Groningen Proton motive force - dependent Hoechst 33342 transport by the ABC transporter LmrA of Lactococcus lactis

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 ∆lmrA ∆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. The bisbenzimide dye Hoechst 33342 is a cell membranepermeant DNA stain that exhibits a bright blue fluorescence upon binding to DNA. In Hoechst 33342-stained murine lymphocyte cells, it was observed that the fluorescence intensity is not always related to DNA content (1). This led to the suggestion that membranes may exhibit different permeabilities for Hoechst 33342. Involvement of drug resistance proteins followed from experiments with colchicine-resistant Chinese hamster ovary cell lines (1). Cell lines with an increased resistance to colchicine concomitantly showed a decreased rate of uptake of the dye. At a later stage, Hoechst 33342 was identified as a substrate for P-glycoprotein (P-gp)1 (2). Hoechst 33342 not only is fluorescent when bound to DNA but also exhibits an increased fluorescence when it partitions into the hydrophobic environment of the lipid membrane. Since Hoechst 33342 is essentially nonfluorescent in an aqueous environment, (net) transport of the compound from the membrane to the aqueous phase will result in a decrease in fluorescence. This has been the basis of Hoechst 33342 transport assays performed with reconstituted proteoliposomes and membrane vesicles derived from cells. Transport of Hoechst 33342 can be continuously monitored by the changes in fluorescence. The Hoechst 33342 transport assay was first used to show transport activity by reconstituted P-gp (2) and with plasma membrane vesicles derived from highly multidrug-resistant cells that express high levels of P-gp (3-5). The assay has been widely used for the analysis of bacterial multidrug transporters, like BmrA from Bacillus subtilis (6), HorA from Lactobacillus breVis (7), MdfA from Escherichia coli (8), NorA from Staphylococcus aureus (9), and LmrP, LmrA, and LmrCD from Lactococcus lactis (10-12). Likewise, Hoechst 33342 can also be used for binding studies (13). To study the self-association of the LmrA dimer and the presumed cooperativity between the nucleotide binding domains, LmrA mutants defective in ATPase activity have been constructed. As expected, these mutations resulted in an inactivation of ATPase activity. Remarkably, the ATPase mutants of LmrA showed Hoechst 33342 transport. Here we show that LmrA mediates transmembrane passage of Hoechst 33342 in a pH-dependent manner. EXPERIMENTAL PROCEDURES Bacterial Strains, Plasmids, and Growth Conditions. L. lactis NZ9000 (∆lmrA), an lmrA deletion strain (14), and † This work was supported by the Dutch Cancer Society. * To whom correspondence should be addressed. Telephone: 3150-3632164. Fax: 31-50-3632154. E-mail: [email protected]. ‡ Current address: Department of Infectious Diseases, Centre for Molecular Microbiology and Infection, Imperial College London, Flowers Building, Armstrong Road, London SW7 2AZ, United Kingdom. 1 Abbreviations: ABC, ATP-binding cassette; DCCD, N,N′-dicyclohexylcarbodiimide; DDM, n-dodecyl â-D-maltoside; MDR, multidrug resistance; NBD, nucleotide binding domain; NEM, N-ethylmaleimide; P-gp, P-glycoprotein; pmf, proton motive force; TMS, transmembrane segment. 16931 Biochemistry 2005, 44, 16931-16938 10.1021/bi051497y CCC: $30.25 © 2005 American Chemical Society Published on Web 11/25/2005 NZ9000 (∆lmrA ∆lmrCD) (12a) were used as a host for pNZ8048-derived plasmids that permit expression of wildtype and mutant His-tagged LmrA and Strep-tagged LmrCD proteins under control of the nisin system (NICE) (Table 1) (15). Cells were grown in M17 medium (Difco) supplemented with 0.5% (w/v) glucose and 5 μg/mL chloramphenicol. DNA Manipulation. General procedures for cloning and DNA manipulation were performed essentially as described by Sambrook et al. (16). For site-directed mutagenesis to create the nucleotide binding site mutants G490A and E512Q of LmrA, the following primers were used: 5′-ggagtcaaaatttctggtgcacaaagacaacg-3′ (G490A-FWD), 5′-cgttgtctttgtgcaccagaaattttgactcc-3′ (G490A-REV), 5′-ctaatgcttgatcaagcaacagc-3′ (E512Q-FWD), 5′-gctgttgcttgatcaagcattag-3′ (E512Q-REV), 5′-gattttgcttacgatgattctgaacaaatattgc-3′ [“upstream” primer (EcoRV)], and 5′-ggttttctaattttggttcaaagaaagcttgagc-3′ [“downstream” primer (XbaI)]. The PCR overlap extension method (17) was used to introduce the G490A (codon gca instead of gga on the DNA level) and E512Q (codon caa instead of gaa on the DNA level) mutations into the lmrA gene on the expression plasmid pNHLmrA, yielding pHLA490A and pHLA512Q, respectively (Table 1). Preparation of Inside-Out Membrane Vesicles. Inside-out membrane vesicles were prepared from L. lactis NZ9000 ∆lmrA cells harboring pNZ8048-based expression or control vectors. Cells were