A melibiose transporter and an operon containing its gene in Enterobacter cloacae.

We detected inducible melibiose transport activity in cells of Enterobacter cloacae IID977. H+, but not Na+, was found to be the coupling cation for this transporter. We cloned and sequenced the gene encoding the melibiose transporter. A homology search of a protein sequence database revealed that this melibiose transporter has high sequence similarity with the lactose transporter (LacY) and the raffinose transporter (RafB) and has some similarity with the melibiose transporter (MelB) of Escherichia coli.     

Degradation and biosynthesis of the glucose transporter protein in chicken embryo fibroblasts transformed by the src oncogene.

The rate of glucose transport in cultured fibroblasts is regulated to a number of physiological variables, including malignant transformation by src, glucose starvation, and stimulation with mitogens. Much of this transport regulation can be accounted for by variations in the amount of transporter protein in the cells. To determine the mechanisms by which levels of the transporter are regulated, we measured the rates of synthesis and degradation of the transporter by pulse-chase experiments and immunoprecipitation of the transporter. We found that transformation by the src oncogene results in a large decrease in the rate at which the transporter protein is degraded but that it does not appreciably increase the rate of transporter biosynthesis. On the other hand, glucose starvation and mitogen stimulation increase the rate of transporter biosynthesis, although a role for control of degradation is possible in these circumstances also. Variations in the rate of glucose transport or the amount of the transporter are not associated with phosphorylation of the transporter protein.     

Insulin action on activity and cell surface disposition of human HepG2 glucose transporters expressed in Chinese hamster ovary cells

Complementary DNA encoding a facilitative glucose transporter was isolated from a human hepatoma cell line (HepG2) cDNA library and subcloned into a metal-inducible mammalian expression vector, pLEN (California Biotechnology) containing human metallothionein gene II promoter sequences. Chinese hamster ovary (CHO) cells transfected with this transporter expression vector, pLENGT, exhibited a 2-17-fold increase in immunoreactive HepG2-type glucose transporter protein, as measured by protein immunoblotting with antipeptide antibodies directed against the HepG2-type glucose transporter C-terminal domain. Expression of the human glucose transporter was verified by protein immunoblotting with a mouse polyclonal antiserum that recognizes the human but not the rodent HepG2-type transporter. 2-Deoxy-D-glucose uptake was increased 2-7-fold in transfected cell lines. Polyclonal antisera directed against purified red blood cell glucose transporter were raised in several rabbits. Antiserum from one rabbit, delta, was found to bind to the surface of intact red cells but not to inside-out red cell ghosts. Using this delta-antiserum in intact cell-binding assays, 1.6-9-fold increases in cell surface expression of the human glucose transporter were measured in CHO-K1 cell lines transfected with the transporter expression vector. Measurements of total cellular glucose transporter immunoreactive protein using anti-HepG2 transporter C-terminal peptide serum, cell surface glucose transporter protein using delta-antiserum and 2-deoxyglucose uptake revealed proportional relationships among these parameters in transfected cell lines expressing different levels of transporter protein. Insulin increased 2-deoxyglucose uptake 40% in control CHO-K1 cells and in CHO-K1 cells expressing modest levels of the human glucose transporter protein. However, stimulation of sugar-uptake by insulin was only 10% in cells overexpressing human glucose transporter protein 9-fold, and no effect of insulin on sugar uptake was detected in several cell lines expressing very high levels (12-17-fold over controls) of human HepG2 glucose transporter protein. No insulin stimulation of anti-cell surface glucose transporter antibody binding was detected in any control or transfected CHO-K1 cell lines. These data indicate that a glucose transporter protein that is insensitive to insulin in HepG2 cells is regulated by insulin when expressed at low but not at high levels in insulin-response CHO-K1 cells. Additionally, the results suggest that insulin does not increase 2-deoxyglucose uptake by increasing the number of cell surface HepG2-type glucose transporters in CHO-K1 fibroblasts.     

