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• W2069235280 abstract "Transporters essential for neurotransmission in mammalian organisms and bacterial multidrug transporters involved in antibiotic resistance are evolutionarily related. To understand in more detail the evolutionary aspects of the transformation of a bacterial multidrug transporter to a mammalian neurotransporter and to learn about mechanisms in a milieu amenable for structural and biochemical studies, we identified, cloned, and partially characterized bacterial homologues of the rat vesicular monoamine transporter (rVMAT2). We performed preliminary biochemical characterization of one of them, Brevibacillus brevis monoamine transporter (BbMAT), from the bacterium B. brevis. BbMAT shares substrates with rVMAT2 and transports them in exchange with >1H+, like the mammalian transporter. Here we present a homology model of BbMAT that has the standard major facilitator superfamily fold; that is, with two domains of six transmembrane helices each, related by 2-fold pseudosymmetry whose axis runs normal to the membrane and between the two halves. The model predicts that four carboxyl residues, a histidine, and an arginine are located in the transmembrane segments. We show here that two of the carboxyls are conserved, equivalent to the corresponding ones in rVMAT2, and are essential for H+-coupled transport. We conclude that BbMAT provides an excellent experimental paradigm for the study of its mammalian counterparts and bacterial multidrug transporters. Transporters essential for neurotransmission in mammalian organisms and bacterial multidrug transporters involved in antibiotic resistance are evolutionarily related. To understand in more detail the evolutionary aspects of the transformation of a bacterial multidrug transporter to a mammalian neurotransporter and to learn about mechanisms in a milieu amenable for structural and biochemical studies, we identified, cloned, and partially characterized bacterial homologues of the rat vesicular monoamine transporter (rVMAT2). We performed preliminary biochemical characterization of one of them, Brevibacillus brevis monoamine transporter (BbMAT), from the bacterium B. brevis. BbMAT shares substrates with rVMAT2 and transports them in exchange with >1H+, like the mammalian transporter. Here we present a homology model of BbMAT that has the standard major facilitator superfamily fold; that is, with two domains of six transmembrane helices each, related by 2-fold pseudosymmetry whose axis runs normal to the membrane and between the two halves. The model predicts that four carboxyl residues, a histidine, and an arginine are located in the transmembrane segments. We show here that two of the carboxyls are conserved, equivalent to the corresponding ones in rVMAT2, and are essential for H+-coupled transport. We conclude that BbMAT provides an excellent experimental paradigm for the study of its mammalian counterparts and bacterial multidrug transporters. Vesicular neurotransmitter transporters mediate the uptake and storage of neurotransmitter molecules in synaptic vesicles and are essential for regulated synaptic release (1Eiden L.E. The vesicular neurotransmitter transporters: current perspectives and future prospects.FASEB J. 2000; 14: 2396-2400Crossref PubMed Scopus (70) Google Scholar, 2Schuldiner S. Shirvan A. Linial M. Vesicular neurotransmitter transporters: from bacteria to humans.Physiol. Rev. 1995; 75: 369-392Crossref PubMed Scopus (266) Google Scholar, 3Chaudhry F.A. Edwards R.H. Fonnum F. Vesicular neurotransmitter transporters as targets for endogenous and exogenous toxic substances.Annu. Rev. Pharmacol. Toxicol. 2008; 48: 277-301Crossref PubMed Scopus (61) Google Scholar, 4Edwards R. The transport of neurotransmitters into synaptic vesicles.Curr. Opin. Neurobiol. 