The homo-dimeric bacterial membrane protein EmrE effluxes polyaromatic cationic substrates in a proton-coupled manner to cause multidrug resistance. We recently determined the structure of substrate-bound EmrE in phospholipid bilayers by measuring hundreds of protein-ligand HN–F distances for a fluorinated substrate, 4-fluoro-tetraphenylphosphonium (F4-TPP+), using solid-state NMR. This structure was solved at low pH where one of the two proton-binding Glu14 residues is protonated. Here, to understand how substrate transport depends on pH, we determine the structure of the EmrE-TPP complex at high pH, where both Glu14 residues are deprotonated. The high-pH complex exhibits an elongated and hydrated binding pocket in which the substrate is similarly exposed to the two sides of the membrane. In contrast, the low-pH complex asymmetrically exposes the substrate to one side of the membrane. These pH-dependent EmrE conformations provide detailed insights into the alternating-access model, and suggest that the high-pH conformation may facilitate proton binding in the presence of the substrate, thus accelerating the conformational change of EmrE to export the substrate.

Small multidrug resistance (SMR) transporters contribute to antibiotic resistance through proton-coupled efflux of toxic compounds. Previous biophysical studies of the E. coli SMR transporter EmrE suggest that it should also be able to perform proton/toxin symport or uniport, leading to toxin susceptibility rather than resistance in vivo. Here we show EmrE does confer susceptibility to several previously uncharacterized small-molecule substrates in E. coli, including harmane. In vitro electrophysiology assays demonstrate that harmane binding triggers uncoupled proton flux through EmrE. Assays in E. coli are consistent with EmrE-mediated dissipation of the transmembrane pH gradient as the mechanism underlying the in vivo phenotype of harmane susceptibility. Furthermore, checkerboard assays show this alternative EmrE transport mode can synergize with some existing antibiotics, such as kanamycin. These results demonstrate that it is possible to not just inhibit multidrug efflux, but to activate alternative transport modes detrimental to bacteria, suggesting a strategy to address antibiotic resistance.

Highlights d Mutation of Hsh155 enables chemical inhibition of pre-mRNA splicing in living yeast d Only a single point mutation in Hsh155 is required for inhibition in cells d Mutations in Hsh155 can synergize with one another to enhance inhibitor sensitivity d Mutations that enable inhibition increase splicing of introns with weak branch sites

The interaction between splicing factors and the transcriptional machinery provides an intriguing link between the coupled processes of transcription and splicing. Here, we show that two components of the SF3B complex that forms part of the U2 small nuclear ribonucleoprotein particle (snRNP), SF3B3 and SF3B5, are also subunits of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional coactivator complex in Drosophila melanogaster. Whereas SF3B3 had previously been identified as a human SAGA subunit, SF3B5 had not been identified as a component of SAGA in any species. We show that SF3B3 and SF3B5 bind to SAGA independent of RNA, and interact with multiple SAGA subunits including Sgf29 and Spt7 in a yeast two-hybrid assay. Through analysis of sf3b5 mutant flies, we show that SF3B5 is necessary for proper development and cell viability, but not for histone acetylation. Although SF3B5 does not appear to function in SAGA’s histone modifying activities, SF3B5 is still required for expression of a subset of SAGA-regulated genes independent of splicing. Thus, our data support an independent function of SF3B5 in SAGA’s transcription coactivator activity that is separate from its role in splicing.

Small multidrug resistance (SMR) transporters perform coupled antiport of protons and toxic substrates, contributing to antibiotic resistance through efflux of these compounds from the bacterial cytoplasm. Extensive biophysical studies of the molecular transport mechanism of the E. coli SMR transporter EmrE indicate that it should also be capable of performing proton/drug symport or uniport, either of which will lead to drug susceptibility rather than drug resistance in vivo. Here we show that EmrE does indeed confer susceptibility to some small molecule substrates in the native E. coli in addition to conferring resistance to known polyaromatic cation substrates. In vitro experiments show that substrate binding at a secondary site triggers uncoupled proton uniport that leads to susceptibility. These results suggest that the SMR transporters provide one avenue for bacterial-selective dissipation of the proton-motive force. This has potential for antibiotic development and disruption of antibiotic resistance due to drug efflux more broadly.

Small multidrug resistance (SMR) transporters efflux toxic substrates from bacterial cells and were recently divided into two subfamilies: specific toxic metabolite transporters and promiscuous drug exporters. These drug exporters are thought to function similarly to EmrE, the model system for this subfamily of SMR transporters. Studies of EmrE homologs indicate that they are able to confer resistance to EmrE substrates in E. coli and in their native organisms. Recent work from our lab showed that functional EmrE can confer resistance or susceptibility in vivo depending on the drug substrate. Here, we test whether this functional promiscuity of EmrE extends to SMR transporters from three additional human or animal pathogens: SAsmr from Staphylococcus aureus, PAsmr from Pseudomonas aeruginosa, and FTsmr from Francisella tularensis. We find that these SMR homologs can confer either resistance or susceptibility to different toxic substrates in E. coli. This demonstrates that the ability of a single transporter to lead to opposite biological outcomes when transporting different substrates is a general property of the promiscuous multidrug transporters in the SMR family. It also suggests the potential for novel antibiotic development targeting these transporters with small molecules that trigger susceptibility. Such a strategy does not require that the target be the primary mode for antibiotic resistance because the goal is not simple inhibition of activity, but rather activation of an alternative transport function that is detrimental to bacteria.

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EmrE is a multidrug resistance transporter found in the inner membrane of E. coli. EmrE has been shown to efflux many different toxic compounds, most notably quaternary ammonium compounds (QACs), such as ethidium bromide (EtBr), and other polyaromatic cations. While this protein has been extensively studied, the native substrate and function of EmrE is unknown. To understand the range of EmrE activity under various environmental conditions, we tested ethidium efflux under different salt and pH conditions. In these screens, we also noted phenotypic differences arise between different knockout strains of E. coli. Additionally, recent literature suggests that variation in carbon sources may also affect expression and/or function of multidrug efflux pumps, such as EmrE. We have screened EmrE efflux activity in E. coli grown on different carbon sources to determine the phenotypic effect for this particular pump.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.