H. Bolhuis et al., ENERGETICS AND MECHANISM OF DRUG TRANSPORT MEDIATED BY THE LACTOCOCCAL MULTIDRUG TRANSPORTER LMRP, The Journal of biological chemistry, 271(39), 1996, pp. 24123-24128
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
.