Rapid transmembrane movement of phosphatidylcholine in small unilamellar lipid vesicles formed by detergent removal

Kramer, Ruth M.; Hasselbach, Hans-Joachim; Semenza, Giorgio

doi:10.1016/0005-2736(81)90236-4

Cited by 26 publications

(5 citation statements)

References 37 publications

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“…Since we and others have previously shown that lipid vesicles prepared by means other than detergent reconstitution are incapable of supporting rapid lipid translocation, the effect that we observed with the octylglucoside and cholate-based preparations is likely due to small amounts of residual detergent or trace contaminants in the detergent. Similar results have been previously reported in which, for example, phosphatidylcholine liposomes reconstituted by dialysis of a cholate extract were shown to be able to translocate DPPC (19). To explore other reconstitution possibilities, we used extracts prepared with Triton X-100 and reconstituted vesicles according to procedures described by Lévy et al (21).…”

Section: Discussionsupporting

confidence: 69%

Reconstitution and Partial Characterization of Phospholipid Flippase Activity from Detergent Extracts of the Bacillus subtilis Cell Membrane

Hrafnsdóttir

Menon

2000

J Bacteriol

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In bacteria, phospholipids are synthesized on the inner leaflet of the cytoplasmic membrane and must translocate to the outer leaflet to propagate a bilayer. Transbilayer movement of phospholipids has been shown to be fast and independent of metabolic energy, and it is predicted to be facilitated by membrane proteins (flippases) since transport across protein-free membranes is negligible. However, it remains unclear as to whether proteins are required at all and, if so, whether specific proteins are needed. To determine whether bacteria contain specific proteins capable of translocating phospholipids across the cytoplasmic membrane, we reconstituted a detergent extract of Bacillus subtilis into proteoliposomes and measured import of a watersoluble phospholipid analog. We found that the proteoliposomes were capable of transporting the analog and that transport was inhibited by protease treatment. Active proteoliposome populations were also able to translocate a long-chain phospholipid, as judged by a phospholipase A 2 -based assay. Protein-free liposomes were inactive. We show that manipulation of the reconstitution mixture by prior chromatographic fractionation of the detergent extract, or by varying the protein/phospholipid ratio, results in populations of vesicles with different specific activities. Glycerol gradient analysis showed that the majority of the transport activity sedimented at ϳ4S, correlating with the presence of specific proteins. Recovery of activity in other gradient fractions was low despite the presence of a complex mixture of proteins. We conclude that bacteria contain specific proteins capable of facilitating transbilayer translocation of phospholipids. The reconstitution methodology that we describe provides the basis for purifying a facilitator of transbilayer phospholipid translocation in bacteria.Transbilayer movement of phospholipids is a rapid process in many, but not all, biological membranes (7,8,26,43). Since flip-flop is very slow in pure lipid bilayers (18), it is generally assumed that biological membranes are equipped with some mechanism to accelerate transport. The plasma membrane of eucaryotes possesses a number of distinct lipid translocation activities that are dependent on ATP or Ca 2ϩ . These include the aminophospholipid translocase (8,6,23,36), products of certain members of the multidrug resistance (mdr) gene family (31,34,38,39), and a unique Ca 2ϩ -stimulated, nonspecific lipid translocase (the phospholipid scramblase) that has been purified and cloned (1,44). In contrast to the progress in identifying lipid translocators situated in eucaryotic plasma membranes, very little is known about lipid translocators in biogenic (self-synthesizing) membranes such as the endoplasmic reticulum and the cytoplasmic membrane of bacteria. Phospholipid biosynthesis occurs on the cytoplasmic face of these membranes (2,29,42), and in order to form a bilayer the phospholipids must translocate to the opposite leaflet at a rate compatible with cell growth. Flip-flop in these membranes h...