3T3-L1 adipocyte glucose transporter (HepG2 class): sequence and regulation of protein and mRNA expression by insulin, differentiation, and glucose starvation

A glucose transporter cDNA (GLUT) clone was isolated from mouse 3T3-L1 adipocytes and sequenced. The nucleotide and deduced amino acid sequences were, respectively, 95 and 99% homologous to those of the rat brain transporter. The mouse cDNA and a polyclonal antibody recognizing the corresponding in vitro translation product were used to compare changes in transporter mRNA and protein levels during differentiation, glucose starvation, and chronic insulin exposure of 3T3-L1 preadipocytes. The respective cellular content of transporter mRNA and protein were increased 6.6- and 7.8-fold during differentiation, and 3.8- and 2.5-fold from chronic insulin exposure of differentiated adipocytes. Glucose starvation increased transporter mRNA and protein levels 2.2- and 3.5-fold in undifferentiated preadipocytes and 1.8- and 3.1-fold in differentiated adipocytes. Starvation of undifferentiated cells completely converted the native transporter to an incompletely glycosylated form, while increasing basal transport rates 4.5-fold. Either full glycosylation is not required to produce a functionally active transporter, or starvation causes a unique predifferentiation induction of the normally absent "responsive" transporter. The changes in transporter protein expression elicited by differentiation were attributed primarily to increased rates of transporter synthesis, while the disproportionate changes in mRNA and protein expression from chronic insulin treatment and starvation suggested these conditions increase synthesis and decrease turnover rates in regulating transporter protein expression. Although chronic insulin exposure and glucose starvation each raised the expression of transporter protein greater than 3-fold and basal transport rates 2.5- to 4.5-fold, no significant increase in the insulin responsiveness of 3T3-L1 preadipocytes or differentiated adipocytes was observed. Thus, the changes in the transporter mRNA and protein expression observed in this study were most consistent with their being associated with the regulated expression of a basal or low level insulin-responsive transporter.     

Quantitation of Rat Dopamine Transporter mRNA: Effects of Cocaine Treatment and Withdrawal

Dopamine transporter mRNA levels in the rat substantia nigra were quantified using a sensitive nuclease protection assay with a highly homologous human dopamine transporter cDNA clone. The same probe was also used to visualize dopamine transporter mRNA in the substantia nigra by in situ hybridization. Repeated cocaine administration (15 mg/kg, twice a day for 6.5 days) resulted in a greater than 40% decrease in nigral dopamine transporter mRNA levels. In contrast, dopamine transporter mRNA levels were unchanged after either acute treatment (4 h before death) or repeated cocaine treatment followed by a 72-h withdrawal period. Thus, blockade of the dopamine transporter by repeated cocaine administration may result in the down-regulation of dopamine transporter gene expression in dopamine neurons.     

Cytochalasin B interferes with conformational changes of the human erythrocyte glucose transporter induced by internal and external sugar binding.

To gain insight into the mechanism of facilitated sugar transport and possible mechanisms by which glucose transporter intrinsic activity might be altered, we have investigated conformational changes of the human erythrocyte glucose transporter induced by internal and external sugar binding and by the transporter inhibitor, cytochalasin B. Changes in the ability of thermolysin to digest glucose transporters present in erythrocyte ghosts were used to monitor conformational changes of the glucose transporter. The degree of protease digestion was determined by the amount of undigested glucose transporter remaining after the protease treatment, as assessed in Western blots using the glucose transporter specific monoclonal antibody 7F7.5. D-Glucose, the physiological substrate of the transporter, increased the transporter's susceptibility to cleavage by thermolysin. Nontransportable glucose analogues which bind specifically to either an internal or external glucose transporter sugar binding site also altered susceptibility of the transporter to thermolysin. Both methyl and propyl glucoside, which preferentially bind the internal sugar site, increased thermolysin susceptibility of the glucose transporter in a manner similar to that of D-glucose. In contrast, 4,6-O-ethylideneglucose, which preferentially binds the external sugar site, protected the transporter from thermolysin digestion. These results suggest that sugar binding to internal and external sugar sites induces distinct conformational changes and that the observed D-glucose effect on the susceptibility of the glucose transporter to thermolysin is due to D-glucose at equilibrium predominantly forming a complex with the internal sugar site. The protection from cleavage by thermolysin caused by external sugar binding is attenuated by the addition of an internally binding sugar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental regulation of rat brain/Hep G2 glucose transporter gene expression

