VMAT structures reveal exciting targets for drug development.

Vesicular monoamine transporter (VMAT)-2 has a crucial role in the neurotransmission of biogenic amines. Recently, Dalton et al., Pidathala et al.

On the link between antibiotic resistance, diabetes, and wastewater.

The study by Lucero et al. (https://doi.org/10.

Acidification of Cytoplasm in Escherichia coli Provides a Strategy to Cope with Stress and Facilitates Development of Antibiotic Resistance.

Awareness of the problem of antimicrobial resistance (AMR) has escalated, and drug-resistant infections are named among the most urgent issues facing clinicians today. Bacteria can acquire resistance to antibiotics by a variety of mechanisms that, at times, involve changes in their metabolic status, thus altering diverse biochemical reactions, many of them pH-dependent. In this work, we found that modulation of the cytoplasmic pH (pH) of Escherichia coli provides a thus far unexplored strategy to support resistance.

Deletion of the major Escherichia coli multidrug transporter AcrB reveals transporter plasticity and redundancy in bacterial cells.

Multidrug Transporters (MDTs) are major contributors to the acquisition and maintenance of Antimicrobial Resistance (AMR), a growing public health threat of broad concern. Despite the large number of MDTs, the overwhelming majority of the studies performed thus far in Gram-negative bacteria emphasize the supremacy of the AcrAB-TolC complex. To unveil the potential role of other MDTs we studied the behavior of a null AcrB Escherichia coli strain when challenged with chloramphenicol, a bacteriostatic antibiotic.

The ins and outs of vesicular monoamine transporters.

The H-coupled vesicular monoamine transporter (VMAT) is a transporter essential for life. VMAT mediates packaging of the monoamines serotonin, dopamine, norepinephrine, and histamine from the neuronal cytoplasm into presynaptic vesicles, which is a key step in the regulated release of neurotransmitters. However, a detailed understanding of the mechanism of VMAT function has been limited by the lack of availability of high-resolution structural data.

The Escherichia coli effluxome.

Multidrug transporters function in a coordinated mode to provide an essential first-line defense mechanism that prevents antibiotics from reaching lethal concentrations, until a number of stable efficient adaptations occur that allow survival. Single-component efflux transporters remove the toxic compounds from the cytoplasm to the periplasmic space where TolC-dependent transporters expel them from the cell. The close interaction between the two types of transporters ensures handling of a wide range of xenobiotics and prevents rapid leak of the hydrophobic substrates back into the cell.

Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis.

Neurotransporters located in synaptic vesicles are essential for communication between nerve cells in a process mediated by neurotransmitters. Vesicular monoamine transporter (VMAT), a member of the largest superfamily of transporters, mediates transport of monoamines to synaptic vesicles and storage organelles in a process that involves exchange of two H per substrate. VMAT transport is inhibited by the competitive inhibitor reserpine, a second-line agent to treat hypertension, and by the noncompetitive inhibitor tetrabenazine, presently in use for symptomatic treatment of hyperkinetic disorders.

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.

A liposomal method for evaluation of inhibitors of H(+)-coupled multidrug transporters.

Introduction: This paper describes a novel technique, Fluorosomes, applied to investigating the interaction of antimicrobials with proton driven microbial efflux transporters. These transporters remove toxic compounds from the cytoplasm, including antibiotics and are involved in antibiotic resistance.

Methods: To assess transporter activity we developed a methodology to generate a proton gradient across Fluorosome membranes into which selected purified fully active efflux transporters were reconstituted.

Specificity determinants in small multidrug transporters.

Multiple-antibiotic resistance has become a major global public health concern, and to overcome this problem, it is necessary to understand the resistance mechanisms that allow survival of the microorganisms at the molecular level. One mechanism responsible for such resistance involves active removal of the antibiotic from the pathogen cell by MDTs (multidrug transporters). A prominent MDT feature is their high polyspecificity allowing for a single transporter to confer resistance against a range of drugs.

Functionally important carboxyls in a bacterial homologue of the vesicular monoamine transporter (VMAT).

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.

Competition as a way of life for H(+)-coupled antiporters.

Antiporters are ubiquitous membrane proteins that catalyze obligatory exchange between two or more substrates across a membrane in opposite directions. Some utilize proton electrochemical gradients generated by primary pumps by coupling the downhill movement of one or more protons to the movement of a substrate. Since the direction of the proton gradient usually favors proton movement toward the cytoplasm, their function results in removal of substrates other than protons from the cytoplasm, either into acidic intracellular compartments or out to the medium.

