Two glucose/xylose transporter genes from the yeast Candida intermedia: first molecular characterization of a yeast xylose-H+ symporter

Candida intermedia PYCC 4715 was previously shown to grow well on xylose and to transport this sugar by two different transport systems: high-capacity and low-affinity facilitated diffusion and a high-affinity xylose-proton symporter, both of which accept glucose as a substrate. Here we report the isolation of genes encoding both transporters, designated GXF1 (glucose/xylose facilitator 1) and GXS1 (glucose/xylose symporter 1) respectively. Although GXF1 was isolated by functional complementation of an HXT-null (where Hxt refers to hexose transporters) Saccharomyces cerevisiae strain, isolation of the GXS1 cDNA required partial purification and micro-sequencing of the transporter, identified by its relative abundance in cells grown on low xylose concentrations. Both genes were expressed in S. cerevisiae and the kinetic parameters of glucose and xylose transport were determined. Gxs1 is the first yeast xylose/glucose-H+ symporter to be characterized at the molecular level. Comparison of its amino acid sequence with available sequence data revealed the existence of a family of putative monosaccharide-H+ symporters encompassing proteins from several yeasts and filamentous fungi.     

Properties of a reconstituted eukaryotic hexose/proton symporter solubilized by structurally related non-ionic detergents: specific requirement of phosphatidylcholine for permease stability

Overexpression of the hexose/proton symporter HUP1 from Chlorella kessleri in S. cerevisiae permits a one-step purification via a biotinylation domain. Milligram amounts of the protein are obtained starting from 2 l of yeast culture. The HUP1 protein is used as a model eukaryotic membrane protein of the 'major facilitator superfamily' (MFS) to study specific lipid requirements for activity and stability. Testing two series of detergents revealed that n-nonyl-beta-D-glucoside (NG) and n-octyl-beta-D-glucoside (OG) solubilize the HUP1 protein efficiently. Only the use of NG resulted in long-term stabilization of the HUP1 protein in the absence of external lipids. When affinity purified protein was extracted with organic solvents, a stoichiometric amount of phosphatidyl choline, phosphatidyl ethanolamine and ergosterol in the ratio of close to 2:1 was detected. These lipids were only observed, however, when the protein purification was carried out in the presence of NG; no lipids were copurified with the HUP1 protein in the presence of OG. Of the three lipids copurified, phosphatidyl choline showed a crucial role in ensuring maximal HUP1 permease activity and stability when added back to the OG-protein. The requirement of phosphatidylcholine documents a specific effect of lipids on vectorial transport mediated by a eukaryotic membrane protein of the MFS family.     

Lipid raft-based membrane compartmentation of a plant transport protein expressed in Saccharomyces cerevisiae

The hexose-proton symporter HUP1 shows a spotty distribution in the plasma membrane of the green alga Chlorella kessleri. Chlorella cannot be transformed so far. To study the membrane localization of the HUP1 protein in detail, the symporter was fused to green fluorescent protein (GFP) and heterologously expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In these organisms, the HUP1 protein has previously been shown to be fully active. The GFP fusion protein was exclusively targeted to the plasma membranes of both types of fungal cells. In S. cerevisiae, it was distributed nonhomogenously and concentrated in spots resembling the patchy appearance observed previously for endogenous H(+) symporters. It is documented that the Chlorella protein colocalizes with yeast proteins that are concentrated in 300-nm raft-based membrane compartments. On the other hand, it is completely excluded from the raft compartment housing the yeast H(+)/ATPase. As judged by their solubilities in Triton X-100, the HUP1 protein extracted from Chlorella and the GFP fusion protein extracted from S. cerevisiae are detergent-resistant raft proteins. S. cerevisiae mutants lacking the typical raft lipids ergosterol and sphingolipids showed a homogenous distribution of HUP1-GFP within the plasma membrane. In an ergosterol synthesis (erg6) mutant, the rate of glucose uptake was reduced to less than one-third that of corresponding wild-type cells. In S. pombe, the sterol-rich plasma membrane domains can be stained in vivo with filipin. Chlorella HUP1-GFP accumulated exactly in these domains. Altogether, it is demonstrated here that a plant membrane protein has the property of being concentrated in specific raft-based membrane compartments and that the information for its raft association is retained between even distantly related organisms.     

