Multidrug resistance in Lactococcus lactis: evidence for ATP‐dependent drug extrusion from the inner leaflet of the cytoplasmic membrane

• Published in: The EMBO journal Vol. 15; no. 16; pp. 4239 - 4245
• Main Authors: Bolhuis, H., Veen, H. W., Molenaar, D., Poolman, B., Driessen, A. J., Konings, W. N.
• Format: Journal Article
• Language: English
• Published: England 15.08.1996
• Subjects: Adenosine Triphosphate - physiology; ATP-Binding Cassette, Sub-Family B, Member 1 - metabolism; Bacterial Proteins - metabolism; Biological Transport, Active; Cell Membrane - metabolism; Chemical Phenomena; Chemistry, Physical; Diphenylhexatriene - analogs & derivatives; Diphenylhexatriene - metabolism; Drug Resistance, Microbial; Drug Resistance, Multiple; Fluoresceins - metabolism; Lactococcus lactis; Lactococcus lactis - genetics; Lactococcus lactis - metabolism; Lipid Bilayers - metabolism; Models, Biological
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1996.tb00798.x

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.