Generation of chemical gradients across biological membranes of cellular compartments is a hallmark of all living systems. Here we report a proof-of-concept prototype transmembrane pumping system in liposomes. The pump
uses fatty acid to fuel chloride transport, thus generating a transmembranechloride gradient. Addition of fatty acid to phospholipid vesicles generates a transmembrane pH gradient (pHin < pHout), and this electrochemical H+
potential is harnessed by an anionophore to drive chloride efflux via H+/Cl− cotransport. Further addition of fatty acid efficiently fuels the system to continuously drive chloride transport against the concentration gradient, up to
[Cl−] 65 mM | [Cl−] 100 mM, and is 1400 times more efficient than using an in out +− external fuel. Based on our findings from dissecting the H /Cl flux process with the use of different liposomal fluorescence assays, and supported by additional liposome-based 13C NMR and DLS studies; we proposed that the presence of an anionophore can induce asymmetric distribution of fatty acid, and contribute to another Cl− flux mechanism in this system.