title: Proton motive force-dependent Hoechst 33342 transport by the ABC transporter LmrA of Lactococcus lactis.
abstract: The fluorescent compound Hoechst 33342 is a substrate for many multidrug resistance (MDR) transporters and is widely used to characterize their transport activity. We have constructed mutants of the adenosine triphosphate (ATP) binding cassette (ABC)-type MDR transporter LmrA of Lactococcus lactis that are defective in ATP hydrolysis. These mutants and wild-type LmrA exhibited an atypical behavior in the Hoechst 33342 transport assay. In membrane vesicles, Hoechst 33342 transport was shown to be independent of the ATPase activity of LmrA, and it was not inhibited by orthovanadate but sensitive to uncouplers that collapse the proton gradient and to N,N'-dicyclohexylcarbodiimide, an inhibitor of the F0F1-ATPase. In contrast, transport of Hoechst 33342 by the homologous, heterodimeric MDR transporter LmrCD showed a normal ATP dependence and was insensitive to uncouplers of the proton gradient. With intact cells, expression of LmrA resulted in an increased rate of Hoechst 33342 influx while LmrCD caused a decrease in the rate of Hoechst 33342 influx. Cellular toxicity assays using a triple knockout strain, i.e., L. lactis delta lmrA delta lmrCD, demonstrate that expression of LmrCD protects cells against the growth inhibitory effects of Hoechst 33342, while in the presence of LmrA, cells are more susceptible to Hoechst 33342. Our data demonstrate that the LmrA-mediated Hoechst 33342 transport in membrane vesicles is influenced by the transmembrane pH gradient due to a pH-dependent partitioning of Hoechst 33342 into the membrane.
authors.affiliation: Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 NN Haren, The Netherlands.
authors.forename: H Bart
authors.initials: HB
authors.lastname: van den Berg van Saparoea
authors.forename: Jacek
authors.initials: J
authors.lastname: Lubelski
authors.forename: Ronald
authors.initials: R
authors.lastname: van Merkerk
authors.forename: Piotr S
authors.initials: PS
authors.lastname: Mazurkiewicz
authors.forename: Arnold J M
authors.initials: AJ
authors.lastname: Driessen
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001562:Benzimidazoles / Q000378:metabolism* / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007476:Ionophores / Q000494:pharmacology; D013294:Lactococcus lactis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008081:Liposomes; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D008744:Methylamines / Q000494:pharmacology; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009550:Nigericin / Q000494:pharmacology; D018892:Proton-Motive Force / Q000502:physiology*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014634:Valinomycin / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
chemical_list: D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D001426:Bacterial Proteins; D001562:Benzimidazoles; D004024:Dicyclohexylcarbodiimide; D005456:Fluorescent Dyes; D007476:Ionophores; D008081:Liposomes; D026901:Membrane Transport Proteins; D008744:Methylamines; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009550:Nigericin; D006180:Proton-Translocating ATPases; D011994:Recombinant Proteins; D014634:Valinomycin; D014638:Vanadates; C101824:LmrA protein, Lactococcus lactis; C027451:methylamine; C017807:bisbenzimide ethoxide trihydrochloride