Multidrug transport by the ABC transporter Sav1866 from Staphylococcus aureus.
Sav1866 is an ATP-binding cassette (ABC) protein from the pathogen Staphylococcus aureus and is a homologue of bacterial and human multidrug ABC transporters. Recently, the three-dimensional crystal structure of Sav1866 was determined at 3.0 A resolution [Dawson, R. J., and Locher, K. P. (2006) Nature 443, 180-185]. Although this structure is frequently used to homology model human and microbial ABC multidrug transporters by computational methods, the ability of Sav1866 to transport multiple drugs has not been described. We obtained functional expression of Sav1866 in the drug-sensitive, Gram-positive bacterium Lactococcus lactis Delta lmrA Delta lmrCD lacking major endogenous multidrug transporters. Sav1866 displayed a Hoechst 33342, verapamil, tetraphenylphosphonium, and vinblastine-stimulated ATPase activity. In growing cells, Sav1866 expression conferred resistance to Hoechst 33342. In transport assays in intact cells, Sav1866 catalyzed the translocation of amphiphilic cationic ethidium. Additionally, Sav1866 mediated the active transport of Hoechst 33342 in membrane vesicles and proteoliposomes containing purified and functionally reconstituted protein. Sav1866-mediated resistance and transport were inhibited by the human ABCB1 and ABCC1 modulator verapamil. This work represents the first demonstration of multidrug transport by Sav1866 and suggests that Sav1866 can serve as a well-defined model for studies on the molecular bases of drug-protein interactions in ABC transporters. Our methods for the overexpression, purification, and functional reconstitution of Sav1866 are described in detail.
Velamakanni
Saroj
S
Department of Pharmacology, University of Cambridge CB2 1PD, Cambridge, UK.
Yao
Yao
Y
Gutmann
Daniel A P
DA
van Veen
Hendrik W
HW
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001562:Benzimidazoles / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D024901:Drug Resistance, Multiple, Bacterial; D004996:Ethidium / Q000378:metabolism; D006801:Humans; D011510:Proteolipids / Q000378:metabolism; D013211:Staphylococcus aureus / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D001562:Benzimidazoles; D011510:Proteolipids; C020485:proteoliposomes; D000251:Adenosine Triphosphatases; D004996:Ethidium; C017807:bisbenzimide ethoxide trihydrochloride