grown at 30 °C to an OD660 of 0.6-0.8, whereupon 0.5 ng/mL Nisin A was added. Growth was continued for a further 90 min. Cells were harvested by centrifugation, washed with 100 mM Hepes-KOH (pH 7.0), and resuspended in the same buffer. Inside-out membrane vesicles were prepared by French pressure cell treatment as described previously (11). Membrane vesicles suspended in 100 mM Hepes-KOH (pH 7.0) containing 10% (v/v) glycerol were stored at -80 °C until they were used. DCCD Treatment of Membrane Vesicles. To inactivate the F0F1-ATPase, membranes (10 mg/mL total protein) were incubated for 30 min with 10 mM N,N′-dicyclohexylcarbodiimide (DCCD) on ice. Membrane vesicles were collected by centrifugation, resuspended in 50 mM Hepes-KOH (pH 7.0) containing 10% (v/v) glycerol, and stored at -80 °C. Purification of His6-LmrA. Membranes (10-20 mg/mL total protein) bearing overexpressed histidine-tagged wildtype or mutant LmrA were solubilized in 50 mM HepesKOH (pH 8.0) containing 0.3 M NaCl, 10% (v/v) glycerol, and 1% (w/v) n-dodecyl â-D-maltoside (DDM). The suspension was mixed and incubated on ice for 30 min. Insoluble material was removed by centrifugation at 280000g (20 min at 4 °C). Solubilized membrane proteins were mixed with Ni2+-NTA agarose (Qiagen, ∼25 μL of resin/mg of protein) which was pre-equilibrated in buffer A [50 mM Hepes-KOH (pH 8.0), 0.3 M NaCl, 10% (v/v) glycerol, and 0.05% (w/v) DDM] containing 20 mM imidazole. The suspension was incubated for 1 h at 4 °C, transferred to a Bio-spin column (Bio-Rad), washed with 20 column volumes of buffer A containing 20 mM imidazole, and eluted with buffer A (pH 7.0) containing 250 mM imidazole (all at 4 °C). The purified protein was used immediately for reconstitution. Reconstitution of LmrA into Liposomes. E. coli total lipid extract (Avanti Polar Lipids) was washed with an acetone/ ether mixture (18), dried, and resuspended in 50 mM HepesKOH (pH 7.2) at a concentration of 20 mg/mL. The suspension was frozen in liquid nitrogen, slowly thawed at room temperature, and sonicated on ice using a tip sonicator at an intensity of 4 μm (peak to peak) for four cycles of sonication for 15 s and resting for 45 s. Aliquots of 2 mL were frozen in liquid nitrogen and slowly thawed at room temperature. This freeze-thaw step was repeated once, and aliquots of the liposome suspension were frozen in liquid nitrogen and stored at -80 °C. To obtain unilamellar liposomes with a relatively homogeneous size, the frozen liposome suspension was slowly thawed at room temperature and extruded 11 times through a 400 nm polycarbonate filter. After dilution to 4 mg of lipid/mL, liposomes were saturated with DDM and solubilization was monitored at OD450 as described by Paternostre et al. (19). Purified LmrA was mixed with DDM-saturated liposomes (1 μmol of DDM/ mg of lipid) at a 1/20 ratio (w/w) and incubated for 30 min at room temperature under gentle agitation. The detergent was removed by two successive extractions with polystyrene beads (Bio-Beads SM-2, Bio-Rad, extensively washed with methanol, ethanol, and water), first at a wet weight of 80 mg/mL of liposome suspension for 2 h at room temperature and then at a wet weight of 160 mg/mL of liposome suspension overnight at 4 °C, under gentle agitation. The proteoliposomes were harvested by centrifugation (280000g for 30 min at 4 °C), resuspended in 50 mM Hepes-KOH (pH 7.4) at a final concentration of 1 mg/mL, frozen in liquid nitrogen, and stored at -80 °C. ATPase Assay. The ATPase activity of reconstituted LmrA was determined using the colorimetric assay of Lanzetta et al. (20). Proteoliposomes were incubated at 30 °C in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NH4Cl, 5 mM MgSO4, 15 mM MgCl2, and 1 mM ATP. At regular time intervals, samples of 30 μL were transferred to a 96well microplate and 150 μL of malachite green molybdate reagent was added. After 5 min, 34% citric acid was added. Absorbance at 600 nm was measured after incubation for 50 min at room temperature and compared with that of a phosphorus standard. Hoechst 33342 Transport Assays. The transport activity of LmrA and LmrCD was assayed by means of the drug Hoechst 33342 [2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole, Molecular Probes] (11, 12). Table 1: L. lactis Plasmids plasmid relevant characteristics ref pNHLmrA pNZ8048 harboring the lmrA gene with upstream regions encoding an N-terminal six-histidine tag and an enterokinase cleavage site 11 pHLA490A pNHLmrA encoding the G490A ATPase mutant of LmrA this study pHLA512Q pNHLmrA encoding the E512Q ATPase mutant of LmrA this study pNSGA pNZ8048 harboring the LmrCD gene with a downstream region encoding a Streptag II 12 control vector pNZ8048 expression vector carrying the inducible PnisA, Cmr 15 16932 Biochemistry, Vol. 44, No. 51, 2005 van den Berg van Saparoea et al.