Proton-coupled Symport Research Articles

Co-conversion of lignocellulose-derived glucose, xylose, and aromatics to polyhydroxybutyrate by metabolically engineered Cupriavidus necator

Utilization of all major components of lignocellulose is essential for biomass biorefining. Glucose, xylose, and lignin-derived aromatics can be generated from cellulose, hemicellulose, and lignin of lignocellulose degradation through pretreatment and hydrolysis. In present work, Cupriavidus necator H16 was engineered to utilize glucose, xylose, p-coumaric acid, and ferulic acid simultaneously by multi-step genetic engineering. Firstly, genetic modification and adaptive laboratory evolution were performed to promote glucose transmembrane transport and metabolism. Xylose metabolism was then engineered by integrating genes xylAB (xylose isomerase and xylulokinase) and xylE (proton-coupled symporter) in the locus of ldh (lactate dehydrogenase) and ackA (acetate kinase) on the genome, respectively. Thirdly, p-coumaric acid and ferulic acid metabolism was achieved by constructing an exogenous CoA-dependent non-β-oxidation pathway. Using corn stover hydrolysates as carbon sources, the resulting engineered strain Reh06 simultaneously converted all components of glucose, xylose, p-coumaric acid, and ferulic acid to produce 11.51 g/L polyhydroxybutyrate.

Genome-Wide Identification and Transcript Analysis Reveal Potential Roles of Oligopeptide Transporter Genes in Iron Deficiency Induced Cadmium Accumulation in Peanut.

The oligopeptide transporter (OPT) family is a group of proton-coupled symporters that play diverse roles, including metal homeostasis. However, little is known about this family of peanuts. To reveal the potential roles of AhOPT genes in Fe/Cd interactions, peanut AhOPT genes were genome-widely identified, and the relationships between gene expression and Cd accumulation were detected in two contrasting peanut cultivars (Fenghua 1 and Silihong) under Fe-sufficient or Fe-deficient conditions. A total of 40 AhOPT genes were identified in peanuts, which were divided into two subfamilies (PT and YS). Most AhOPT genes underwent gene duplication events predominated by whole-genome duplication. Clustered members generally have similar protein structures. However, gene structural divergences occurred in most of the duplicated genes. Transcription analysis revealed that AhOPT3.2/3.4 and AhYSL3.1/3.2 might be responsible for Fe deficiency tolerance, while AhOPT3.1/3.4, AhOPT7.1/7.2, and AhYSL1.1 be involved in Fe/Cd interactions. These genes might be regulated by transcription factors, including ATHB-12, ATHB-6, DIVARICATA, MYB30, NAC02, DOF3.4, IDD7, and LUX. Reduced expressions of AhYSL3.1/3.2 and higher expressions of AhOPT3.4 might contribute to higher Fe-deficiency tolerance in Silihong. Higher expression of AhOPT7.3 and AhOPT6.1 might be responsible for low Cd accumulation in Fenghua 1. Our results confirmed that AhOPT3/6/7 and AhYSL1/3 might be involved in the transport of Fe and/or Cd in peanuts and provided new clues to understanding potential mechanisms of Fe/Cd interactions.

Construction and Functional Evaluation of a Three-Dimensional Blood–Brain Barrier Model Equipped With Human Induced Pluripotent Stem Cell-Derived Brain Microvascular Endothelial Cells

