Funder: China Scholarship Council (CSC); doi: https://doi.org/10.13039/501100004543Funder: Cambridge Commonwealth Trust; doi: https://doi.org/10.13039/501100003342The 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.This research was funded by Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/R00224X/1 (to H.W.V.V). D.G. and Y.T. were funded by China Scholarship Council – Cambridge Trust PhD Scholarships. C.G. was funded by a BBSRC Doctoral Training Partnership (DTP) Targeted PhD studentship (project 2114197). T.N. received a student grant from Christ’s College Cambridge
