A Transporter Interactome Is Essential for the Acquisition of Antimicrobial Resistance to Antibiotics.

Awareness of the problem of antimicrobial resistance (AMR) has escalated and drug-resistant infections are named among the most urgent problems facing clinicians today. Our experiments here identify a transporter interactome and portray its essential function in acquisition of antimicrobial resistance. By exposing E.

Topology determination of untagged membrane proteins.

The topology of integral membrane proteins with a weak topological tendency can be influenced when fused to tags, such as these used for topological determination or protein purification. Here, we describe a technique for topology determination of an untagged membrane protein. This technique, optimized for bacterial cells, allows the visualization of the protein in the native environment and incorporates the substituted-cysteine accessibility method.

New substrates on the block: clinically relevant resistances for EmrE and homologues.

Transporters of the small multidrug resistance (SMR) family are small homo- or heterodimers that confer resistance to multiple toxic compounds by exchanging substrate with protons. Despite the wealth of biochemical information on EmrE, the most studied SMR member, a high-resolution three-dimensional structure is missing. To provide proteins that are more amenable to biophysical and structural studies, we identified and partially characterized SMR transporters from bacteria living under extreme conditions of temperature and radiation.

Topologically random insertion of EmrE supports a pathway for evolution of inverted repeats in ion-coupled transporters.

Inverted repeats in ion-coupled transporters have evolved independently in many unrelated families. It has been suggested that this inverted symmetry is an essential element of the mechanism that allows for the conformational transitions in transporters. We show here that small multidrug transporters offer a model for the evolution of such repeats.

Parallel topology of genetically fused EmrE homodimers.

EmrE is a small H+-coupled multidrug transporter in Escherichia coli. Claims have been made for an antiparallel topology of this homodimeric protein. However, our own biochemical studies performed with detergent-solubilized purified protein support a parallel topology of the protomers.

Identification of tyrosine residues critical for the function of an ion-coupled multidrug transporter.

Aromatic residues may play several roles in integral membrane proteins, including direct interaction with substrates. In this work, we studied the contribution of tyrosine residues to the activity of EmrE, a small multidrug transporter from Escherichia coli that extrudes various drugs across the plasma membrane in exchange with protons. Each of five tyrosine residues was replaced by site-directed mutagenesis.

Characterization of bacterial drug antiporters homologous to mammalian neurotransmitter transporters.

Multidrug transporters are ubiquitous proteins, and, based on amino acid sequence similarities, they have been classified into several families. Here we characterize a cluster of archaeal and bacterial proteins from the major facilitator superfamily (MFS). One member of this family, the vesicular monoamine transporter (VMAT) was previously shown to remove both neurotransmitters and toxic compounds from the cytoplasm, thereby conferring resistance to their effects.

Exploring the binding domain of EmrE, the smallest multidrug transporter.

EmrE is a small multidrug transporter in Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons, thereby rendering cells resistant to these compounds. Biochemical experiments indicate that the basic functional unit of EmrE is a dimer where the common binding site for protons and substrate is formed by the interaction of an essential charged residue (Glu14) from both EmrE monomers. Previous studies implied that other residues in the vicinity of Glu14 are part of the binding domain.

Substrate-induced tryptophan fluorescence changes in EmrE, the smallest ion-coupled multidrug transporter.

Tryptophan residues may play several roles in integral membrane proteins including direct interaction with substrates. In this work we studied the contribution of tryptophan residues to substrate binding in EmrE, a small multidrug transporter of Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons. Each of the four tryptophan residues was replaced by site-directed mutagenesis.

In vitro synthesis of fully functional EmrE, a multidrug transporter, and study of its oligomeric state.

EmrE is a small multidrug transporter from Escherichia coli that provides a unique model for the study of polytopic membrane proteins. Here, we show its synthesis in a cell-free system in a fully functional form. The detergent-solubilized protein binds substrates with high affinity and, when reconstituted into proteoliposomes, transports substrate in a Deltamicro(H)(+)-dependent fashion.

Killing of intraerythrocytic Plasmodium falciparum by lysosomotropic amino acid esters.

Esters of amino acids are known to penetrate into cells by simple diffusion. Subsequently, they are hydrolyzed by hydrolases to release the parent amino acid. Due to the abundance of hydrolases in phagolysosomes, amino acids accumulate, there because the rate of influx and hydrolysis exceed the rate of amino acid efflux through specific carriers.

An amino acid cluster around the essential Glu-14 is part of the substrate- and proton-binding domain of EmrE, a multidrug transporter from Escherichia coli.

EmrE is a small multidrug transporter (110 amino acids long) from Escherichia coli that extrudes various drugs in exchange with protons, thereby rendering bacteria resistant to these compounds. Glu-14 is the only charged membrane-embedded residue in EmrE and is evolutionarily highly conserved. This residue has an unusually high pK and is an essential part of the binding domain, shared by substrates and protons.

Crosslinking of membrane-embedded cysteines reveals contact points in the EmrE oligomer.

EmrE is a small multidrug transporter that extrudes various drugs in exchange with protons, thereby rendering Escherichia coli cells resistant to these compounds. In this study, relative helix packing in the EmrE oligomer solubilized in detergent was probed by intermonomer crosslinking analysis. Unique cysteine replacements in transmembrane domains were shown to react with organic mercurials but not with sulfhydryl reagents, such as maleimides and methanethiosulfonates.

