sites is hydrophilic and corresponds to the two SH groups labeled readily by N-ethylmaleimide; the second class of sites is only reached by hydrophobic probes. (10,3)MSL binds equally well to the two classes of sites on rhodopsin, whereas (1 , I 4)MSL, more hydrophobic, binds preferentially to the hydrophobic sites. Apparently a third class of SH groups can be labeled if a very large excess of (m,n)MSL is employed, but proteins may be denatured in this latter case. Labels not covalently bound are removed from the membranes by incubation with fatty acid free bovine serum albumin. However, it is found that the probes do not bind only to rhodopsin in the disc membranes. (m,n)MSL also binds covalently to phosphatidylethanolamine in the rod outer segments or in liposomes. This covalent binding to phospholipids is demonstrated by lipid extraction and thin-layer chromatographic analysis. In order to obtain the pure EPR spectra of the spin-labeled fatty acids bound to the protein, the spectra corresponding to phospholipid-bound spin labels have been I t is often admitted that intrinsic membrane proteins are surrounded by a boundary layer or "annulus" of rigidly bound lipid. The immobilization of this shell of lipid has been deduced essentially from EPR experiments involving spin-labeled fatty acids incorporated into reconstituted systems containing variable lipid to protein ratios. Jost et al. (1973) were the first to propose from spin-label experiments the model of a boundary layer of lipid surrounding an intrinsic membrane protein, namely cytochrome oxidase. Later, Hesketh et al. (1976) reported similar experiments with Ca2+-ATPase, while Chapman et al. (1977) showed that gramcidin A can lead to the same EPR results, if this polypeptide is dissolved in a small amount of lipid.Rhodopsin was also tested with spin-labeled fatty acids. Pontus and Delmelle (1975) found evidence of a rigid boundary layer around this hydrophobic protein. However, previously, Hong & Hubbell (1972) had reached a different conclusion from spin-label experiments with rhodopsin reincorporated into phospholipid; they suggested that the acerage viscosity of the membranes was dependent on the lipid to protein ratio. This is in good agreement with recent results put foward by Cherry et al. (1977) from very different experiments involving bacteriorhodopsin. Finally, using proton manuscript receiced A'ocember 21, 1978. This investigation was supported by research grants from the Centre National de la Recherche Scientifique (ERA 690) and the DelEgation G&n&rale a la Recherche Scientifique et Technique (Comm...

Deuterium nuclear magnetic resonance (2H NMR) was used to study the interaction of cytochrome c (from horse heart) with bilayers of mixed dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylserine (DMPS). Three types of labeled lipids were used: chain-perdeuterated phosphatidylcholine (DMPC-d54), chain-perdeuterated phosphatidylserine (DMPS-d54), and phosphatidylserine labeled at the alpha-position of the head group (DMPS-d2). Liposomes containing equimolar mixtures of DMPC and DMPS were found to bind cytochrome c with a maximum ratio of about 1 mg of cytochrome c per 1 mg of DMPS. The 2H NMR spectra of equimolar mixtures of DMPC-d54-DMPS and DMPC-DMPS-d54 were examined with and without cytochrome c. No change of the NMR spectra of either DMPC or DMPS could be detected after protein addition, for temperatures both above and below the phospholipid phase transition region. On the other hand, in the liquid-crystalline state, the transverse relaxation time, T2e, was reduced by 30-40% after protein addition. Measurements of the spin-lattice relaxation time, T1, showed, under all circumstances, multiple components. For simplicity, we have examined the shape of the relaxation curves at short and long times. Addition of protein increased by 2-fold the value of the slow T1 component of DMPS-d54 but not that of DMPC-d54. Partially relaxed spectroscopy allowed us to assign this slow component (at least in part) to the methyl group and C2H2 groups near the methyl end of the chains, i.e., far from the binding sites of the extrinsic protein.(ABSTRACT TRUNCATED AT 250 WORDS)

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