PurposeThe purpose of this study was to construct and validate an in vitro three-dimensional blood–brain barrier (3DBBB) model system equipped with brain microvascular endothelial cells derived from human induced pluripotent stem cells (hiPS-BMECs).MethodsThe 3D-BBB system was constructed by seeding hiPS-BMECs onto the capillary lane of a MIMETAS OrganoPlate® 3-lane coated with fibronectin/collagen IV. hiPS-BMECs were incubated under continuous switchback flow with an OrganoFlow® for 2 days. The 3D capillary structure and expression of tight-junction proteins and transporters were confirmed by immunocytochemistry. The mRNA expression of transporters in the 3D environment was determined using qRT-PCR, and the permeability of endogenous substances and drugs was evaluated under various conditions.Results and DiscussionThe expression of tight-junction proteins, including claudin-5 and ZO-1, was confirmed by immunohistochemistry. The permeability rate constant of lucifer yellow through hiPS-BMECs was undetectably low, indicating that paracellular transport is highly restricted by tight junctions in the 3D-BBB system. The mRNA expression levels of transporters and receptors in the 3D-BBB system differed from those in the 2D-culture system by 0.2- to 5.8-fold. The 3D-cultured hiPS-BMECs showed asymmetric transport of substrates of BCRP, CAT1 and LAT1 between the luminal (blood) and abluminal (brain) sides. Proton-coupled symport function of MCT1 was also confirmed.ConclusionThe 3D-BBB system constructed in this study mimics several important characteristics of the human BBB, and is expected to be a useful high-throughput evaluation tool in the development of CNS drugs.

Mechanics of Nramp-family transition metal transporters

Transition metal ions (like Mn2+, Fe2+ and Co2+) are nutrients crucial to metabolism in all living cells. The natural resistance associated macrophage protein (Nramp) transporters, found across the tree of life, enable cells to take up a range of essential divalent transition metals. Human Nramps are required for the dietary uptake and endosomal recycling of non-heme iron, and in the innate immune response to intracellular pathogens. Nramps are pH dependent and co-transport protons, and thus have been labeled as proton-coupled symporters.

Uniport, Not Proton-Symport, in a Non-Mammalian SLC23 Transporter

SLC23 family members are transporters of either nucleobases or ascorbate. While the mammalian SLC23 ascorbate transporters are sodium-coupled, the non-mammalian nucleobase transporters have been proposed, but not formally shown, to be proton-coupled symporters. This assignment is exclusively based on in vivo transport assays using protonophores. Here, by establishing the first in vitro transport assay for this protein family, we demonstrate that a representative member of the SLC23 nucleobase transporters operates as a uniporter instead. We explain these conflicting assignments by identifying a critical role of uracil phosphoribosyltransferase, the enzyme converting uracil to UMP, in driving uracil uptake in vivo. Detailed characterization of uracil phosphoribosyltransferase reveals that the sharp reduction of uracil uptake in whole cells in presence of protonophores is caused by acidification-induced enzyme inactivation. The SLC23 family therefore consists of both uniporters and symporters in line with the structurally related SLC4 and SLC26 families that have previously been demonstrated to accommodate both transport modes as well.

Molecular mechanism of sugar transport in plants unveiled by structures of glucose/H+ symporter STP10.

Sugars are essential sources of energy and carbon and also function as key signalling molecules in plants. Sugar transport proteins (STP) are proton-coupled symporters responsible for uptake of glucose from the apoplast into plant cells. They are integral to organ development in symplastically isolated tissues such as seed, pollen and fruit. Additionally, STPs play a vital role in plant responses to stressors such as dehydration and prevalent fungal infections like rust and mildew. Here we present a structure of Arabidopsis thaliana STP10 in the inward-open conformation at 2.6 Å resolution and a structure of the outward-occluded conformation at improved 1.8 Å resolution, both with glucose and protons bound. The two structures describe key states in the STP transport cycle. Together with molecular dynamics simulations that establish protonation states and biochemical analysis, they pinpoint structural elements, conserved in all STPs, that clarify the basis of proton-to-glucose coupling. These results advance our understanding of monosaccharide uptake, which is essential for plant organ development, and set the stage for bioengineering strategies in crops.