Identification of conformationally sensitive residues essential for inhibition of vesicular monoamine transport by the noncompetitive inhibitor tetrabenazine.

Vesicular monoamine transporter 2 (VMAT2) transports monoamines into storage vesicles in a process that involves exchange of the charged monoamine with two protons. VMAT2 is a member of the DHA12 family of multidrug transporters that belongs to the major facilitator superfamily of secondary transporters. Tetrabenazine (TBZ) is a non-competitive inhibitor of VMAT2 that is used in the treatment of hyperkinetic disorders associated with Huntington disease and Tourette syndrome.

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.

Identification of molecular hinge points mediating alternating access in the vesicular monoamine transporter VMAT2.

Vesicular monoamine transporter 2 (VMAT2) catalyzes transport of monoamines into storage vesicles in a process that involves exchange of the charged monoamine with two protons. VMAT2 is a member of the DHA12 family of multidrug transporters that belongs to the major facilitator superfamily (MFS) of secondary transporters. Here we present a homology model of VMAT2, which has the standard MFS fold, that is, with two domains of six transmembrane helices each which are related by twofold pseudosymmetry and whose axis runs normal to the membrane and between the two halves.

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.

Transforming a drug/H+ antiporter into a polyamine importer by a single mutation.

EmrE, a multidrug antiporter from Escherichia coli, has presented biochemists with unusual surprises. Here we describe the transformation of EmrE, a drug/H(+) antiporter to a polyamine importer by a single mutation. Antibiotic resistance in microorganisms may arise by mutations at certain chromosomal loci.

Undecided membrane proteins insert in random topologies. Up, down and sideways: it does not really matter.

It is usually assumed that to ensure proper function, membrane proteins must be inserted in a unique topology. However, a number of dimeric small multidrug transporters can function in the membrane in various topologies. Thus, the dimers can be a random mixture of NiCi (N and C termini facing the cell cytoplasm) and NoCo (N and C termini facing the outside) orientation.

Expression of neurotransmitter transporters for structural and biochemical studies.

Neurotransmitter transporters play essential roles in the process of neurotransmission. Vesicular neurotransmitter transporters mediate storage inside secretory vesicles in a process that involves the exchange of lumenal H(+) for cytoplasmic transmitter. Retrieval of the neurotransmitter from the synaptic cleft catalyzed by sodium-coupled transporters is critical for the termination of the synaptic actions of the released neurotransmitter.

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.

Directed evolution reveals hidden properties of VMAT, a neurotransmitter transporter.

The vesicular neurotransmitter transporter VMAT2 is responsible for the transport of monoamines into synaptic and storage vesicles. VMAT2 is the target of many psychoactive drugs and is essential for proper neurotransmission and survival. Here we describe a new expression system in Saccharomyces cerevisiae that takes advantage of the polyspecificity of VMAT2.

In vitro unfolding and refolding of the small multidrug transporter EmrE.

The composition of the lipid bilayer is increasingly being recognised as important for the regulation of integral membrane protein folding and function, both in vivo and in vitro. The folding of only a few membrane proteins, however, has been characterised in different lipid environments. We have refolded the small multidrug transporter EmrE in vitro from a denatured state to a functional protein and monitored the influence of lipids on the folding process.

A coordinated network of transporters with overlapping specificities provides a robust survival strategy.

Multidrug transporters provide a survival strategy for living organisms. As expected given their central role in survival, these transporters are ubiquitous, and in many genomes, several genes coding for putative transporters have been identified. However, in an organism such as Escherichia coli mutations in genes coding for transporters other than the major AcrAB-TolC multidrug efflux transporter have only a marginal effect on phenotype.

EmrE, a model for studying evolution and mechanism of ion-coupled transporters.

EmrE is a small (110 residues) SMR transporter from Escherichia coli that extrudes positively charged aromatic drugs in exchange for two protons, thus rendering bacteria resistant to a variety of toxic compounds. Due to its size, stability and retention of its function upon solubilization in detergent, EmrE provides a unique experimental paradigm for the biochemical and biophysical studies of membrane based ion-coupled transporters. In addition, EmrE has been in center stage in the past two years because it provides also a paradigm for the study of the evolution of membrane proteins.

Identification of a glycine motif required for packing in EmrE, a multidrug transporter from Escherichia coli.

Glycine residues may play functional and structural roles in membrane proteins. In this work we studied the role of glycine residues in EmrE, a small multidrug transporter from Escherichia coli. EmrE extrudes various drugs across the plasma membrane in exchange with protons and, as a result, confers resistance against their toxic effects.