Multidrug resistance in Lactococcus lactis: evidence for ATP-dependent drug extrusion from the inner leaflet of the cytoplasmic membrane.
Lactococcus lactis possesses an ATP-dependent drug extrusion system which shares functional properties with the mammalian multidrug resistance (MDR) transporter P-glycoprotein. One of the intriguing aspects of both transporters is their ability to interact with a broad range of structurally unrelated amphiphilic compounds. It has been suggested that P-glycoprotein removes drugs directly from the membrane. Evidence is presented that this model is correct for the lactococcal multidrug transporter through studies of the extrusion mechanism of BCECF-AM and cationic diphenylhexatriene (DPH) derivatives from the membrane. The non-fluorescent probe BCECF-AM can be converted intracellularly into its fluorescent derivative, BCECF, by non-specific esterase activities. The development of fluorescence was decreased upon energization of the cells. These and kinetic studies showed that BCECF-AM is actively extruded from the membrane before it can be hydrolysed intracellularly. The increase in fluorescence intensity due to the distribution of TMA-DPH into the phospholipid bilayer is a biphasic process. This behaviour reflects the fast entry of TMA-DPH into the outer leaflet followed by a slower transbilayer movement to the inner leaflet of the membrane. The initial rate of TMA-DPH extrusion correlates with the amount of probe associated with the inner leaflet. Taken together, these results demonstrate that the lactococcal MDR transporter functions as a 'hydrophobic vacuum cleaner', expelling drugs from the inner leaflet of the lipid bilayer. Thus, the ability of amphiphilic substrates to partition in the inner leaflet of the membrane is a prerequisite for recognition by multidrug transporters.
Bolhuis
H
H
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
van Veen
H W
HW
Molenaar
D
D
Poolman
B
B
Driessen
A J
AJ
Konings
W N
WN
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000502:physiology*; D001426:Bacterial Proteins / Q000378:metabolism*; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000378:metabolism*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004161:Diphenylhexatriene / Q000031:analogs & derivatives / Q000378:metabolism; D004352:Drug Resistance, Microbial; D018432:Drug Resistance, Multiple; D005452:Fluoresceins / Q000378:metabolism; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism; D008954:Models, Biological
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001426:Bacterial Proteins; D005452:Fluoresceins; D008051:Lipid Bilayers; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; D004161:Diphenylhexatriene; C033628:1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene; D000255:Adenosine Triphosphate