Crystal structure of the plant symporter STP10 illuminates sugar uptake mechanism in monosaccharide transporter superfamily

Plants are dependent on controlled sugar uptake for correct organ development and sugar storage, and apoplastic sugar depletion is a defense strategy against microbial infections like rust and mildew. Uptake of glucose and other monosaccharides is mediated by Sugar Transport Proteins, proton-coupled symporters from the Monosaccharide Transporter (MST) superfamily. We present the 2.4 Å structure of Arabidopsis thaliana high affinity sugar transport protein, STP10, with glucose bound. The structure explains high affinity sugar recognition and suggests a proton donor/acceptor pair that links sugar transport to proton translocation. It contains a Lid domain, conserved in all STPs, that locks the mobile transmembrane domains through a disulfide bridge, and creates a protected environment which allows efficient coupling of the proton gradient to drive sugar uptake. The STP10 structure illuminates fundamental principles of sugar transport in the MST superfamily with implications for both plant antimicrobial defense, organ development and sugar storage.

Nutrient exchange in arbuscular mycorrhizal symbiosis from a thermodynamic point of view.

Summary To obtain insights into the dynamics of nutrient exchange in arbuscular mycorrhizal (AM) symbiosis, we modelled mathematically the two‐membrane system at the plant–fungus interface and simulated its dynamics.In computational cell biology experiments, the full range of nutrient transport pathways was tested for their ability to exchange phosphorus (P)/carbon (C)/nitrogen (N) sources.As a result, we obtained a thermodynamically justified, independent and comprehensive model of the dynamics of the nutrient exchange at the plant–fungus contact zone. The predicted optimal transporter network coincides with the transporter set independently confirmed in wet‐laboratory experiments previously, indicating that all essential transporter types have been discovered.The thermodynamic analyses suggest that phosphate is released from the fungus via proton‐coupled phosphate transporters rather than anion channels. Optimal transport pathways, such as cation channels or proton‐coupled symporters, shuttle nutrients together with a positive charge across the membranes. Only in exceptional cases does electroneutral transport via diffusion facilitators appear to be plausible. The thermodynamic models presented here can be generalized and adapted to other forms of mycorrhiza and open the door for future studies combining wet‐laboratory experiments with computational simulations to obtain a deeper understanding of the investigated phenomena.

Genome Mining of Plant NPFs Reveals Varying Conservation of Signature Motifs Associated With the Mechanism of Transport.

Nitrogen is essential for all living species and may be taken up from the environment in different forms like nitrate or peptides. In plants, members of a transporter family named NPFs transport nitrate and peptides across biological membranes. NPFs are phylogenetically related to a family of peptide transporters (PTRs) or proton-coupled oligopeptide transporters (POTs) that are evolutionarily conserved in all organisms except in Archaea. POTs are present in low numbers in bacteria, algae and animals. NPFs have expanded in plants and evolved to transport a wide range of substrates including phytohormones and glucosinolates. Functional studies have shown that most NPFs, like POTs, operate as symporters with simultaneous inwardly directed movement of protons. Here we focus on four structural features of NPFs/POTs/PTRs that have been shown by structural and functional studies to be essential to proton-coupled symport transport. The first two features are implicated in proton binding and transport: a conserved motif named ExxER/K, located in the first transmembrane helix (TMH1) and a D/E residue in TMH7 that has been observed in some bacterial and algal transporters. The third and fourth features are two inter-helical salt bridges between residues on TMH1 and TMH7 or TMH4 and TMH10. To understand if the mechanism of transport is conserved in NPFs with the expansion to novel substrates, we collected NPFs sequences from 42 plant genomes. Sequence alignment revealed that the ExxER/K motif is not strictly conserved and its conservation level is different in the NPF subfamilies. The proton binding site on TMH7 is missing in all NPFs with the exception of two NPFs from moss. The two moss NPFs also have a positively charged amino acid on TMH1 that can form the salt bridge with the TMH7 negative residue. None of the other NPFs we examined harbor residues that can form the TMH1–TMH7 salt bridge. In contrast, the amino acids required to form the TMH4–TMH10 salt bridge are highly conserved in NPFs, with some exceptions. These results support the need for further biochemical and structural studies of individual NPFs for a better understanding of the transport mechanism in this family of transporters.