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Section: Discussionsupporting

confidence: 69%

Reconstitution and Partial Characterization of Phospholipid Flippase Activity from Detergent Extracts of the Bacillus subtilis Cell Membrane

Hrafnsdóttir

Menon

2000

J Bacteriol

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“…But vesicles of diameter 80 nm prepared by detergent dialysis gave a ti of 10.2 h, whereas much larger vesicles (160 nm diameter2) prepared by reverse-phase evaporation gave a ti of 5.5 h (McLean & Phillips, 1984;Fugler et al, 1985). (contaminants such as detergents and organic solvents have in fact been shown to influence cholesterol exchange, phospholipid exchange and phospholipid 'Flip Flop' (Kramer et al, 1981;Clejan & Bittman, 1984;Nichols, 1986). Thus, in order to ascertain the effect ofcurvature, a reinvestigation using vesicles of various sizes prepared by a single and fairly reproducible method was necessary.…”

Section: Discussionmentioning

confidence: 99%

Effect of surface curvature on the rate of cholesterol transfer between lipid vesicles

Thomas

Poznansky

1988

Biochemical Journal

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The effect of surface curvature on the spontaneous movement of cholesterol between membranes was investigated by measuring the rates of cholesterol transfer from donor vesicles of various sizes to a common acceptor vesicle. Donor vesicles of size in the range 40-240 nm were prepared by extruding multilamellar dispersions through polycarbonate filters of different pore sizes under pressure. The smallest donor vesicle and the acceptor vesicles were obtained by the normal sonication procedures. The rate of cholesterol transfer, as measured by the movement of [3H]cholesterol, decreases with increasing size of the donor vesicle in an almost linear fashion. The extrapolation of the results gave a half-time (t1/2) of 16-20 h of the desorption of cholesterol from a planar bilayer, and this can be considered as a reference value for most cellular membranes which are characterized by very low curvatures. Our earlier studies have shown that the t1/2 for cholesterol efflux is influenced by the presence of gangliosides and phosphatidylethanolamine, and the asymmetric distribution of these lipids in the plasma membrane could partially account for the large difference in the rates of cholesterol movement from the two sides of the plasma membrane. The small differences in rates arising from asymmetric distribution will be magnified by the longer t1/2 obtained here for membranes of low curvatures, so that the large difference in rates might be a coupled effect of lipid asymmetry and low curvature of the plasma membrane. This, in turn, may have a role in maintaining the large differences in cholesterol/phospholipid molar ratios observed between plasma membrane and intracellular membranes.

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“…The failure to observe separate choline methyl peaks in DOC-L mixtures may result from the omission of an equilibration period or the fact that DOC promotes the formation of large vesicles or mixed micelles. An alternate explanation involves the possibility of enhanced transbilayer flip-flop rates (Kramer et al, 1981): even if vesicle structures remain intact, BS-accelerated exchange of lecithin molecules in inner and outer environments may result in a single NMR signal.…”

Section: Discussionmentioning

confidence: 99%

Influence of dilution on the physical state of model bile systems: NMR and quasi-elastic light-scattering investigations

Stark¹,

Gosselin²,

Donovan³

et al. 1985

Biochemistry

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Multinuclear (1H and 31P) nuclear magnetic resonance (NMR) spectroscopy and quasi-elastic light scattering have been used to characterize molecular aggregates formed in dilute sodium taurocholate--egg lecithin solutions. When mixed micelles (1.25 g/dL) are diluted with 150 mM aqueous sodium chloride, light-scattering measurements suggest a transformation from mixed micelles to unilamellar vesicle species. Decreased 1H NMR line widths for bile salt resonances are consistent with predominance of a monomer form. The concurrent appearance of a second phospholipid choline methyl resonance indicates two types of phospholipid environment in slow chemical exchange: this behavior is consistent with small unilamellar vesicles. The appearance of bilayer vesicles in dilute model bile solutions is confirmed by addition of a lanthanide shift reagent (Pr3+), which splits the 1H or 31P head-group peak into two components with distinct chemical shift sensitivities. These mixed micelle and vesicle aggregates are also distinguished by their susceptibility to the lipolytic enzyme phospholipase A2 from cobra venom.

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Rapid transmembrane movement of phosphatidylcholine in small unilamellar lipid vesicles formed by detergent removal

Cited by 26 publications

References 37 publications

Reconstitution and Partial Characterization of Phospholipid Flippase Activity from Detergent Extracts of the Bacillus subtilis Cell Membrane

Reconstitution and Partial Characterization of Phospholipid Flippase Activity from Detergent Extracts of the Bacillus subtilis Cell Membrane

Effect of surface curvature on the rate of cholesterol transfer between lipid vesicles

Influence of dilution on the physical state of model bile systems: NMR and quasi-elastic light-scattering investigations

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