Glucose increases the expression of the ATP/ADP translocator and the glyceraldehyde-3-phosphate dehydrogenase genes in Chlorella.

A presumably full-length cDNA clone of the mitochondrial ATP/ADP translocator (AAT) of Chlorella kessleri has been isolated and sequenced. The expression of the AAT gene is highly increased in the presence of D-glucose (14 mM). At least nine more genes are activated when autotrophically grown Chlorella cells switch to heterotrophic growth. Among these is the HUP1 gene coding for the hexose transporter (Sauer, N., Caspari, T., Klebl, F., and Tanner, W. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7949-7952) and, as also shown in this paper, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. When glucose or the nonmetabolizable analogue 6-deoxyglucose is added to the cells, an increased expression of GAPDH or AAT is observed after 10 or 30 min, respectively. Hexose uptake mutants (HUP1-) do not respond to sugars in this way, which indicates that either the inducer has to be internalized or that the HUP1 translocator is part of the signal transduction mechanism.     

A phloem-specific sucrose-H+ symporter from Plantago major L. supports the model of apoplastic phloem loading

In this paper the cloning of a full-length cDNA clone encoding the PmSUC2 sucrose-H⁺ symporter from Plantago major is described. This plant allows the simple preparation of vascular bundles from the basal regions of fully developed source leaves and thus a separation of vascular and non-vascular tissue. A cDNA library was constructed from poly(A)⁺ RNA isolated from vascular bundles and used for the subsequent cloning of cDNAs. The respective mRNA is specifically expressed in the vascular bundles as shown on Northern blots of total RNA from vascular and non-vascular tissues. The PmSUC2 protein has 12 putative transmembrane helices and is highly homologous to other plant sucrose transporters. Substrate specificity and energy dependence of the transporter encoded by this cDNA were determined by expression in baker's yeast Saccharomyces cerevisiae. The PmSUC2 protein catalyses the transport of sucrose into transgenic yeast cells. Invertase null mutants of yeast expressing PmSUC2 accumulate sucrose more than 200-fold. This transport was sensitive to uncouplers or SH-group inhibitors. Plasma membranes from yeast cells expressing the PmSUC2 protein were purified and fused to proteoliposomes containing cytochrome-c-oxidase. In this system sucrose is accumulated only when proton motive force is generated, indicating that PmSUC2 is a sucrose-H⁺ symporter. The apparent molecular weight of the PmSUC2 protein is 35 kDa on 10% SDS-polyacrylamide gels. The presented data strongly support the theory of phloem loading from the apoplastic space by a sucrose-H⁺ symporter.     

Plant sucrose-H+ symporters mediate the transport of vitamin H

A cDNA coding for a vitamin H (biotin) transport protein from Arabidopsis was identified by genetic complementation of a biotin uptake-deficient yeast mutant. Vitamin H transport by this protein was sensitive to the SH-group inhibitor p-chloromercuribenzene sulfonic acid (PCMBS) and to the uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP), suggesting an energy-dependent biotin-H+ symport mechanism. The transport activity could contribute to the so-far uncharacterized plant sucrose-H+ symporter AtSUC5 which mediates the energy-dependent transport of biotin and sucrose, and restores growth of the biotin transport-deficient yeast mutant on medium with low biotin concentrations. Functional comparison of the AtSUC5 transporter with previously characterized plant sucrose or monosaccharide transporters revealed that biotin transport may be a general and specific property of all plant sucrose transporters (sucrose/biotin-H+ symporters). This first report on a transporter with dual substrate specificity for two structurally unrelated molecules has a major impact on general thinking concerning the specificity of membrane transporters. The physiological relevance of this finding is discussed.     