1992; 2: 586-594Crossref PubMed Scopus (88) Google Scholar). The transport of monoamines (serotonin, dopamine, histamine, adrenaline, and noradrenaline) is carried out by the vesicular monoamine transporter (VMAT) 3The abbreviations used are: VMATvesicular monoamine transporterMPP+1-methyl-4-phenylpyridiniumMFSmajor facilitator superfamilyBbMATB. brevis monoamine transporterMDTmultidrug transporterTMtransmembraneTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. family, which includes two isoforms: VMAT1 and VMAT2. The influx of charged substrates is coupled to the opposite movement of two protons (1Eiden L.E. The vesicular neurotransmitter transporters: current perspectives and future prospects.FASEB J. 2000; 14: 2396-2400Crossref PubMed Scopus (70) Google Scholar, 2Schuldiner S. Shirvan A. Linial M. Vesicular neurotransmitter transporters: from bacteria to humans.Physiol. Rev. 1995; 75: 369-392Crossref PubMed Scopus (266) Google Scholar). In addition to their native substrates, VMATs interact with many clinically relevant drugs, including the psychostimulant 3,4-methylene-dioxymethamphetamine and the parkinsonian toxin 1-methyl-4-phenylpyridinium (MPP+) (2Schuldiner S. Shirvan A. Linial M. Vesicular neurotransmitter transporters: from bacteria to humans.Physiol. Rev. 1995; 75: 369-392Crossref PubMed Scopus (266) Google Scholar, 3Chaudhry F.A. Edwards R.H. Fonnum F. Vesicular neurotransmitter transporters as targets for endogenous and exogenous toxic substances.Annu. Rev. Pharmacol. Toxicol. 2008; 48: 277-301Crossref PubMed Scopus (61) Google Scholar, 4Edwards R. The transport of neurotransmitters into synaptic vesicles.Curr. Opin. Neurobiol. 1992; 2: 586-594Crossref PubMed Scopus (88) Google Scholar, 5Yelin R. Schuldiner S. The pharmacological profile of the vesicular monoamine transporter resembles that of multidrug transporters.FEBS Lett. 1995; 377: 201-207Crossref PubMed Scopus (58) Google Scholar, 6Darchen F. Scherman D. Desnos C. Henry J.-P. Characteristics of the transport of the quaternary ammonium 1-methyl-4-phenylpyridinium by chromaffin granules.Biochem. Pharmacol. 1988; 37: 4381-4387Crossref PubMed Scopus (32) Google Scholar). Heterologous expression of VMATs protects mammalian and yeast cells against MPP+ toxicity by sequestering the toxin in vesicles, away from its primary site of action in mitochondria (7Liu Y. Peter D. Roghani A. Schuldiner S. Privé G.G. Eisenberg D. Brecha N. Edwards R.H. A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter.Cell. 1992; 70: 539-551Abstract Full Text PDF PubMed Scopus (523) Google Scholar, 8Gros Y. Schuldiner S. Directed evolution reveals hidden properties of VMAT, a neurotransmitter transporter.J. Biol. Chem. 2010; 285: 5076-5084Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). vesicular monoamine transporter 1-methyl-4-phenylpyridinium major facilitator superfamily B. brevis monoamine transporter multidrug transporter transmembrane N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Phylogenetic analysis reveals that VMAT proteins belong to the drug/H+ antiporters (DHA12) subfamily of the major facilitator superfamily (MFS), i.e. they are evolutionarily related to bacterial drug transporters and multidrug transporters (2Schuldiner S. Shirvan A. Linial M. Vesicular neurotransmitter transporters: from bacteria to humans.Physiol. Rev. 1995; 75: 369-392Crossref PubMed Scopus (266) Google Scholar, 7Liu Y. Peter D. Roghani A. Schuldiner S. Privé G.G. Eisenberg D. Brecha N. Edwards R.H. A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter.Cell. 1992; 70: 539-551Abstract Full Text PDF PubMed Scopus (523) Google Scholar, 9Vardy E. Arkin I.T. Gottschalk K.E. Kaback H.R. Schuldiner S. Structural conservation in the major facilitator superfamily as revealed by comparative modeling.Protein Sci. 2004; 13: 1832-1840Crossref PubMed Scopus (88) Google Scholar, 10Vardy E. Steiner-Mordoch S. Schuldiner S. Characterization of bacterial drug antiporters homologous to mammalian neurotransmitter transporters.J. Bacteriol. 2005; 187: 7518-7525Crossref PubMed Scopus (17) Google Scholar). Moreover, using the power of yeast genetics, we were able to demonstrate that three mutations are sufficient to transform rVMAT2 back into an MDT. These mutations cause rVMAT2 to lose the ability to transport neurotransmitters while still conferring resistance against the toxic substrates MPP+, ethidium, and acriflavine (8Gros Y. Schuldiner S. Directed evolution reveals hidden properties of VMAT, a neurotransmitter transporter.J. Biol. Chem. 2010; 285: 5076-5084Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The above findings led us to study bacterial homologues to try to understand in greater detail the evolutionary aspects of the transformation of a bacterial MDT to a mammalian vesicular neurotransmitter transporter and to unveil mechanisms in a milieu more amenable to structural, biochemical, and biophysical studies. Crystal structures and subsequent mechanistic studies of prokaryotic homologues of mammalian neurotransporters have had a major impact on the field (11Singh S.K. LeuT: a prokaryotic stepping stone on the way to a eukaryotic neurotransmitter transporter structure.Channels. 