Ion-coupled Transporter Research Articles

Exploration of the effects of altitude change on bacteria and fungi in the rumen of yak (Bos grunniens).

The yak (Bos grunniens) is a ruminant animal with strong regional adaptability. However, little is known about the adaptation of the rumen microbial community of yaks at different altitudes and the adaptation mechanism of the host and intestinal microorganisms to the habitat. We investigated the adaptability of the rumen microorganisms of yaks at high and low altitudes. We also compared and analyzed the abundance and diversity of core microorganisms and those that varied between different animals. The aim was to compare the rumen bacterial and fungal communities of grazing yak living at two elevations. Bacteroidetes, Firmicutes, Ascomycota, and Chytridiomycota were the dominant bacteria in the plateau and low-altitude regions. Significant differences between the dominant microorganisms in the rumen of yaks were evident in the two regions. The proportion of fiber-degrading bacteria was significantly different between yaks dwelling at high-altitude and low-altitude regions. The abundance of starch-degrading bacteria was not significantly different with altitude. Species clustering similarity analysis showed that the rumen microorganisms in the two areas were obviously isolated and clustered into branches. Functional prediction showed significant differences in rumen microbial methane metabolism, starch and sucrose metabolism, ion-coupled transporter and bacterial secretion system at different altitudes. Overall, the results of this study improved our understanding of the abundance and composition of microorganisms in the rumen of yak at different altitudes.

Abstract 2832: Comparison of bacteriome in plaque dental, saliva and tumor tissue in oral squamus carcinoma

Abstract Introduction: Bacterial microbiota found in the oral cavity, facilitates or promotes its carcinogenesis to Oral Squamous Cell Carcinoma (OSCC), by persistent inflammatory and production of acetaldehyde. Objective: To determine and compare the microbiome and functional potential of dental plaque, saliva and tumor tissue of patients with OSCC with that found in dental plaque and saliva of healthy people. Methods: After signed the informed consent 10 patients diagnosed with OSCC were selected. Samples were saliva, dental plaque and tumor tissue; sociodemographic data were collected from each patient, which were used to search for their corresponding control as age, gender, alcohol and tobacco consumptions. Subsequently, DNA was extracted from all the samples and 16S amplification and sequencing by ion-torrent. Once the data assembled paired-end reads were clustered into Operational Taxonomic Units (OTUs) algorithm with the open reference OTU picking pipeline in QIIME 1.9.1. The taxonomic assignment was Greengenes Database version 13.8. The α diversity and β diversity were calculated. To determine the metabolic potential of the oral microbiota, we used the PICRUSt significance was less 0.05. Results: It was observed that the greatest bacterial diversity in dental plaque samples compared to the samples of saliva and tumor tissue. Multiple comparisons were made between the microbiome present between the different types of samples and between the two groups (cancer vs control). A total of 25 bacterial genera were found in exclusively in patients with OSCC and in more than 30% of patients who were Geobacillus, Anaerotruncus, Comamonas, Sphingomonas, Sphaerochaeta, Dorea, Delftia, Pseudoxanthomonas, Luteibacter, Pseudomonas, Peptoniphilus, Novosphingobium, Bradyrhizobium, Faecalibacterium, Clostridium, Dechloromonas, Methylobacterium, Proteus, Finegoldia, Enterococcus, Turicibacter, Desulfovibrio, Pyramidobacter, Morganella and Staphylococcus, and 2 those found exclusively in healthy people in more than 30% of controls that were Odoribacter and Helicobacter. Regarding the functional potential, statistically significant differences were observed between patients with OSCC and healthy people, increased ion-coupled transporter signaling pathways in patients with OSCC and the signaling pathways related to increased replication, recombination and repair in the group of healthy people. Conclusions: Bacterial genera associated with OSCC and associated with health were described and propose them as non-invasive biomarkers. Additionally, signaling pathways increased by the microbiota associated with oral cancer are related to transporters associated with ions, while signaling pathways increased by the microbiota associated with health are related to replication, recombination and repair. Citation Format: Dabeiba A. García Robayo, Herlinto Alveiro Tupaz Erira, Fredy Omar Gamboa Jaimes. Comparison of bacteriome in plaque dental, saliva and tumor tissue in oral squamus carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2832.

Divalent metal-ion transporter DMT1 mediates both H+ -coupled Fe2+ transport and uncoupled fluxes