Refactoring the upper sugar metabolism of Pseudomonas putida for co-utilization of cellobiose, xylose, and glucose

Given its capacity to tolerate stress, NAD(P)H/ NAD(P) balance, and increased ATP levels, the platform strain Pseudomonas putida EM42, a genome-edited derivative of the soil bacterium P. putida KT2440, can efficiently host a suite of harsh reactions of biotechnological interest. Because of the lifestyle of the original isolate, however, the nutritional repertoire of P. putida EM42 is centered largely on organic acids, aromatic compounds and some hexoses (glucose and fructose). To enlarge the biochemical network of P. putida EM42 to include disaccharides and pentoses, we implanted heterologous genetic modules for D-cellobiose and D-xylose metabolism into the enzymatic complement of this strain. Cellobiose was actively transported into the cells through the ABC complex formed by native proteins PP1015-PP1018. The knocked-in β-glucosidase BglC from Thermobifida fusca catalyzed intracellular cleavage of the disaccharide to D-glucose, which was then channelled to the default central metabolism. Xylose oxidation to the dead end product D-xylonate was prevented by deleting the gcd gene that encodes the broad substrate range quinone-dependent glucose dehydrogenase. Intracellular intake was then engineered by expressing the Escherichia coli proton-coupled symporter XylE. The sugar was further metabolized by the products of E. coli xylA (xylose isomerase) and xylB (xylulokinase) towards the pentose phosphate pathway. The resulting P. putida strain co-utilized xylose with glucose or cellobiose to complete depletion of the sugars. These results not only show the broadening of the metabolic capacity of a soil bacterium towards new substrates, but also promote P. putida EM42 as a platform for plug-in of new biochemical pathways for utilization and valorization of carbohydrate mixtures from lignocellulose processing.

Putative role of the H(+)/sucrose symporter SLC45A3 as an osmolyte transporter in the kidney.

The solute carrier family 45 a3 member (SLC45A3), known also as prostein, has been implicated with prostate cancer and the regulation of lipid metabolism in oligodendrocytes. Recently, we expressed SLC45A3 in yeast cells and characterised it as a proton-coupled sucrose symporter. However, the physiological functions of SLC45A3 were still unknown. Here, we report that SLC45A3 occurs in the kidney and is highly expressed in the medullary collecting duct (IMCD), a part of the kidney responsible for final urine concentration and faced to hyperosmotic environment. Moreover, messenger RNA (mRNA) expression of endogenous SLC45A3 in rat IMCD cells as well as in NRK52E cells increased up to four-fold under hyperosmotic conditions at 600mOsmol/kg. Using NRK52E cells as an experimental model, we investigated the proton-coupled sugar transport and found that the uptake of sucrose or glucose was enhanced by hyperosmolarity. Down-regulation of expression by small interfering RNA (siRNA) decreased the osmotically inducible part of sucrose uptake and confirmed the involvement of SLC45A3 in this process. Furthermore, we observed an up to four-fold elevation of sucrose uptake triggered by hyperosmolarity across the apical membrane of NRK52E cells, while uptake across the basolateral membrane was not affected. Due to this finding, we conclude that SLC45A3 may occur at the luminal side of kidney epithelial cells and thus may take up solutes from the tubular fluid. Altogether, we show that SLC45A3 is a novel sugar transporter in kidney and hypothesise that the disaccharide sucrose, and probably the monosaccharides glucose and fructose, may serve as compatible osmolytes in urine.

Functional Analysis of Two l-Arabinose Transporters from Filamentous Fungi Reveals Promising Characteristics for Improved Pentose Utilization in Saccharomyces cerevisiae.

