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Significance Biological membranes define cellular boundaries, allow compartmentalization, and represent a prerequisite for life. In gram-negative bacteria, the outer membrane (OM) prevents entry of toxic substances, conferring intrinsic resistance against many antibiotics. This barrier function requires unequal distribution of lipids across the OM bilayer, yet how such lipid asymmetry is maintained is not well understood. In this study, we established the directionality of lipid transport for a conserved membrane protein complex and uncovered mechanistic insights into how ATP powers such transport from the OM to the inner membrane. Our work provides fundamental understanding of lipid trafficking within the gram-negative double-membrane envelope in the context of OM lipid asymmetry and highlights the importance of targeting lipid transport processes for future antibiotics development.

ATP disrupts lipid-binding equilibrium to drive retrograde transport critical for bacterial outer membrane asymmetry