Functional analysis of novel multidrug transporters from human pathogens.

Proteins of the Smr family are the smallest multidrug transporters, about 110 amino acids long, that extrude various drugs in exchange with protons, thereby rendering bacteria resistant to these compounds. One of these proteins, EmrE, is an Escherichia coli protein, which has been cloned based on its ability to confer resistance to ethidium and methyl viologen and which has been extensively characterized. More than 60 genes coding for Smr proteins have been identified in several bacteria based on amino acid sequence similarity to the emrE gene. In this work we have analyzed the sequence similarity among these homologues and identified some distinct signature sequence elements and several fully conserved residues. Five of these homologues, from human pathogens Mycobacterium tuberculosis, Bordetella pertussis, and Pseudomonas aeruginosa and from Escherichia coli, were cloned into an E. coli expression system. The proteins were further characterized and show varying degrees of methyl viologen uptake into proteoliposomes and [(3)H]TPP binding in solubilized membranes. The homologues can also form mixed oligomers with EmrE that exhibit intermediate binding characteristics. A comparative study of various homologous proteins provides a tool for deciphering structure-function relationship and monomer-monomer interaction in multidrug transporters and in membrane proteins in general.

Small is mighty: EmrE, a multidrug transporter as an experimental paradigm.

EmrE is a multidrug transporter from Escherichia coli that functions as a homooligomer and is unique in its small size. In each monomer there are four tightly packed transmembrane segments and one membrane-embedded charged residue. This residue provides the basis for the coupling mechanism as part of a binding site "time shared" by substrates and protons.

Precious things come in little packages.

The 110-amino acid multidrug transporter from E. coli, EmrE, is a member of the family of MiniTexan or Smr drug transporters. EmrE can transport acriflavine, ethidium bromide, tetraphenylphosphonium (TPP+), benzalkonium and several other drugs with relatively high affinities. EmrE is an H+/drug antiporter, utilizing the proton electrochemical gradient generated across the bacterial cytoplasmic membrane by exchanging two protons with one substrate molecule. The EmrE multidrug transporter is unique in its small size and hydrophobic nature. Hydropathic analysis of the EmrE sequence predicts four alpha-helical transmembrane segments. This model is experimentally supported by FTIR studies that confirm the high alpha-helicity of the protein and by high-resolution heteronuclear NMR analysis of the protein structure. The TMS of EmrE are tightly packed in the membrane without any continuous aqueous domain, as was shown by Cysteine scanning experiments. These results suggest the existence of a hydrophobic pathway through which the substrates are translocated. EmrE is functional as a homo-oligomer as suggested by several lines of evidence, including co-reconstitution experiments of wild-type protein with inactive mutants in which negative dominance has been observed. EmrE has only one membrane embedded charged residue, Glu-14, that is conserved in more than fifty homologous proteins and it is a simple model system to study the role of carboxylic residues in ion-coupled transporters. We have used mutagenesis and chemical modification to show that Glu-14 is part of the substrate-binding site. Its role in proton binding and translocation was shown by a study of the effect of pH on ligand binding, uptake, efflux and exchange reactions. We conclude that Glu-14 is an essential part of a binding site, common to substrates and protons. The occupancy of this site is mutually exclusive and provides the basis of the simplest coupling of two fluxes. Because of some of its properties and its size, EmrE provides a unique system to understand mechanisms of substrate recognition and translocation.

High lung uptake of 99mTc-multilamellar liposomes after intravenous administration in rats and rabbits.

The study presents the results of our efforts to prepare liposomes with high lung uptake by modifying the liposome composition and size. Multilamellar liposomes composed of soya phosphatidylcholine and cholesterol (molar ratio 1:1), negatively charged with 0.1 mol sodium cholate were initially prepared. They were treated with SnF2 before (Lp) and after (Lp2) lyophilization and both types were labelled with 99mTc-NaTcO4 in saline solution. A fraction of small 99mTc-Lp2 was obtained by extrusion of negatively charged 99mTc liposomes through membrane filter (type 10 PCTE membranes) pore size 0.4 micron. The biodistribution of the 99mTc-Lp and 99mTc-Lp2 was compared after i.v. injection to rats [0.1 ml/2 mg lipid (3-4 MBq) per 100 g body weight]. After decapitation blood, liver and lung samples were collected and the relative concentrations of activity (RC) were obtained. 99mTc-Lp2 showed more prolonged blood circulation than 99mTc-Lp with a secondary moderate increase of the activity about 3 h and about 6 h postdose for 99mTc-Lp and 99mTc-Lp2, respectively. The lung localization of the 99mTc-Lp2 was clearly more intensive than the 99mTc-Lp localization (ratio of their maximal RC values 7:1). On the contrary, the 99mTc-Lp accumulated more intensively in the liver than the 99mTc-Lp2 (ratio of the RC values 5:1). The higher affinity of the smaller liposomes (99mTc-Lp2) to the lung was observed also in rabbits after i.v. administration of the extruded 99mTc-Lp2 fraction in the ear vein [0.1 ml/2 mg lipid (3-4 MBq) per 100 g body weight].(ABSTRACT TRUNCATED AT 250 WORDS)