2008; 2: 380-389Crossref PubMed Scopus (31) Google Scholar, 12Gouaux E. Review: the molecular logic of sodium-coupled neurotransmitter transporters.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2009; 364: 149-154Crossref PubMed Scopus (40) Google Scholar). Notably, in the case of the plasma membrane transporters, they have served as structural paradigms for the interpretation of a wealth of biochemical and electrophysiological data available on their eukaryotic counterparts (12Gouaux E. Review: the molecular logic of sodium-coupled neurotransmitter transporters.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2009; 364: 149-154Crossref PubMed Scopus (40) Google Scholar, 13Forrest L.R. Rudnick G. The rocking bundle: a mechanism for ion-coupled solute flux by symmetrical transporters.Physiology. 2009; 24: 377-386Crossref PubMed Scopus (216) Google Scholar, 14Boudker O. Verdon G. Structural perspectives on secondary active transporters.Trends Pharmacol Sci. 2010; 31: 418-426Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). We identified over a dozen candidate transporters and chose to biochemically characterize one from Brevibacillus brevis (B. brevis monoamine transporter, BbMAT). It displays high sequence homology (25% identity and 52% similarity) to and shares some of the substrates (MPP+ and acriflavine) with rVMAT2, using exchange for multiple protons as the driving force. Because these bacterial multidrug transporters translocate toxic compounds and antibiotics away from the cytoplasm, they confer resistance against these toxins, and they have been associated with the phenomenon of resistance to multiple antibiotics. Antimicrobial resistance has been identified recently as one of the most serious health threats in a 2013 Centers for Disease Control and Prevention report (15Center for Disease Control (2013) Annual Report of the Centers for Disease Control. USA Center for Disease Control and Prevention, Atlanta, GA.Google Scholar). Therefore, study of these homologues should also aid in understanding basic mechanisms of antibiotic resistance. Despite exciting recent progress, three-dimensional structures are available for only 13 MFS proteins, none of which have been shown to function as an H+-coupled antiporter (16Yan N. Structural advances for the major facilitator superfamily (MFS) transporters.Trends Biochem. Sci. 2013; 38: 151-159Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Here we present a homology model of BbMAT on the basis of the crystal structure of YajR, a putative proton-driven MFS transporter from Escherichia coli (17Jiang D. Zhao Y. Wang X. Fan J. Heng J. Liu X. Feng W. Kang X. Huang B. Liu J. Zhang X.C. Structure of the YajR transporter suggests a transport mechanism based on the conserved motif A.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 14664-14669Crossref PubMed Scopus (128) Google Scholar). As expected, the model contains 12 transmembrane (TM) helices, arranged in two domains of six TMs each, which are related by 2-fold pseudosymmetry with an axis that runs normal to the membrane and between the two halves. According to this model, there are four membrane-embedded carboxylic amino acids, but we show that only two, Asp-25 (TM1) and Glu-229 (TM7), are essential for transport. Given that the equivalent residues in rVMAT2 are crucial for transport activity, we suggest analogous roles for these residues in the two proteins. A BLAST search using the rVMAT2 sequence as a query against the non-redundant database of the available microbial genomes was performed using the NCBI server. The rVMAT2 and BbMAT sequences were aligned using Clustal Omega (18Goujon M. McWilliam H. Li W. Valentin F. Squizzato S. Paern J. Lopez R. A new bioinformatics analysis tools framework at EMBL-EBI.Nucleic Acids Res. 2010; 38: W695-W699Crossref PubMed Scopus (1309) Google Scholar). Conservation analysis was carried out using 500 homologues of BbMAT obtained using three iterations of PSI-BLAST with BbMAT as a query and aligned using Muscle (19Edgar R.C. MUSCLE: multiple sequence alignment with high accuracy and high throughput.Nucleic Acids Research. 