Primary structure and properties of the Na+/glucose symporter (Sg1S) of Vibrio parahaemolyticus.

Previously, we cloned and sequenced a DNA fragment from Vibrio parahaemolyticus and found four open reading frames (ORFs). Here, we clearly demonstrate that one of the ORFs, ORF1, is the gene (sglS) encoding a Na+/glucose symporter (SglS). We characterize the Na+/glucose symporter produced in Escherichia coli mutant (JM1100) cells which lack original glucose transport activity and galactose transport activity. We also show that phlorizin, a potent inhibitor of the SGLT1 Na+/glucose symporter of animal cells, inhibited glucose transport, but not galactose transport, via the SglS system.     

Primary structure, genomic organization and heterologous expression of a glucose transporter from Arabidopsis thaliana.

Both genomic and full length cDNA clones of an Arabidopsis thaliana sugar carrier, STP1, have been obtained using a cDNA clone of the H+/hexose cotransporter from the green alga Chlorella kessleri as hybridization probe. The peptide predicted from these sequences in 522 amino acids long and has a molecular weight of 57,518 kd. This higher plant sugar carrier contains 12 putative transmembrane segments and is highly homologous to the H+/hexose cotransporter from Chlorella, with an overall identity in the amino acid sequence of 47.1%. It is also homologous to the human HepG2 glucose transporter (28.4%), and other sugar carriers from man, rat, yeast and Escherichia coli. The definite proof for the function of the STP1 protein as a hexose transporter and data on its substrate specificity were obtained by heterologous expression in the fission yeast Schizosaccharomyces pombe. Transformed yeast cells transport D-glucose with a 100-fold lower KM value than control cells. Moreover only the transformed cells were able to accumulate the non-metabolizable D-glucose analogue 3-O-methyl-D-glucose, indicating that the Arabidopsis carrier catalyses an energy dependent, active uptake of hexoses. Expression of STP1 mRNA is low in heterotrophic tissues like roots or flowers. High levels of expression are found in leaves.     

Expression of eukaryotic plasma membrane transporter HUP1 from Chlorella kessleri in Escherichia coli

To study the effect of sterols on the activity of the eukaryotic plasma membrane transporter, the hexose-proton symporter HUP1 from the unicellular alga Chlorella kessleri was expressed in Escherichia coli, a prokaryotic microorganism containing virtually no sterols. Under certain conditions, the recombinant protein was partially active in this prokaryotic organism. The heterologously produced HUP1p was purified from membrane fractions of E. coli and reconstituted in an in vitro system. The presence of ergosterol during solubilization, purification and reconstitution resulted in an increased activity of the reconstituted protein. Its activity, however, was 5-6 times lower as compared to the activity of HUP1p produced in Saccharomyces cerevisiae membranes and solubilized, purified, and reconstituted under the same conditions as above.     

The conserved sequence NXX[S/T]HX[S/T]QDXXXT of the lactate/pyruvate:H+ symporter subfamily defines the function of the substrate translocation pathway

In Saccharomyces cerevisiae Jen1p is a lactate/proton symporter belonging to the lactate/pyruvate:H⁺ symporter subfamily (TC#2.A.1.12.2) of the Major Facilitator Superfamily. We investigated structure-function relationships of Jen1p using a rational mutational analysis based on the identification of conserved amino acid residues. In particular, we studied the conserved sequence ³⁷⁹NXX[S/T]HX[S/T]QDXXXT³⁹¹. Substitution of amino acid residues N379, H383 or D387, even with very similar amino acids, resulted in a dramatic reduction of lactate and pyruvate uptake, but conserved measurable acetate transport. Acetate transport inhibition assays showed that these mutants conserve the ability to bind, but do not transport, lactate and pyruvate. More interestingly, the double mutation H383D/D387H, while behaving as a total loss-of-function allele for lactate and pyruvate uptake, can fully restore the kinetic parameters of Jen1p for acetate transport. Thus, residues N379, H383 or D387 affect both the transport capacity and the specificity of Jen1p. Substitutions of Q386 and T391 resulted in no or moderate changes in Jen1p transport capacities for lactate, pyruvate and acetate. On the other hand, Q386N reduces the binding affinities for all Jen1p substrates, while Q386A increases the affinity specifically for pyruvate. We also tested Jen1p specificity for a range of monocarboxylates. Several of the mutants studied showed altered inhibition constants for these acids. These results and 3D in silico modelling by homology threading suggest that the conserved motif analyzed is part of the substrate translocation pathway in the lactate/pyruvate:H⁺ symporter subfamily.     

