Abstract
The ABC multidrug exporter MsbA mediates the translocation of lipopolysaccharides and phospholipids across the plasma membrane in Gram-negative bacteria. Although MsbA is structurally well characterised, the energetic requirements of lipid transport remain unknown. Here, we report that, similar to the transport of small-molecule antibiotics and cytotoxic agents, the flopping of physiologically relevant long-acyl-chain 1,2-dioleoyl (C18)-phosphatidylethanolamine in proteoliposomes requires the simultaneous input of ATP binding and hydrolysis and the chemical proton gradient as sources of metabolic energy. In contrast, the flopping of the large hexa-acylated (C12-C14) Lipid-A anchor of lipopolysaccharides is only ATP dependent. This study demonstrates that the energetics of lipid transport by MsbA is lipid dependent. As our mutational analyses indicate lipid and drug transport via the central binding chamber in MsbA, the lipid availability in the membrane can affect the drug transport activity and vice versa.
Highlights
The ATP-binding cassette (ABC) multidrug exporter MsbA mediates the translocation of lipopolysaccharides and phospholipids across the plasma membrane in Gram-negative bacteria
Examples of mammalian phospholipid transporters include (i) the calcium-activated nhTMEM16 scramblase that catalyses the shuffling of phospholipids between the inner and outer leaflets of the plasma membrane independent of metabolic energy[3], (ii) the type IV P-type ATPase flippases that catalyse the inward translocation of phospholipids from the outer leaflet to the inner leaflet of the plasma membrane[4], and (iii) the liver ATP-binding cassette (ABC) floppase ABCB45 for which in vivo data indicate a role in the outward translocation of phosphatidylcholine (PC) and its biliary secretion from hepatocytes into the canaliculi[6]
As (i) a previous study detected the transport of phospholipids and Lipid-A by MsbA in a 32Pi and 14C-acetate labelling approach in E. coli cells[16], (ii) PE is the predominant phospholipid in E. coli making up 70–80% of the total phospholipid pool[17,18], and (iii) advanced studies by native mass spectrometry detected the binding of PE to MsbA19, we established a transport assay for physiologically relevant long-acylchain (2 × C18) PE in proteoliposomes in which MsbA was incorporated in an inside-out fashion (Fig. 1a)
Summary
The ABC multidrug exporter MsbA mediates the translocation of lipopolysaccharides and phospholipids across the plasma membrane in Gram-negative bacteria. 1234567890():,; The passive movement of phospholipids between the leaflets in a membrane bilayer is extremely slow, in the order of hours to days, as shown in model membranes[1,2] This is, at least in part, due to the energetically unfavourable movement of the hydrophilic headgroup across the hydrophobic core of the bilayer. The Lipid-A (endotoxin) domain of LPS is a unique, glucosamine-based phospholipid that serves as the hydrophobic anchor of LPS and is the bioactive component of the molecule that is associated with Gram-negative septic shock. After it is formed, LPS is transported across the periplasm into the outer leaflet of the outer membrane by the LPS translocase LptB2FGCADE9. We study the energetics of lipid transport by purified MsbA in proteoliposomes and compare the lipid and drug transport activities in a mutagenesis approach
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