The developmental regulation of rat brain-derived/Hep G2 glucose transporter gene expression was studied by means of Northern blot hybridization, using a rat brain glucose transporter cDNA probe, in order to directly quantify steady state glucose transporter mRNA levels. The results obtained showed different tissue-specific patterns of glucose transporter mRNA levels during ontogenesis; while in brain there was a sustained increase in the levels of the message from 20 days embryogenesis until 50 days postnatal, other organs such as heart, lung, liver, and muscle expressed maximal levels of the glucose transporter mRNA in 20-day fetuses and 1-day neonates, decreasing subsequently to very low levels. The relative expression of the glucose transporter mRNA in the different tissues, at both fetal and adult stages, was analyzed using a solution hybridization-RNase protection assay. This approach revealed that, while the heart expresses the highest levels of glucose transporter mRNA at 20 days of fetal life, the brain shows the highest levels at the adult stage. These results indicate a tissue-specific ontogenic pattern of glucose transporter gene expression, suggesting a developmental role for this glucose transporter gene product.     

Characterization of antisera to a synthetic carboxyl-terminal peptide of the glucose transporter protein

Peptides corresponding to amino acid residues 1-12 of the amino terminal and 480-492 of the carboxyl terminal of the deduced sequence of the glucose transporter were synthesized and used to produce site-specific polyclonal antipeptide sera. In a solid-phase radioimmunoassay, antiserum to the carboxyl terminal recognizes peptide 480-492 and purified human erythrocyte glucose transporter, but not peptide 1-12. Antiserum to the amino terminal recognizes peptide 1-12 but neither peptide 480-492 nor the erythrocyte transporter. The antiserum to the carboxyl terminal specifically immunoblots the Mr 55,000 glucose transporter in erythrocyte membranes and the purified erythrocyte transporter. It also recognizes a Mr 40,000-60,000 polypeptide in membranes of cells derived from different mammalian species and tissues including insulin-sensitive rat adipocytes as well as a Mr 20,000 tryptic fragment of the transporter which contains the site for photolabeling by cytochalasin B. Antiserum to the carboxyl terminal of the transporter binds specifically to leaky erythrocyte membranes but not to intact erythrocytes. This binding is saturable and competitively inhibited by peptide 480-492. Using immunofluorescence microscopy, this antiserum detects glucose transporter protein in permeabilized erythrocytes, but not in intact erythrocytes. These studies provide immunochemical evidence in support of the predicted cytoplasmic orientation of the carboxyl terminus of the glucose transporter, allow us to suggest a spatial relationship of the cytochalasin B binding site to the carboxyl terminal of the glucose transporter and suggest that antisera directed to the carboxyl terminal domain of the protein may be useful for the immunocytochemical localization of the glucose transporter.     

Immunological identification of the human erythrocyte monosaccharide transporter

Antibodies to the purified cytochalasin B binding component of the human erythrocyte glucose transporter were prepared in rabbits. They precipitated detergent-solubilized transporter, and partially inhibited its binding of cytochalasin B. The antibodies were used to locate the transporter polypeptide in SDS-polyacrylamide gels of erythrocyte membranes prepared from freshly drawn blood in the presence of protease inhibitors. They labelled only the region of the gel corresponding to that occupied by the purified transporter, with an apparent molecular weight range of 45,000–75,000. These findings indicate that the isolated transporter does not arise by proteolytic degradation of a larger polypeptide, either during the storage of blood or during purification of the transporter.     

Functional coupling of serotonin and noradrenaline transporters

Re-uptake of the neurotransmitters serotonin and noradrenaline out of the synaptic cleft is mediated by selective transporter proteins, the serotonin transporter and the noradrenaline transporter respectively. Both are integral membrane proteins that are have a high degree of homology and represent members of a larger neurotransmitter transporter superfamily. Several studies have indicated that the serotonin transporter has an an oligomeric structure. To determine whether monoamine transporters can also function in oligomeric structures in situ, we constructed a concatenate consisting of one molecule of serotonin transporter covalently linked to one molecule of noradrenaline transporter. Heterologous expression of this hybrid construct allowed us to analyse the function, i.e. transport activity, and the structure, i.e. the molecular weight of the total construct and of its single components, at the same time. We showed that serotonin-noradrenaline transporter fusion proteins are fully active and exhibit the pharmacological profile of both their individual components. These findings support the hypothesis that monoamine transporters are expressed and may function as oligomeric proteins composed of non-interacting monomers.     