2004; 32: 1792-1797Crossref PubMed Scopus (30435) Google Scholar). To build a molecular model of BbMAT, we searched for a template structure using the following search tools: PSI-BLAST v2.2.29 (20Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59879) Google Scholar) on the non-redundant PDB database, I-Tasser v4.0 (21Roy A. Kucukural A. Zhang Y. I-TASSER: a unified platform for automated protein structure and function prediction.Nat. Protoc. 2010; 5: 725-738Crossref PubMed Scopus (4695) Google Scholar) on the basis of multiple-threading alignments obtained from the LOMETS (22Wu S. Zhang Y. LOMETS: a local meta-threading-server for protein structure prediction.Nucleic Acids Res. 2007; 35: 3375-3382Crossref PubMed Scopus (633) Google Scholar) metaserver, and HHblits (from HH-suite v2.0) (23Remmert M. Biegert A. Hauser A. Söding J. HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment.Nat. Methods. 2012; 9: 173-175Crossref Scopus (1321) Google Scholar) using a non-redundant PDB sequence database clustered at 70% sequence identity. Databases were dated July 12, 2014. The crystal structure of a putative proton-driven E. coli transporter, YajR, in an outward-facing conformation (PDB code 3WDO) (17Jiang D. Zhao Y. Wang X. Fan J. Heng J. Liu X. Feng W. Kang X. Huang B. Liu J. Zhang X.C. Structure of the YajR transporter suggests a transport mechanism based on the conserved motif A.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 14664-14669Crossref PubMed Scopus (128) Google Scholar) exhibited the highest sequence identity (∼20%) and excellent coverage (∼90%), with E-values from PSI-BLAST and HHblits of 6 × 10−38 and 8.6 × 10−34, respectively. The structure of YajR was therefore selected as the most suitable template. In preparation for modeling, a preliminary pair-wise sequence alignment of YajR and BbMAT was constructed using the AlignMe PST mode on the AlignMe server v1.1 (24Stamm M. Staritzbichler R. Khafizov K. Forrest L.R. AlignMe: a membrane protein sequence alignment web server.Nucleic Acids Res. 2014; 42: W246-W251Crossref PubMed Scopus (64) Google Scholar). Conserved MFS and DHA12 sequence motifs were aligned correctly (10Vardy E. Steiner-Mordoch S. Schuldiner S. Characterization of bacterial drug antiporters homologous to mammalian neurotransmitter transporters.J. Bacteriol. 2005; 187: 7518-7525Crossref PubMed Scopus (17) Google Scholar, 17Jiang D. Zhao Y. Wang X. Fan J. Heng J. Liu X. Feng W. Kang X. Huang B. Liu J. Zhang X.C. Structure of the YajR transporter suggests a transport mechanism based on the conserved motif A.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 14664-14669Crossref PubMed Scopus (128) Google Scholar, 25Yaffe D. Radestock S. Shuster Y. Forrest L.R. Schuldiner S. Identification of molecular hinge points mediating alternating access in the vesicular monoamine transporter VMAT2.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: E1332-E1341Crossref PubMed Scopus (25) Google Scholar). Alternative sequence matches were identified on the basis of a multiple sequence alignment obtained using the PRALINETM server (26Simossis V.A. Heringa J. PRALINE: a multiple sequence alignment toolbox that integrates homology-extended and secondary structure information.Nucleic Acids Res. 2005; 33: W289-W294Crossref PubMed Scopus (364) Google Scholar) and on a multiple structure alignment generated using MAMMOTH-mult (27Lupyan D. Leo-Macias A. Ortiz A.R. A new progressive-iterative algorithm for multiple structure alignment.Bioinformatics. 