Identification of a H+/glucose and galactose symporter gene glt from Xanthomonas oryzae pv. oryzae

We identified a glucose and galactose transporter gene from the plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae. Sequence analysis indicated that the gene, named glt, encoded a polypeptide of 592 amino acid residues and the product was significantly homologous with members of the Na+/glucose cotransporter (SGLT) family from mammalian and bacterial origin, especially with vSGLT from Vibrio parahaemolyticus (50% identity). GLT functioned as a glucose and galactose transporter in an Escherichia coli mutant deficient in glucose and galactose transport activity. A protonophore inhibited the transport activity, suggesting that GLT is a H+-coupled glucose/galactose symporter.     

A member of the sugar transporter family, Stl1p is the glycerol/H+ symporter in Saccharomyces cerevisiae

Glycerol and other polyols are used as osmoprotectants by many organisms. Several yeasts and other fungi can take up glycerol by proton symport. To identify genes involved in active glycerol uptake in Saccharomyces cerevisiae we screened a deletion mutant collection comprising 321 genes encoding proteins with 6 or more predicted transmembrane domains for impaired growth on glycerol medium. Deletion of STL1, which encodes a member of the sugar transporter family, eliminates active glycerol transport. Stl1p is present in the plasma membrane in S. cerevisiae during conditions where glycerol symport is functional. Both the Stl1 protein and the active glycerol transport are subject to glucose-induced inactivation, following identical patterns. Furthermore, the Stl1 protein and the glycerol symporter activity are strongly but transiently induced when cells are subjected to osmotic shock. STL1 was heterologously expressed in Schizosaccharomyces pombe, a yeast that does not contain its own active glycerol transport system. In S. pombe, STL1 conferred the ability to take up glycerol against a concentration gradient in a proton motive force-dependent manner. We conclude that the glycerol proton symporter in S. cerevisiae is encoded by STL1.     

The conserved sequence NXX[S/T]HX[S/T]QDXXXT of the lactate/pyruvate:H(+) symporter subfamily defines the function of the substrate translocation pathway

In Saccharomyces cerevisiae Jen1p is a lactate/proton symporter belonging to the lactate/pyruvate:H(+) symporter subfamily (TC#2.A.1.12.2) of the Major Facilitator Superfamily. We investigated structure-function relationships of Jen1p using a rational mutational analysis based on the identification of conserved amino acid residues. In particular, we studied the conserved sequence (379)NXX[S/T]HX[S/T]QDXXXT(391). Substitution of amino acid residues N379, H383 or D387, even with very similar amino acids, resulted in a dramatic reduction of lactate and pyruvate uptake, but conserved measurable acetate transport. Acetate transport inhibition assays showed that these mutants conserve the ability to bind, but do not transport, lactate and pyruvate. More interestingly, the double mutation H383D/D387H, while behaving as a total loss-of-function allele for lactate and pyruvate uptake, can fully restore the kinetic parameters of Jen1p for acetate transport. Thus, residues N379, H383 or D387 affect both the transport capacity and the specificity of Jen1p. Substitutions of Q386 and T391 resulted in no or moderate changes in Jen1p transport capacities for lactate, pyruvate and acetate. On the other hand, Q386N reduces the binding affinities for all Jen1p substrates, while Q386A increases the affinity specifically for pyruvate. We also tested Jen1p specificity for a range of monocarboxylates. Several of the mutants studied showed altered inhibition constants for these acids. These results and 3D in silico modelling by homology threading suggest that the conserved motif analyzed is part of the substrate translocation pathway in the lactate/pyruvate:H(+) symporter subfamily.     

