ENERGETICS AND MECHANISM OF DRUG TRANSPORT MEDIATED BY THE LACTOCOCCAL MULTIDRUG TRANSPORTER LMRP

The gene encoding the secondary multidrug transporter LmrP of Lactococ cus lactis was heterologously expressed in Escherichia coli. The energ etics and mechanism of drug extrusion mediated by LmrP were studied in membrane vesicles of E. coli, LmrP-mediated extrusion of tetraphenyl phosphonium (TPP+) from right-side-out membrane vesicles and uptake of the fluorescent membrane probe -(trimethylamino)phenyl]-6-phenylhexa- 1,3,5-triene (TMA-DPH) into inside-out membrane vesicles are driven by the membrane potential (Delta psi) and the transmembrane proton gradi ent (Delta pH), pointing to an electrogenic drug/proton antiport mecha nism, Ethidium bromide, a substrate for LmrP, inhibited the LmrP-media ted TPP+ extrusion from right-side-out membrane vesicles, showing that LmrP is capable of transporting structurally unrelated drugs. Kinetic analysis of LmrP-mediated TMA-DPH transport revealed a direct relatio n between the transport rate and the amount of TMA-DPH associated with the cytoplasmic leaflet of the lipid bilayer. This observation indica tes that drugs are extruded from the inner leaflet of the cytoplasmic membrane into the external medium. This is the first report that shows that drug extrusion by a secondary multidrug resistance (MDR) transpo rter occurs by a ''hydrophobic vacuum cleaner'' mechanism in a similar way as was proposed for the primary lactococcal MDR transporter, LmrA .