Limited uptake is one of the bottlenecks for l-arabinose fermentation from lignocellulosic hydrolysates in engineered Saccharomyces cerevisiae. This study characterized two novel l-arabinose transporters, LAT-1 from Neurospora crassa and MtLAT-1 from Myceliophthora thermophila. Although the two proteins share high identity (about 83%), they display different substrate specificities. Sugar transport assays using the S. cerevisiae strain EBY.VW4000 indicated that LAT-1 accepts a broad substrate spectrum. In contrast, MtLAT-1 appeared much more specific for l-arabinose. Determination of the kinetic properties of both transporters revealed that the Km values of LAT-1 and MtLAT-1 for l-arabinose were 58.12 ± 4.06 mM and 29.39 ± 3.60 mM, respectively, with corresponding Vmax values of 116.7 ± 3.0 mmol/h/g dry cell weight (DCW) and 10.29 ± 0.35 mmol/h/g DCW, respectively. In addition, both transporters were found to use a proton-coupled symport mechanism and showed only partial inhibition by d-glucose during l-arabinose uptake. Moreover, LAT-1 and MtLAT-1 were expressed in the S. cerevisiae strain BSW2AP containing an l-arabinose metabolic pathway. Both recombinant strains exhibited much faster l-arabinose utilization, greater biomass accumulation, and higher ethanol production than the control strain. In conclusion, because of higher maximum velocities and reduced inhibition by d-glucose, the genes for the two characterized transporters are promising targets for improved l-arabinose utilization and fermentation in S. cerevisiae.

Amino Acid Positions Important For Substrate Specificity in Plant Sucrose Transporters

Plant sucrose transporters (SUTs) are members of the GPH family within the Major Facilitator Superfamily (MFS). They are proton‐coupled symporters in the plasma membrane or vacuole (lysosome) membrane and function in sucrose uptake into the cytoplasm. SUTs function throughout the plant but are especially important for sucrose uptake into vascular tissue. Plants contain small gene families of SUTs that can be classified into three basic types (I, II, III) both by their amino acid sequence and by their substrate specificity. Type I SUTs are less selective and transport a range of beta glucosides, including the fluorescent coumarin glucosides esculin and fraxin, in addition to sucrose. Type II SUTs are more selective for sucrose and do not transport esculin or fraxin. To identify amino acid positions that control substrate specificity, we used gene shuffling between a Type II SUT OsSUT1 and a non‐functional version of OsSUT1 in which 64 positions had been mutated to the corresponding positions in the Type I SUT StSUT1. Gene shuffling libraries were expressed in yeast and assayed for their ability to confer uptake of esculin using fluorescence activated cell sorting (FACS). Mutation of five amino acids in OsSUT1 resulted in esculin transport. The amino acid changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was analyzed by expression in Xenopus oocytes and electrophysiology and was almost identical to that of type I SUTs. We are now investigating the role of these positions in more detail.

Structural basis for polyspecificity in the POT family of proton-coupled oligopeptide transporters.

An enigma in the field of peptide transport is the structural basis for ligand promiscuity, as exemplified by PepT1, the mammalian plasma membrane peptide transporter. Here, we present crystal structures of di- and tripeptide-bound complexes of a bacterial homologue of PepT1, which reveal at least two mechanisms for peptide recognition that operate within a single, centrally located binding site. The dipeptide was orientated laterally in the binding site, whereas the tripeptide revealed an alternative vertical binding mode. The co-crystal structures combined with functional studies reveal that biochemically distinct peptide-binding sites likely operate within the POT/PTR family of proton-coupled symporters and suggest that transport promiscuity has arisen in part through the ability of the binding site to accommodate peptides in multiple orientations for transport.

Structural Basis for Dynamic Mechanism of Proton-Coupled Symport by the Peptide Transporter POT

Membrane transporters transport their substrates across the membrane, thereby contributing to the maintenance of the cellular environments. One of the largest superfamily of the membrane transporters is Major Facilitator Superfamily (MFS), and many of MFS transporters are involved in the cellular uptake of various compounds utilizing the proton motive force across the membrane. Although several crystal structures of MFS transporters have been reported so far, their active transport mechanism has still remained elusive. Proton-dependent oligopeptide transporter, POT, is a member of MFS, and is involved in the uptake of oligopeptides as well as peptide-like drugs. In this article, we explain the active transporter mechanism by POT based on our recent results of the structural and computational analyses.

Structural basis for dynamic mechanism of proton-coupled symport by the peptide transporter POT.