2005; 21: 3255-3263Crossref PubMed Scopus (106) Google Scholar) of 36 MFS proteins. These proteins were identified with the search tools used to find the template. Adjustments to the AlignMe alignment were made in the following residues of the BbMAT sequence: 42–43, 113–116, 133–134, 199–206, 241–301, and 324–334. The adjustments were selected to optimize the agreement with PSIPRED v3.2 (28Jones D.T. Protein secondary structure prediction based on position-specific scoring matrices.J. Mol. Biol. 1999; 292: 195-202Crossref PubMed Scopus (4464) Google Scholar) secondary structure and TOPCONS (29Bernsel A. Viklund H. Hennerdal A. Elofsson A. TOPCONS: consensus prediction of membrane protein topology.Nucleic Acids Res. 2009; 37: W465-W468Crossref PubMed Scopus (426) Google Scholar) transmembrane predictions and to reduce the number of residues in generously allowed and disallowed regions of the Ramachandran plot as evaluated by PROCHECK v3.5.4 (30Laskowski R.A. Macarthur M.W. Moss D.S. Thornton J.M. Procheck: a program to check the stereochemical quality of protein structures.J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). For each adjustment, the effect on the per-residue and total ProQM (31Ray A. Lindahl E. Wallner B. Model quality assessment for membrane proteins.Bioinformatics. 2010; 26: 3067-3074Crossref PubMed Scopus (60) Google Scholar) scores was tracked. In the final alignment, ∼17% of the residues are identical, and the similarity reaches ∼44%, as estimated over the whole sequence but excluding residues 1–6 in the N terminus and residues 389–405 in the C terminus. The four helices that contain the pore-lining residues investigated in this study, namely TM1, TM4, TM7, and TM11, exhibit a similarity with the template of 45, 54, 43, and 63%, respectively. The final alignment (Fig. 3B) was used to build a total of 300 homology models of BbMAT on the basis of YajR using MODELLER (version 9.13) (32Eswar N. Webb B. Marti-Renom M.A. Madhusudhan M.S. Eramian D. Shen M.Y. Pieper U. Sali A. Comparative protein structure modeling using MODELLER.Curr. Protoc. Protein Sci. 2007; 50: 2.9.1-2.9.31Crossref Google Scholar). α Helix constraints were applied for residues 113–119 and 311–315 of TM4 and TM10, respectively. The best BbMAT model (Fig. 3A) was selected as that with the lowest Molpdf energy value of MODELLER (32Eswar N. Webb B. Marti-Renom M.A. Madhusudhan M.S. Eramian D. Shen M.Y. Pieper U. Sali A. Comparative protein structure modeling using MODELLER.Curr. Protoc. Protein Sci. 2007; 50: 2.9.1-2.9.31Crossref Google Scholar) and the highest PROCHECK (30Laskowski R.A. Macarthur M.W. Moss D.S. Thornton J.M. Procheck: a program to check the stereochemical quality of protein structures.J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and global ProQM (31Ray A. Lindahl E. Wallner B. Model quality assessment for membrane proteins.Bioinformatics. 2010; 26: 3067-3074Crossref PubMed Scopus (60) Google Scholar) scores. The final model is of excellent quality according to PROCHECK (30Laskowski R.A. Macarthur M.W. Moss D.S. Thornton J.M. Procheck: a program to check the stereochemical quality of protein structures.J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar), with zero residues in the generously allowed and disallowed regions of the Ramachandran plot. The ProQM score was 0.707, which compares well with that of the template structure (0.741). The model of BbMAT is available upon request. E. coli JM109 (33Yanisch-Perron C. Vieira J. Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.Gene. 1985; 33: 103-119Crossref PubMed Scopus (11461) Google Scholar), BW25113 ΔemrE ΔmdfA (34Tal N. Schuldiner S. A coordinated network of transporters with overlapping specificities provides a robust survival strategy.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9051-9056Crossref PubMed Scopus (137) Google Scholar), and C41 (35Miroux B. Walker J.E. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels.J. Mol. Biol. 1996; 260: 289-298Crossref PubMed Scopus (1577) Google Scholar) were used throughout this work. The desired gene was cloned into pT7-7-Myc-His6 plasmid by PCR using genomic DNA from B. brevis as the template (DSM30, ATCC, catalog no. 8246). Primers were designed to overlap the ends of the gene and included restriction sites for NdeI and EcoRI. The plasmid was named pT7-7 BbMAT. For simplicity, the BbMAT-Myc-His6 protein is termed BbMAT. Site-directed mutagenesis was accomplished with the QuikChange® II site-directed mutagenesis kit (Stratagene), and the sequences of all constructs were verified by DNA sequencing. The sequence of BbMAT