Abstract
The MsbA protein is an essential ABC (ATP-binding-cassette) superfamily member in Gram-negative bacteria. This 65 kDa membrane protein is thought to function as a homodimeric ATP-dependent lipid translocase or flippase that transports lipid A from the inner to the outer leaflet of the cytoplasmic membrane. We have previously shown that purified MsbA from Escherichia coli displays high ATPase activity, and binds to lipids and lipid-like molecules, including lipid A, with affinity in the low micromolar range. Bacterial membrane vesicles isolated from E. coli overexpressing His6-tagged MsbA displayed ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species. Purified MsbA was reconstituted into proteoliposomes of E. coli lipid and its ability to translocate NBD-labelled lipid derivatives was characterized. In this system, the protein displayed maximal lipid flippase activity of 7.7 nmol of lipid translocated per mg of protein over a 20 min period for an acyl chain-labelled PE (phosphatidylethanolamine) derivative. The protein showed the highest rates of flippase activity when reconstituted into an E. coli lipid mixture. Substantial flippase activity was also observed for a variety of other NBD-labelled phospholipids and glycolipids, including molecules labelled on either the headgroup or the acyl chain. Lipid flippase activity required ATP hydrolysis, and was dependent on the concentration of ATP and NBD–lipid. Translocation of NBD–PE was inhibited by the presence of the putative physiological substrate lipid A. The present paper represents the first report of a direct measurement of the lipid flippase activity of purified MsbA in a reconstituted system.
Highlights
Gram-negative bacteria such as Escherichia coli possess both an inner membrane and an outer membrane, separated by a layer of peptidoglycan and the periplasmic space
The cytoplasmic membrane lipids are distributed asymmetrically, with the inner leaflet of Gram-positive bacteria being enriched in PE and the outer leaflet enriched in PG ([1]; bacterial lipid topology is reviewed in [2])
In previous work [14], we have shown that the ATPase activity of purified MsbA is modulated by a variety of lipids and lipid-based molecules, including lipid A, the LPS precursors RaLPS (LPS from the Ra mutant of E. coli) and deep rough chemotype LPS (ReLPS), and phospholipid-based amphipathic drugs
Summary
Gram-negative bacteria such as Escherichia coli possess both an inner (cytoplasmic) membrane and an outer membrane, separated by a layer of peptidoglycan and the periplasmic space. Proteoliposomes of E. coli lipid containing MsbA and 0.3 % NBD–PE (16:0, 6:0) were incubated at 37 ◦C in the presence or absence of ATP and a regenerating system for 20 min. E. coli membrane vesicles, purified MsbA in detergent, and reconstituted protein in proteoliposomes displayed a high level of ATPase activity that was stimulated and inhibited by the presence of substrates [14], suggesting that the protein was functional.
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