Proton-dependent oligopeptide transporters (POTs) are major facilitator superfamily (MFS) proteins that mediate the uptake of peptides and peptide-like molecules, using the inwardly directed H(+) gradient across the membrane. The human POT family transporter peptide transporter 1 is present in the brush border membrane of the small intestine and is involved in the uptake of nutrient peptides and drug molecules such as β-lactam antibiotics. Although previous studies have provided insight into the overall structure of the POT family transporters, the question of how transport is coupled to both peptide and H(+) binding remains unanswered. Here we report the high-resolution crystal structures of a bacterial POT family transporter, including its complex with a dipeptide analog, alafosfalin. These structures revealed the key mechanistic and functional roles for a conserved glutamate residue (Glu310) in the peptide binding site. Integrated structural, biochemical, and computational analyses suggested a mechanism for H(+)-coupled peptide symport in which protonated Glu310 first binds the carboxyl group of the peptide substrate. The deprotonation of Glu310 in the inward open state triggers the release of the bound peptide toward the intracellular space and salt bridge formation between Glu310 and Arg43 to induce the state transition to the occluded conformation.

Metabolic engineering of raffinose-family oligosaccharides in the phloem reveals alterations in carbon partitioning and enhances resistance to green peach aphid

Many plants employ energized loading strategies to accumulate osmotically-active solutes into the phloem of source organs to accentuate the hydrostatic pressure gradients that drive the flow of water, nutrients and signals from source to sinks. Proton-coupled symport of sugars from the apoplasm into the phloem symplasm is the best studied phloem-loading mechanism. As an alternative, numerous species use a polymer trapping mechanism to load through symplasm: sucrose enters the phloem through specialized plasmodesmata and is converted to raffinose-family oligosaccharides (RFOs) which accumulate because of their larger size. In this study, metabolic engineering was used to generate RFOs at the inception of the translocation stream of Arabidopsis thaliana, which loads from the apoplasm and transports predominantly sucrose, and the fate of the sugars throughout the plant determined. Three genes, GALACTINOL SYNTHASE, RAFFINOSE SYNTHASE and STACHYOSE SYNTHASE, were expressed from promoters specific to the companion cells of minor veins. Two transgenic lines homozygous for all three genes (GRS63 and GRS47) were selected for further analysis. Three-week-old plants of both lines had RFO levels approaching 50% of total soluble sugar. RFOs were also identified in exudates from excised leaves of transgenic plants whereas levels were negligible in exudates from wild type (WT) leaves. Differences in starch accumulation between WT and GRS63 and GRS47 lines were not observed. Similarly, there were no differences in vegetative growth between WT and engineered plants, but the latter flowered slightly earlier. Finally, since the sugar composition of the translocation stream appeared altered, we tested for an impact on green peach aphid (Myzus persicae Sulzer) feeding. When given a choice between WT and transgenic plants, green peach aphids preferred settling on the WT plants. Furthermore, green peach aphid fecundity was lower on the transgenic plants compared to the WT plants. When added to an artificial diet, RFOs did not have a negative effect on aphid fecundity, suggesting that although aphid resistance in the transgenic plants is enhanced, it is not due to direct toxicity of RFO toward the insect.

SLC36A4 (hPAT4) Is a High Affinity Amino Acid Transporter When Expressed in Xenopus laevis Oocytes