cloned and sequenced from the above strain differs from Uniprot entry C0ZB03 as follows: K42M, I59V, M75V, G102A, T137A, K194R, V196E, M260L, G270S, Y275F, K276Q, M283I, W294L, I308V, G321A, T394A, S395R, S397C, and I398T (the first letter denotes the amino acid in the Uniprot entry C0ZB03 sequence, the number is the position in the sequence, and the second letter is the amino acid as sequenced in our laboratory). The new, experimentally determined sequence was used in all our studies. Resistance to toxic compounds was assessed essentially as described in Ref. 36Rotem D. Schuldiner S. EmrE, a multidrug transporter from Escherichia coli, transports monovalent and divalent substrates with the same stoichiometry.J. Biol. Chem. 2004; 279: 48787-48793Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 37Elbaz Y. Salomon T. Schuldiner S. Identification of a glycine motif required for packing in EmrE, a multidrug transporter from Escherichia coli.J. Biol. Chem. 2008; 283: 12276-12283Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, using E. coli JM109 cells transformed with the indicated plasmids. For experiments on solid medium, cells were diluted to an A600 of 0.2, and 4.5 μl of a series of 10-fold dilutions were spotted on LB plates containing 30 mm BisTris propane (pH 7) and 100 μg/ml ampicillin with or without the addition of the indicated concentration of the toxic compound. Growth was analyzed after overnight incubation at 37 °C. For experiments in liquid medium, cells were diluted to an A600 of 0.01 and grown for 12 h in LB medium containing 30 mm BisTris propane (pH 7) and 100 μg/ml ampicillin with or without the addition of an increasing concentration of the toxic compound. The cells were grown in a Synergy 2 Alpha microplate reader (BioTek) at 37 °C, and A600 was measured automatically by the device. IC50 was calculated from growth observed after 6 h. Overexpression, purification, and reconstitution of BbMAT were performed essentially as described for EmrE, with the following modifications (38Soskine M. Adam Y. Schuldiner S. Direct evidence for substrate-induced proton release in detergent-solubilized EmrE, a multidrug transporter.J. Biol. Chem. 2004; 279: 9951-9955Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). E. coli C41 cells bearing the pT7-7 plasmid with either wild-type BbMAT or different mutants were grown in LB medium to an A600 of 0.5. Protein expression was induced by adding isopropyl 1-thio-β-d-galactopyranoside to a final concentration of 300 μm, followed by a 10- to 12-h incubation at 20 °C. Cells were harvested by centrifugation and washed twice with lysis buffer (150 mm NaCl, 15 mm Tris (pH 7.5), and 250 mm sucrose) before further handling or storage at −70 °C. Cells were then resuspended in sodium-sucrose buffer containing 2.5 mm MgSO4, 15 μg/liter culture DNase, and 1 mm PMSF and lysed with a LV1 microfluidizer (Microfluidics) at 16,000 psi. Unbroken cells were sedimented by centrifugation, and the membrane fraction was collected by ultracentrifugation at 245,000 × g for 2 h at 4 °C and resuspended in sodium-sucrose buffer. The membranes were rapidly frozen in liquid nitrogen and stored at −70 °C. Reconstitution was performed by solubilizing membranes containing ∼10 μg of His-tagged protein in sodium buffer (150 mm NaCl, 15 mm Tris (pH 7.5)) containing 0.5% lauryl maltose neopentyl glycol (Anatrace) and 0.5 mm PMSF. After 60 min at 4 °C, unsolubilized material was pelleted by centrifugation (208,000 × g for 15 min), imidazole was added to 10 mm, and the His-tagged protein was incubated with 10 μl Ni2+-nitrilotriacetic acid beads (Qiagen) for 1 h at 4 °C. Beads were then washed once with 10 volumes of sodium buffer with 0.005% lauryl maltose neopentyl glycol and 20 mm imidazole and washed three times with 10 volumes of the same solution but with 1% octyl-glucoside (Anatrace) instead of lauryl maltose neopentyl glycol. The protein was eluted with 500 μl of the octyl-glucoside buffer containing 450 mm imidazole and mixed with 5 mg of E. coli polar lipid extract (Avanti) suspended in 500 μl of sodium buffer and 1.2% octyl-glucoside. Eluted protein and phospholipids were sonicated together in a bath-type sonicator to clarity. To remove detergent, the mixture was dialyzed (dialysis membrane molecular weight cutoff 12–14 kDa) overnight at 4 °C against 300 volumes of the appropriate buffer (140 mm (NH4)2-SO4 with 15 mm Tris-SO4 (pH 7.4)). Dialysis buffer was exchanged with fresh buffer for an additional 2 h, and then the mixture was ultracentrifuged for 70 min at 200,000 × g. The liposome pellet was resuspended in 150 μl of the above buffer, divided into 25-μl aliquots, and stored at −70 °C. To quantify the amount of protein, liposomes were solubilized in a denaturing buffer (15 mm Tris-Cl (pH 7.5), 150 mm NaCl, 2% SDS, and 6 m urea) at room temperature for 20 min. The solubilized liposomes were then centrifuged (208,000 × g, 20 min) to remove the insoluble fraction. The supernatant was incubated at 4 °C with Ni2+-nitrilotriacetic acid-agarose beads for 1 h, and the beads were subsequently washed with denaturing buffer. Protein was eluted with sample buffer containing 450 mm imidazole, samples were separated by SDS-PAGE on 15% Laemmli gels (39Laemmli U. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207159) Google Scholar), stained with Coomassie Brilliant Blue, and digitally analyzed with Image Gauge 3.46 Fujifilm software. To evaluate expression levels, membranes were solubilized, separated via SDS-PAGE, and analyzed as described above. Liposomes were thawed and sonicated to clarity in a bath-type sonicator. Uptake was measured in reaction buffer containing 140 mm K2SO4, 15 mm Tricine, 15 mm glycine, and 5 mm MgCl2 (pH 9). Liposomes (1.5 μl) were diluted into 200 μl of reaction buffer with 50 nm valinomycin (unless indicated otherwise) and the indicated concentrations of the radiolabeled MPP+, usually 1 μm. Nonspecific accumulation of [3H]MPP+ was measured in the presence of the ionophore nigericin (15 μm) and subtracted from total transport. The reaction was terminated at the indicated time points by diluting the mixture in 2 ml of ice-cold buffer, filtering through 0.2-μm Supor®-200 filters (PALL), and washing with an additional 2 ml of ice-cold buffer. Radioactivity in the liposomes was measured via liquid scintillation. To assess inhibition by different compounds, liposomes were incubated in the presence of increasing concentrations of the indicated compound together with [3H]MPP+ (1 μm). Data were analyzed with Origin 8.6 (OriginLab). All experiments were performed in duplicate and repeated at least twice (the lowest R-squared value is 0.95). Transport of ethidium in whole cells can be followed conveniently because of the intense increase in ethidium fluorescence after binding with DNA. In this assay, the endogenous energy sources and the proton electrochemical gradient are depleted by treatment with an uncoupler. Under these conditions, ethidium transport is driven only by its chemical gradient. The proton electrochemical gradient can be restored quickly, after removal of the uncoupler, by the addition of glucose. The protocol used for this assay was essentially as described previously (40Yerushalmi H. Lebendiker M. Schuldiner S. EmrE, an Escherichia coli 12-kDa multidrug transporter, exchanges toxic cations and H+ and is soluble in organic solvents.J. Biol. Chem. 1995; 270: 6856-6863Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar), with the following modifications. EmrE and MdfA are the primary endogenous ethidium transporters in E. coli, so we used the knockout strain BW251113 ΔemrEΔmdfA, which has very low background activity (34Tal N. Schuldiner S. A coordinated network of transporters with overlapping specificities provides a robust survival strategy.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9051-9056Crossref PubMed Scopus (137) Google Scholar). Cells transformed with “empty” pT7-7 plasmid (negative control) or one coding for the either the wild-type BbMAT or the indicated mutants were grown at 37 °C in minimal medium A supplemented with 100 μg/ml ampi" @default.
• W2069235280 created "2016-06-24" @default.
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• W2069235280 date "2014-12-01" @default.
• W2069235280 modified "2023-10-17" @default.
• W2069235280 title "Functionally Important Carboxyls in a Bacterial Homologue of the Vesicular Monoamine Transporter (VMAT)" @default.
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