Cloning of a rabbit brain glucose transporter cDNA and alteration of glucose transporter mRNA during tissue development

A full-length cDNA clone that codes for glucose transporter protein was isolated from a rabbit brain cDNA library by using synthetic oligonucleotide probe derived from the sequence of human glucose transporter cDNA. The coding region shared 93.2% nucleotide and 97.0% amino-acid similarities with those of human glucose transporter and 89.4% nucleotide and 97.4% amino-acid similarities with those of rat transporter. Northern blot analysis revealed that glucose transporter mRNA is most abundant in the placenta and that it is also abundant in the brain. The fat tissue, heart, liver, and skeletal muscle of adult rats contained a very small amount of mRNA, while heart, liver, skeletal muscle and kidney of fetal rats contained a very high amount of glucose transporter mRNA. These results suggest that this type of glucose transporter might be closely related with cell proliferation and tissue development.     

Insulin-stimulated translocation of the HepG2/erythrocyte-type glucose transporter expressed in 3T3-L1 adipocytes

Insulin stimulates glucose transport into adipocytes, at least in part, via the translocation of intracellular transporters to the plasma membrane. The human HepG2-type transporter, which is not insulin-responsive in its native cell type, was expressed in 3T3-L1 adipocytes by infection with recombinant retrovirus harboring the HepG2 transporter cDNA in order to determine whether glucose transporter translocation in adipocytes is restricted to a distinct insulin-sensitive transporter species. The distributions of the endogenous murine and the HepG2 transporters were estimated by quantitative immunoblot analysis of subcellular fractions probed with either a monoclonal antibody that recognized only the human transporter or a polyclonal antibody that recognized both transporter species. In the basal state, the intracellular membrane fraction comprised approximately 50% of the total of each transporter type. Insulin decreased the content of both transporter species in the intracellular membranes by approximately 50% and increased the plasma membrane content of both species by approximately 1.5-2-fold. The similar insulin-mediated increase in the plasma membrane content of endogenous murine and HepG2 glucose transporters was verified by labeling of cell surface glycoproteins with [3H]NaBH4 followed by immunoprecipitation with glucose transporter antibodies. These data indicate that insulin-mediated translocation in 3T3-L1 adipocytes is not restricted to a tissue-specific insulin-responsive glucose transporter species and suggest that other tissue-specific factors regulate the translocation process.     

Secretion of the Serratia marcescens HasA protein by an ABC transporter.

We previously identified a Serratia marcescens extracellular protein, HasA, able to bind heme and required for iron acquisition from heme and hemoglobin by the bacterium. This novel type of extracellular protein does not have a signal peptide and does not show sequence similarities to other proteins. HasA secretion was reconstituted in Escherichia coli, and we show here that like many proteins lacking a signal peptide, HasA has a C-terminal targeting sequence and is secreted by a specific ATP binding cassette (ABC) transporter consisting of three proteins, one inner membrane protein with a conserved ATP binding domain, called the ABC; a second inner membrane protein; and a third, outer membrane component. Since the three S. marcescens components of the HasA transporter have not yet been identified, the reconstituted HasA secretion system is a hybrid. It consists of the two S. marcescens inner membrane-specific components, HasD and HasE, associated with an outer membrane component coming from another bacterial ABC transporter, such as the E. coli TolC protein, the outer membrane component of the hemolysin transporter, or the Erwinia chrysanthemi PrtF protein, the outer membrane component of the protease transporter. This hybrid transporter was first shown to allow the secretion of the S. marcescens metalloprotease and the E. chrysanthemi metalloproteases B and C. On account of that, the two S. marcescens components HasD and HasE were previously named PrtDSM and PrtESM, respectively. However, HasA is secreted neither by the PrtD-PrtE-PrtF transporter (the genuine E. chrysanthemi protease transporter) nor by the HlyB-HlhD-TolC transporter (the hemolysin transporter). Moreover, HasA, coexpressed in the same cell, strongly inhibits the secretion of proteases B and C by their own transporter, indicating that the E. chrysanthemi transporter recognizes HasA. Since PrtF could replace TolC in the constitution of the HasA transporter, this indicates that the secretion block does not take place at the level of the outer membrane component but rather at an earlier step of interaction between HasA and the inner membrane components.     