The SLC36 family of transporters consists of four genes, two of which, SLC36A1 and SLC36A2, have been demonstrated to code for human proton-coupled amino acid transporters or hPATs. Here we report the characterization of the fourth member of the family, SLC36A4 or hPAT4, which when expressed in Xenopus laevis oocytes also encodes a plasma membrane amino acid transporter, but one that is not proton-coupled and has a very high substrate affinity for the amino acids proline and tryptophan. hPAT4 in Xenopus oocytes mediated sodium-independent, electroneutral uptake of [(3)H]proline, with the highest rate of uptake when the uptake medium pH was 7.4 and an affinity of 3.13 μM. Tryptophan was also an excellently transported substrate with a similarly high affinity (1.72 μM). Other amino acids that inhibited [(3)H]proline were isoleucine (K(i) 0.23 mM), glutamine (0.43 mM), methionine (0.44 mM), and alanine (1.48 mM), and with lower affinity, glycine, threonine, and cysteine (K(i) >5 mM for all). Of the amino acids directly tested for transport, only proline, tryptophan, and alanine showed significant uptake, whereas glycine and cysteine did not. Of the non-proteogenic amino acids and drugs tested, only sarcosine produced inhibition (K(i) 1.09 mM), whereas γ-aminobutyric acid (GABA), β-alanine, L-Dopa, D-serine, and δ-aminolevulinic acid were without effect on [(3)H]proline uptake. This characterization of hPAT4 as a very high affinity/low capacity non-proton-coupled amino acid transporter raises questions about its physiological role, especially as the transport characteristics of hPAT4 are very similar to the Drosophila orthologue PATH, an amino acid "transceptor" that plays a role in nutrient sensing.

FSY1, a horizontally transferred gene in the Saccharomyces cerevisiae EC1118 wine yeast strain, encodes a high-affinity fructose/H+ symporter

Transport of glucose and fructose in the yeast Saccharomyces cerevisiae plays a crucial role in controlling the rate of wine fermentation. In S. cerevisiae, hexoses are transported by facilitated diffusion via hexose carriers (Hxt), which prefer glucose to fructose. However, utilization of fructose by wine yeast is critically important at the end of fermentation. Here, we report the characterization of a fructose transporter recently identified by sequencing the genome of the commercial wine yeast strain EC1118 and found in many other wine yeasts. This transporter is designated Fsy1p because of its homology with the Saccharomyces pastorianus fructose/H(+) symporter Fsy1p. A strain obtained by transformation of the V5 hxt1-7Δ mutant with FSY1 grew well on fructose, but to a much lesser extent on glucose as the sole carbon source. Sugar uptake and symport experiments showed that FSY1 encodes a proton-coupled symporter with high affinity for fructose (K(m) 0.24±0.04mM). Using real-time RT-PCR, we also investigated the expression pattern of FSY1 in EC1118 growing on various carbon sources. FSY1 was repressed by high concentrations of glucose or fructose and was highly expressed on ethanol as the sole carbon source. The characteristics of this transporter indicate that its acquisition could confer a significant advantage to S. cerevisiae during the wine fermentation process. This transporter is a good example of acquisition of a new function in yeast by horizontal gene transfer.

Evolutionary ancestry and novel functions of the mammalian glucose transporter (GLUT) family

BackgroundIn general, sugar porters function by proton-coupled symport or facilitative transport modes. Symporters, coupled to electrochemical energy, transport nutrients against a substrate gradient. Facilitative carriers transport sugars along a concentration gradient, thus transport is dependent upon extracellular nutrient levels. Across bacteria, fungi, unicellular non-vertebrates and plants, proton-coupled hexose symport is a crucial process supplying energy under conditions of nutrient flux. In mammals it has been assumed that evolution of whole body regulatory mechanisms would eliminate this need. To determine whether any isoforms bearing this function might be conserved in mammals, we investigated the relationship between the transporters of animals and the proton-coupled hexose symporters found in other species.ResultsWe took a comparative genomic approach and have performed the first comprehensive and statistically supported phylogenetic analysis of all mammalian glucose transporter (GLUT) isoforms. Our data reveals the mammalian GLUT proteins segregate into five distinct classes. This evolutionary ancestry gives insight to structure, function and transport mechanisms within the groups. Combined with biological assays, we present novel evidence that, in response to changing nutrient availability and environmental pH, proton-coupled, active glucose symport function is maintained in mammalian cells.ConclusionsThe analyses show the ancestry, evolutionary conservation and biological importance of the GLUT classes. These findings significantly extend our understanding of the evolution of mammalian glucose transport systems. They also reveal that mammals may have conserved an adaptive response to nutrient demand that would have important physiological implications to cell survival and growth.