Restrictive use of detergents in the functional reconstitution of the secondary multidrug transporter LmrP.
The histidine-tagged secondary multidrug transporter LmrP was overexpressed in Lactococcus lactis, using a novel protein expression system for cytotoxic proteins based on the tightly regulated, nisin-inducible nisA promoter. LmrP-mediated H+/drug antiport activity in inside-out membrane vesicles was inhibited by detergents, such as Triton X-100, Triton X-114, and Tween 80, at low concentrations that did not affect the magnitude or composition of the proton motive force. The inhibition of the activity of LmrP by detergents restricted the range of compounds that could be used for the solubilization and reconstitution of the protein because low concentrations of detergent are retained in proteoliposomes. Surprisingly, dodecyl maltoside did not modulate the activity of LmrP. Therefore, LmrP was solubilized with dodecyl maltoside, purified by nickel-chelate affinity chromatography, and reconstituted in dodecyl maltoside-destabilized, preformed liposomes prepared from Escherichia coli phospholipids and egg phosphatidylcholine. Reconstituted LmrP mediated the transport of multiple drugs in response to an artificially imposed pH gradient, demonstrating that the protein functions as a proton motive force-dependent multidrug transporter, independent of accessory proteins. These observations are relevant for the effective solubilization and reconstitution of multidrug transporters belonging to the major facilitator superfamily, which, in view of their broad drug specificity, may strongly interact with detergents.
Putman
M
M
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
van Veen
H W
HW
Poolman
B
B
Konings
W N
WN
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000378:metabolism; D013294:Lactococcus lactis; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D026901:Membrane Transport Proteins; D017830:Octoxynol / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D011136:Polysorbates / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis; D012995:Solubility
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003902:Detergents; C096807:LmrP protein, Lactococcus lactis; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011136:Polysorbates; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D006639:Histidine; D017830:Octoxynol; C010615:Nonidet P-40