Genetic deletion of vesicular monoamine transporter-2 (VMAT2) reduces dopamine transporter activity in mesencephalic neurons in primary culture

Our aim was to investigate whether a defect in vesicular monoamine transporter-2 (VMAT2) activities would affect dopaminergic cell functions or not. We examined mesencephalon dopaminergic cultures prepared from VMAT2 wild-type, heterozygous or homozygous knockout (KO) 14-day-old mouse fetuses to determine the number of tyrosine hydroxylase (TH)-positive cells and dopamine transporter activity. The number of TH-positive cells remained unchanged in the VMAT2-KO cultures. Of interest, the dopamine transporter activity in the homozygous cells was significantly decreased, but not in the heterozygous cells, suggesting that complete deletion of VMAT2 inhibited dopamine transporter function. Furthermore, dopamine transporter activity was prominently decreased in the synaptosomal fraction of neonatal homozygous VMAT2-KO mice compared with that of wild-type/heterozygous VMAT2-KO ones, indicating that VMAT2 activity might be one of the factors regulating dopamine transporter activities. To test this possibility, we used reserpine, a VMAT2 inhibitor. Reserpine (1muM) decreased dopamine transporter activity (approx. 50%) in wild-type and heterozygous VMAT2-KO cultures but not in homozygous ones, indicating that blockade of VMAT2 activity reduced dopamine transporter activity. To investigate possible mechanisms underlying the decreased dopamine transporter activity in VMAT2-KO mice, we measured dopamine transporter activities after 24-48h exposure of primary cultures of mesencephalic neurons to dopamine receptor antagonists, PKC inhibitor, PI(3)K inhibitor, and l-DOPA. Among these drugs, l-DOPA slightly reduced the dopamine transporter activities of all genotypes, but the other drugs could not. Since the ratios of reduction in dopamine transporter activity of each genotype treated with l-DOPA were similar, substrate inhibition of dopamine transporters was not the main mechanism underlying the reduced dopamine transporter activity due to genetic deletion of VMAT2. Our results demonstrate that genetic deletion of VMAT2 did not induce immediate cell death but did markedly inhibit dopamine transporter activity.     

Choline transport into rat liver mitochondria. Characterization and kinetics of a specific transporter.

Rat liver mitochondria possess a specific choline transporter in the inner membrane. The transporter shows saturable kinetics at high membrane potential with a Km of 220 microM and a Vmax of 0.4 nmol/mg of protein/min at pH 7.0 and 25 degrees C. At physiological concentrations of choline, the rate of choline uptake by the transporter shows a linear dependence on membrane potential; uptake is distinct from the nonspecific cation diffusion process. Hemicholinium-3, hemicholinium-15, quinine, and quinidine, all analogues of choline, are high affinity competitive inhibitors of choline transport with Ki values of 17, 55, 15, and 127 microM, respectively. The choline transporter is distinct from other known mitochondrial transporters. Rat heart mitochondria do not appear to possess a choline transporter. Evidence suggests that the transporter is an electrophoretic uniporter. Analogue studies have shown that the hydroxyl and the quaternary ammonium groups of choline are necessary for binding to the transporter. A comparison of molecular models of choline and the high affinity inhibitors has provided evidence for the preferred conformation of choline for binding to the transporter. The presence of a choline transporter in the mitochondrial inner membrane provides a potential site for control of choline oxidation and hence supply of endogenous betaine.     

Phorbol Esters Increase Dopamine Transporter Phosphorylation and Decrease Transport Vmax

Abstract: Sodium- and chloride-coupled transport of dopamine from synapses into presynaptic terminals plays a key role in terminating dopaminergic neurotransmission. Regulation of the function of the dopamine transporter, the molecule responsible for this translocation, is thus of interest. The primary sequence of the dopamine transporter contains multiple potential phosphorylation sites, suggesting that the function of the transporter could be regulated by phosphorylation. Previous work from this laboratory has documented that phorbol ester activation of protein kinase C (PKC) decreases dopamine transport V _max in transiently expressing COS cells. In the present report, we document in vivo phosphorylation of the rat dopamine transporter stably expressed in LLC-PK₁ cells and show that phosphorylation is increased threefold by phorbol esters. Dopamine uptake is also regulated by phorbol esters in these cells; phorbol 12-myristate 13-acetate (PMA) reduces transport V _max by 35%. Parallels between the time course, concentration dependency, and staurosporine sensitivity of alterations in transporter phosphorylation and transporter V _max suggest that dopamine transporter phosphorylation involving PKC could contribute to this decreased transporter function. Phosphorylation of the dopamine transporter by PKC or by a PKC-activated kinase could be involved in rapid neuroadaptive processes in dopaminergic neurons.     

