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

Abstract

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 water-soluble 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 ∼4S, 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.