The purified and functionally reconstituted multidrug transporter LmrA of Lactococcus lactis mediates the transbilayer movement of specific fluorescent phospholipids

Lactococcus lactis possesses an ATP-binding cassette transporter, LmrA, whi ch is a homolog of the mammalian multidrug resistance (MDR) P-glycoprotein, and is able to transport a broad range of structurally unrelated amphiphil ic drugs. A histidine tag was introduced at the N-terminus of LmrA to facil itate purification by nickel affinity chromatography. The histidine-tagged protein was overexpressed in L. lactis using a novel protein expression sys tem for cytotoxic proteins based on the tightly regulated, nisin-inducible nisA promoter, This system allowed us to get functional overexpression of L mrA up to a level of 30% of total membrane protein. For reconstitution, Lmr A was solubilized with dodecylmaltoside, purified by nickel-chelate affinit y chromatography, and reconstituted in dodecylmaltoside-destabilized, prefo rmed liposomes prepared from L, lactis phospholipids. The detergent was rem oved by adsorption onto polystyrene beads. The LmrA protein was reconstitut ed in a functional form, and mediated the ATP-dependent transport of the fl uorescent substrate Hoechst-33342 into the proteoliposomes. Interestingly, reconstituted LmrA also catalyzed the ATP-dependent transport of fluorescen t phosphatidylethanolamine, but not of fluorescent phosphatidylcholine. The se data demonstrate that LmrA activity is independent of accessory proteins and support the notion that LmrA may be involved in the transport of speci fic lipids or lipid-linked precursors in L. lactis.