The H(+) -coupled divalent metal-ion transporter DMT1 serves as both the primary entry point for iron into the body (intestinal brush-border uptake) and the route by which transferrin-associated iron is mobilized from endosomes to cytosol in erythroid precursors and other cells. Elucidating the molecular mechanisms of DMT1 will therefore increase our understanding of iron metabolism and the etiology of iron overload disorders. We expressed wild type and mutant DMT1 in Xenopus oocytes and monitored metal-ion uptake, currents and intracellular pH. DMT1 was activated in the presence of an inwardly directed H(+) electrochemical gradient. At low extracellular pH (pH(o)), H(+) binding preceded binding of Fe(2+) and its simultaneous translocation. However, DMT1 did not behave like a typical ion-coupled transporter at higher pH(o), and at pH(o) 7.4 we observed Fe(2+) transport that was not associated with H(+) influx. His(272) --> Ala substitution uncoupled the Fe(2+) and H(+) fluxes. At low pH(o), H272A mediated H(+) uniport that was inhibited by Fe(2+). Meanwhile H272A-mediated Fe(2+) transport was independent of pH(o). Our data indicate (i) that H(+) coupling in DMT1 serves to increase affinity for Fe(2+) and provide a thermodynamic driving force for Fe(2+) transport and (ii) that His-272 is critical in transducing the effects of H(+) coupling. Notably, our data also indicate that DMT1 can mediate facilitative Fe(2+) transport in the absence of a H(+) gradient. Since plasma membrane expression of DMT1 is upregulated in liver of hemochromatosis patients, this H(+) -uncoupled facilitative Fe(2+) transport via DMT1 can account for the uptake of nontransferrin-bound plasma iron characteristic of iron overload disorders.

A Structural Model of EmrE, a Multi-Drug Transporter from Escherichia coli

Using a recently reported computational method, we describe an approach to model the structure of EmrE, a proton coupled multi-drug transporter of Escherichia coli. EmrE is the smallest ion-coupled transporter known; it functions as an oligomer and each monomer comprises four transmembrane segments. Because of its size, EmrE provides a unique experimental paradigm. The computational method does not afford a unique solution for the monomer. The experimental constraints available were used to select the most likely structure and to dock two monomers together to yield a dimer. The model is further validated by modeling of Hsmr, an EmrE homolog with a remarkable amino acid composition with over 40% of Ala and Val. The Hsmr model is similar to that of EmrE, with the majority of the Ala or Val residues facing the lipid. In addition, the model of EmrE features a putative substrate-binding site very similar to that observed in BmrR, a transcription activator of multi-drug transporters, with a similar substrate profile. The two crucial residues that couple proton fluxes with substrate binding in the homo-dimer of EmrE, Glu-14, have a spatial arrangement that agrees with proposed molecular mechanisms of transport.

Direct Evidence for Substrate-induced Proton Release in Detergent-solubilized EmrE, a Multidrug Transporter

A novel approach to study coupling of substrate and ion fluxes is presented. EmrE is an H(+)-coupled multidrug transporter from Escherichia coli. Detergent-solubilized EmrE binds substrate with high affinity in a pH-dependent mode. Here we show, for the first time in an ion-coupled transporter, substrate-induced release of protons in a detergent-solubilized preparation. The direct measurements allow for an important quantitation of the phenomenon. Thus, stoichiometry of the release in the wild type and a mutant with a single carboxyl at position 14 is very similar and about 0.8 protons/monomer. The findings demonstrate that the only residue involved in proton release is a highly conserved membrane-embedded glutamate (Glu-14) and that all the Glu-14 residues in the EmrE functional oligomer participate in proton release. Furthermore, from the pH dependence of the release we determined the pK of Glu-14 as 8.5 and for an aspartate replacement at the same position as 6.7. The high pK of the carboxyl at position 14 is essential for coupling of fluxes of protons and substrates.

EmrE, the smallest ion-coupled transporter, provides a unique paradigm for structure-function studies.

EmrE is an Escherichia coli multidrug transporter which confers resistance to a wide variety of toxicants by actively removing them in exchange for hydrogen ions. EmrE is a highly hydrophobic 12 kDa protein which has been purified by taking advantage of its unique solubility in organic solvents. After solubilization and purification, the protein retains its ability to transport as judged from the fact that it can be reconstituted in a functional form. Hydrophobicity analysis of the sequence yielded four putative transmembrane domains of similar sizes. Results from transmission Fourier transform infrared measurements agree remarkably well with this hypothesis and yielded alpha-helical estimates of 78% and 80% for EmrE in CHCl3:MeOH and 1,2-dimyristoyl phosphocholine, respectively. Furthermore, the fact that most of the amide groups in the protein do not undergo amide-proton H/D exchange implies that most (approximately 80%) of the residues are embedded in the bilayer. These observations are only consistent with four transmembrane helices. A domain lined by Cys41 and Cys95 accessible only to substrates such as the organic mercurial 4-(chloromercuri)benzoic acid has been identified. Both residues are asymmetric in their location with respect to the plane of the membrane, Cys95 being closer than Cys41 to the outside face of the membrane. In co-reconstitution experiments of wild-type protein with three different inactive mutants, negative dominance has been observed. This phenomenon suggests that EmrE is functional as a homo-oligomer.