Glycosylation of a vesicular monoamine transporter: a mutation in a conserved proline residue affects the activity, glycosylation, and localization of the transporter.

The role of N-glycosylation in the expression, ligand recognition, activity, and intracellular localization of a rat vesicular monoamine transporter (rVMAT1) was investigated. The glycosylation inhibitor tunicamycin induced a dose-dependent decrease in the rVMAT1-mediated uptake of [3H]serotonin. Part of this effect was due to a general toxic effect of the drug.

Modification of the pH profile and tetrabenazine sensitivity of rat VMAT1 by replacement of aspartate 404 with glutamate.

Vesicular monoamine transporters (VMAT) catalyze transport of serotonin, dopamine, epinephrine, and norepinephrine into subcellular storage organelles in a variety of cells. Accumulation of the neurotransmitter depends on the proton electrochemical gradient (Delta micro H+) across the organelle membrane and involves VMAT-mediated exchange of two lumenal protons with one cytoplasmic amine. Mutagenic analysis of the role of two conserved Asp residues located in transmembrane segments X and XI of rat VMAT type I reveals an important role of these two residues in catalysis.

Histidine-419 plays a role in energy coupling in the vesicular monoamine transporter from rat.

Vesicular monoamine transporters (VMAT) catalyze transport of serotonin, dopamine, epinephrine and norepinephrine into subcellular storage organelles in a variety of cells. Accumulation of the neurotransmitter depends on the proton electrochemical gradient across the organelle membrane and involves VMAT-mediated exchange of two lumenal protons with one cytoplasmic amine. It has been suggested in the past that His residues play a role in H+ movement or in its coupling to active transport in H(+)-symporters and antiporters.

Cloning and functional expression of a tetrabenazine sensitive vesicular monoamine transporter from bovine chromaffin granules.

Using oligonucleotide primers derived from the vesicular monoamine transporters sequences, a cDNA predicted to encode the bovine chromaffin granule amine transporter has been cloned (b-VMAT2). Surprisingly, its structure is more similar to the rat brain transporter (VMAT2), than to the rat adrenal counterpart (VMAT1). Unlike rat VMAT1, bovine VMAT2 appears to be expressed both in the adrenal medulla and the brain, as judged by Northern analysis.

Amphetamine derivatives interact with both plasma membrane and secretory vesicle biogenic amine transporters.

The interaction of fenfluramine, 3,4-methylenedioxymethamphetamine (MDMA), and p-chloroamphetamine (PCA) with the platelet plasma membrane serotonin transporter and the vesicular amine transporter were studied using both transport and binding measurements. Fenfluramine is apparently a substrate for the plasma membrane transporter, and consequently inhibits both serotonin transport and imipramine binding. Moreover, fenfluramine exchanges with internal [3H]serotonin in a plasma membrane transporter-mediated reaction that requires NaCl and is blocked by imipramine.

Homology of a vesicular amine transporter to a gene conferring resistance to 1-methyl-4-phenylpyridinium.

The vesicular amine transporter (VAT) catalyzes transport and storage of catechol and indolamines into subcellular organelles in a wide variety of cells. It plays a central role in neurotransmission and is the primary target for several pharmacological agents. One of the drugs, reserpine, binds very tightly to the transporter and remains bound even after solubilization, a finding that has proven useful for purification of the transporter from bovine adrenal medulla in a fully functional state.

Energetics of reserpine binding and occlusion by the chromaffin granule biogenic amine transporter.

The energetics of reserpine binding to the bovine adrenal biogenic amine transporter suggest that H+ ion translocation converts the transporter to a form which binds reserpine essentially irreversibly. Reserpine binding to bovine adrenal chromaffin granule membrane vesicles is accelerated by generation of a transmembrane pH difference (delta pH) (interior acid) or electrical potential (delta psi) (interior positive). Both components of the electrochemical H+ potential (delta mu H+) must be dissipated to block reserpine binding, and generation of either one stimulates the binding rate.

Function of the delipidated beta-adrenergic receptor appears to require a fatty acid or a neutral lipid in addition to phospholipids.

Detergent-solubilized preparations of the beta-adrenergic receptor (R) and of the guanyl nucleotide binding proteins (Gs) were extensively treated to remove phospholipids and cholesterol. Reconstitution of an R-Gs system was subsequently performed in the presence of a mixture of natural phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine or the synthetic dioleoyl derivatives of the same phospholipids. In both cases, an additional lipid was required for the agonist-dependent activation of Gs.

Free fatty acids decouple oxidative phosphorylation by dissipating intramembranal protons without inhibiting ATP synthesis driven by the proton electrochemical gradient.

Free fatty acids (FFA) uncouple oxidative phosphorylation and reverse electron transport and inhibit ATP-Pi exchange in beef heart submitochondrial particles. In this, they resemble classical uncouplers and ionophores. However, in contrast to the latter agents, FFA do not collapse the substrate generated proton electrochemical potential and do not inhibit ATP synthesis when the latter is driven by artificially imposed delta microH.

Lipid requirements for reconstitution of the delipidated beta-adrenergic receptor and the regulatory protein.

The role of lipids in the interaction of the beta-adrenergic receptor (R) with the regulatory protein (Gs) was investigated. Solubilized preparations of R and of Gs from turkey erythrocytes were delipidated by gel filtration. They were subsequently combined and reconstituted by the addition of various lipids.