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

Abstract

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...

Inhibition of biogenic membrane flippase activity in reconstituted ER proteoliposomes in the presence of low cholesterol levels

“…2, trace a). Time-dependent fluorescence quenching of liposomes follows single phase exponential decay kinetics as reported earlier [10]. Liposomes when reconstituted with ePC and lipids extracted from bovine liver ER and spinach leaf ER showed no flippase activity ( Fig.…”

“…However, biogenic or self-synthesizing membranes are equipped with a special class of protein translocators called biogenic membrane flippases, which are involved in rapid flip-flop of PLs (irrespective of PL head group) and are metabolic energy independent, thereby differing from conventional ATP-dependent flippases which are localized at non-biogenic membranes such as plasma membrane (PM) [9]. Biogenic membrane flippase activity has been shown in bacterial cytoplasmic membranes, and ER of animals and plants [10][11][12]. We hypothesize that cholesterol affects the rapid flip-flop of PLs across the bilayer.…”

ATPase Activity of the MsbA Lipid Flippase of Escherichia coli

“…Recent experiments designed to study phospholipid flip-flop in bacterial membranes and model liposomes have suggested that this process might be energy-independent in bacteria (58,59). In model membranes, lipid flip-flop occurs at near physiological rates in the absence of ATP or a proton motive force, provided that synthetic membrane-spanning peptides are reconstituted together with the lipids (60).…”

Reconstitution of Glucosylceramide Flip-Flop across Endoplasmic Reticulum

“…The present study makes use of a detergent-based vesicle reconstitution protocol in which Triton X-100-solubilized phospholipids and ER membrane proteins are incubated with detergent-adsorbing Bio-Beads. Although greater than 99% of the detergent is removed during this procedure (26) to generate large unilamellar vesicles, we considered whether residual detergent in the vesicles could affect the spontaneous flip-flop of lipids.…”