Characterization and identification of the glucose transporter of human erythrocytes

The glucose transporter was purified from human erythrocytes (Kasahara, M. and Hinkle, P.C. (1977) J. Biol. Chem. 252, 7384–7390). The following results support the conclusion that a major protein in the purified transporter fraction, zone 4.5 is the glucose transporter (or a part of the transporter) and is different from band 3: (1) peptide maps of zone 4.5 were similar throughout the broad band in sodium dodecyl sulfate-gel electrophoresis and were different from those of band 3, (2) specific binding of cytochalasin B was found to the transporter fraction, but not to a band 3 fraction, (3) the N-terminal amino acid analysis of the transporter fraction showed a single N-terminal of lysine, whereas the band 3 fraction showed no clear N-terminal, and (4) the rabbit antibody raised against the transporter fraction formed a precipitation line with the transporter fraction, but not with the band 3 fraction. A filtration apparatus was devised for quick and accurate measurement of cytochalasin B binding, with which results comparable to those from equilibrium dialysis were obtained.     

Comparative analyses of fundamental differences in membrane transport capabilities in prokaryotes and eukaryotes

Whole-genome transporter analyses have been conducted on 141 organisms whose complete genome sequences are available. For each organism, the complete set of membrane transport systems was identified with predicted functions, and classified into protein families based on the transporter classification system. Organisms with larger genome sizes generally possessed a relatively greater number of transport systems. In prokaryotes and unicellular eukaryotes, the significant factor in the increase in transporter content with genome size was a greater diversity of transporter types. In contrast, in multicellular eukaryotes, greater number of paralogs in specific transporter families was the more important factor in the increase in transporter content with genome size. Both eukaryotic and prokaryotic intracellular pathogens and endosymbionts exhibited markedly limited transport capabilities. Hierarchical clustering of phylogenetic profiles of transporter families, derived from the presence or absence of a certain transporter family, showed that clustering patterns of organisms were correlated to both their evolutionary history and their overall physiology and lifestyles.     

Identification of the glucose transporter in rat skeletal muscle

The glucose transporter in the plasma membrane of rat skeletal muscle has been identified by two approaches. In one, the transporter was detected as the polypeptide that was differentially labeled by photolysis with [³H]cytochalasin B in the presence of l- and d-glucose. [³H]Cytochalasin B is a high-affinity ligand for the transporter that is displaced by d-glucose. In the other, the transporter was detected by means of its reaction with rabbit antibodies against the purified glucose transporter from human erythrocytes. By both procedures, the transporter was found to be a polypeptide with a mobility corresponding to a molecular weight of 45,000–50,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Isolation and reconstitution of the chloride transporter of clathrin-coated vesicles

Clathrin-coated vesicle acidification is mediated by an endomembrane proton translocating ATPase. This pump is electrogenic, and significant pH gradient formation requires the parallel movement of chloride through a chloride transporter in order to maintain net electroneutrality. We have solubilized, isolated and achieved 270-fold purification of this chloride transporter by means of selective detergent solubilization with cholate and polyoxyethelene 9-lauryl ether (C12E9), hydroxylapatite chromatography, and glycerol gradient centrifugation. Stabilization of the solubilized transporter requires 5 mM dithiothreitol. The partially purified transporter was co-reconstituted with the purified clathrin-coated vesicle proton translocating complex to yield preparations of proteoliposomes capable of valinomycin-independent proton pumping, as assessed by ATP-generated acridine orange quenching. In addition, the chloride transporter was independently reconstituted and was shown to catalyze diisothiocyano-disulfonic acid stilbene-sensitive 36Cl uptake. The anionic conductive selectivity of the reconstituted transporter (chloride = bromide greater than nitrate) exactly matched that of the transporter of native clathrin-coated vesicles. These studies demonstrate that the chloride transporter of vacuolar acidification systems is structurally and functionally dissociable from co-existing proton pumps and allow for investigations of pump-transporter interactions in a resolved system.