Negative Dominance Studies Demonstrate the Oligomeric Structure of EmrE, a Multidrug Antiporter from Escherichia coli

EmrE, the smallest known ion-coupled transporter, is an Escherichia coli 12-kDa protein 80% helical and soluble in organic solvents. EmrE is a polyspecific antiporter that exchanges hydrogen ions with aromatic toxic cations such as methyl viologen. Since it is many times smaller than the classical consensus 12 transmembrane segments transporters, it was particularly interesting to determine its oligomeric state. For this purpose, a series of nonfunctional mutants has been generated and characterized to test their effect on the activity of the wild-type protein upon mixing. As opposed to the wild type, these mutants do not confer resistance to methyl viologen, ethidium bromide, or a series of other toxicants. Co-expression of each of the nonfunctional mutants with the wild-type protein results in a reduction in the ability of the functional transporter to confer resistance to several toxicants. To perform mixing experiments in vitro, all the mutants have been purified by extraction with organic solvents, reconstituted in proteoliposomes, and found to be inactive. When co-reconstituted with wild-type protein, they inhibit the activity of the latter in a dose-dependent form up to full inhibition. We assume that this inhibition is due to the formation of mixed oligomers in which the presence of one nonfunctional subunit causes full inactivation. A binomial analysis of the results based on the latter assumptions do not provide statistically significant answers but suggests that the oligomer is composed of three subunits. The results described provide the first in vitro demonstration of the functional oligomeric structure of an ion-coupled transporter.

Identification of Residues in the Translocation Pathway of EmrE, a Multidrug Antiporter from Escherichia coli

EmrE is a small, 12-kDa, highly polyspecific antiporter, which exchanges hydrogen ions with aromatic cations such as methyl viologen. EmrE-mediated transport is inhibited by the sulfhydryl-reactive reagent 4-(chloromercuri)benzoic acid (PCMB) but not by a variety of other sulfhydryl reagents. This differential effect is due to the fact that the organic mercurial is a substrate of the transporter and can reach domains otherwise inaccessible to the different reagents. To find out which of the three cysteine residues in EmrE is reacting with PCMB, each was replaced with serine and it was shown that none of them is essential for transport activity. A protein completely devoid of Cys residues (CL) is also capable of substrate accumulation albeit at a slower rate. Mutated proteins in which only one of the native cysteines was left whereas the other changed to serine were also constructed. The use of these proteins demonstrated that two of the three Cys in EmrE, Cys-41 and Cys-95, but not Cys-39, react with PCMB. A related mercurial, 4-(chloromercuri)benzenesulfonic acid (PCMBS), is only a very poor inhibitor, probably because of the negative charge it bears. PCMBS reacts with EmrE in an asymmetric and unique way. It reacts with the mutant bearing a single Cys residue in position 95 (CL-C95) only when the reagent is present in the outside face of the membrane and with the mutant CL-C41 only when allowed to permeate to the cell interior; as expected, it does not react with the mutant protein bearing a single Cys at position 39 (CL-C39). It is concluded that PCMB permeates through the substrate pathway of EmrE and covalently reacts with the two exposed residues, Cys-95 and Cys-41, but not with Cys-39, located on the opposite face of the helix relative to residue 41. In addition, because of the asymmetric reactivity to PCMBS, an inhibitor that does not permeate through the protein, it is concluded that Cys-41 is closer to the cytoplasmic face than Cys-95. The results demonstrate the existence of a domain accessible only to substrates and provide a unique tool for studying the substrate permeation pathway of an ion-coupled transporter.

Mammalian ion‐coupled solute transporters.

Active transport of solutes into and out of cells proceeds via specialized transporters that utilize diverse energy-coupling mechanisms. Ion-coupled transporters link uphill solute transport to downhill electrochemical ion gradients. In mammals, these transporters are coupled to the co-transport of H+, Na+, Cl- and/or to the countertransport of K+ or OH-. By contrast, ATP-dependent transporters are directly energized by the hydrolysis of ATP. The development of expression cloning approaches to select cDNA clones solely based on their capacity to induce transport function in Xenopus oocytes has led to the cloning of several ion-coupled transporter cDNAs and revealed new insights into structural designs, energy-coupling mechanisms and physiological relevance of the transporter proteins. Different types of mammalian ion-coupled transporters are illustrated by discussing transporters isolated in our own laboratory such as the Na+/glucose co-transporters SGLT1 and SGLT2, the H(+)-coupled oligopeptide transporters PepT1 and PepT2, and the Na(+)- and K(+)-dependent neuronal and epithelial high affinity glutamate transporter EAAC1. Most mammalian ion-coupled organic solute transporters studied so far can be grouped into the following transporter families: (1) the predominantly Na(+)-coupled transporter family which includes the Na+/glucose co-transporters SGLT1, SGLT2, SGLT3 (SAAT-pSGLT2) and the inositol transporter SMIT, (2) the Na(+)- and Cl(-)-coupled transporter family which includes the neurotransmitter transporters of gamma-amino-butyric acid (GABA), serotonin, dopamine, norepinephrine, glycine and proline as well as transporters of beta-amino acids, (3) the Na(+)- and K(+)-dependent glutamate/neurotransmitter family which includes the high affinity glutamate transporters EAAC1, GLT-1, GLAST, EAAT4 and the neutral amino acid transporters ASCT1 and SATT1 reminiscent of system ASC and (4) the H(+)-coupled oligopeptide transporter family which includes the intestinal H(+)-dependent oligopeptide transporter PepT1.