title: Functionally important carboxyls in a bacterial homologue of the vesicular monoamine transporter (VMAT).
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.
authors.affiliation: From the Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University, 91904 Jerusalem, Israel.
authors.forename: Dana
authors.initials: D
authors.lastname: Yaffe
authors.affiliation: the Computational Structural Biology Section, NINDS, National Institutes of Health, Bethesda, Maryland 20852, and.
authors.forename: Ariela
authors.initials: A
authors.lastname: Vergara-Jaque
authors.affiliation: From the Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University, 91904 Jerusalem, Israel.
authors.forename: Yonatan
authors.initials: Y
authors.lastname: Shuster
authors.affiliation: From the Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University, 91904 Jerusalem, Israel.
authors.forename: Dina
authors.initials: D
authors.lastname: Listov
authors.affiliation: the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520.
authors.forename: Sitaram
authors.initials: S
authors.lastname: Meena
authors.affiliation: the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520.
authors.forename: Satinder K
authors.initials: SK
authors.lastname: Singh
authors.affiliation: the Computational Structural Biology Section, NINDS, National Institutes of Health, Bethesda, Maryland 20852, and.
authors.forename: Lucy R
authors.initials: LR
authors.lastname: Forrest
authors.affiliation: From the Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University, 91904 Jerusalem, Israel, shimon.schuldiner@huji.ac.il.
authors.forename: Shimon
authors.initials: S
authors.lastname: Schuldiner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015306:Biogenic Monoamines / Q000737:chemistry* / Q000378:metabolism; D058675:Brevibacillus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D009435:Synaptic Transmission / Q000502:physiology; D050493:Vesicular Monoamine Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D015306:Biogenic Monoamines; D002352:Carrier Proteins; D011994:Recombinant Proteins; C493454:Slc18a2 protein, rat; D050493:Vesicular Monoamine Transport Proteins; D006639:Histidine; D001120:Arginine
keywords: Antibiotic Resistance; Membrane Transport; Multidrug Transporter; Neurotransmitter; Neurotransmitter Transport; Proton Transport