Functional characterization of the Arabidopsis AtSUC2 Sucrose/H+ symporter by tissue-specific complementation reveals an essential role in phloem loading but not in long-distance transport

AtSUC2 (At1g22710) encodes a phloem-localized sucrose (Suc)/H(+) symporter necessary for efficient Suc transport from source tissues to sink tissues in Arabidopsis (Arabidopsis thaliana). AtSUC2 is highly expressed in the collection phloem of mature leaves, and its function in phloem loading is well established. AtSUC2, however, is also expressed strongly in the transport phloem, where its role is more ambiguous, and it has been implicated in mediating both efflux and retrieval to and from flanking tissues via the apoplast. To characterize the role of AtSUC2 in controlling carbon partitioning along the phloem path, AtSUC2 cDNA was expressed from tissue-specific promoters in an Atsuc2 mutant background. Suc transport in this mutant is highly compromised, as indicated by stunted growth and the accumulation of large quantities of sugar and starch in vegetative tissues. Expression of AtSUC2 cDNA from the 2-kb AtSUC2 promoter was sufficient to restore growth and carbon partitioning to nearly wild-type levels. The GALACTINOL SYNTHASE promoter of Cucumis melo (CmGAS1p) confers expression only in the minor veins of mature leaves, not in the transport phloem of larger leaf veins and stems. Mutant plants expressing AtSUC2 cDNA from CmGAS1p had intermediate growth and accumulated sugar and starch, but otherwise they had normal morphology. These characteristics support a role for AtSUC2 in retrieval but not efflux along the transport phloem and show that the only vital function of AtSUC2 in photoassimilate distribution is phloem loading. In addition, Atsuc2 mutant plants, although debilitated, do grow, and AtSUC2-independent modes of phloem transport are discussed, including an entirely symplastic pathway from mesophyll cells to sink tissues.     

Functional reconstitution of SdcS, a Na+-coupled dicarboxylate carrier protein from Staphylococcus aureus

In Staphylococcus aureus, the transport of dicarboxylates is mediated in part by the Na+-linked carrier protein SdcS. This transporter is a member of the divalent-anion/Na+ symporter (DASS) family, a group that includes the mammalian Na+/dicarboxylate cotransporters NaDC1 and NaDC3. In earlier work, we cloned and expressed SdcS in Escherichia coli and found it to have transport properties similar to those of its eukaryotic counterparts (J. A. Hall and A. M. Pajor, J. Bacteriol. 187:5189-5194, 2005). Here, we report the partial purification and subsequent reconstitution of functional SdcS into liposomes. These proteoliposomes exhibited succinate counterflow activity, as well as Na+ electrochemical-gradient-driven transport. Examination of substrate specificity indicated that the minimal requirement necessary for transport was a four-carbon terminal dicarboxylate backbone and that productive substrate-transporter interaction was sensitive to substitutions at the substrate C-2 and C-3 positions. Further analysis established that SdcS facilitates an electroneutral symport reaction having a 2:1 cation/dicarboxylate ratio. This study represents the first characterization of a reconstituted Na+-coupled DASS family member, thus providing an effective method to evaluate functional, as well as structural, aspects of DASS transporters in a system free of the complexities and constraints associated with native membrane environments.     

Polyamine regulation of Na+/glucose symporter expression in LLC-PK1 cells

Addition of polyamines or their analogs to newly confluent LLC-PK₁ cells resulted in down-regulation of Na⁺-glucose transport (symport) activity. Polyamines prevented the induction of this symporter by the differentiation inducer hexamethylene bisacetamide (HMBA) but did not influence induction by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Partial depletion of endogenous polyamines after addition of α -difluoromethylornithine (DFMO) resulted in a 4 to 5-fold increase in symporter expression. Symporter induction by either HMBA or DFMO was inhibited by the protein kinase inhibitor H-7 but H-7 did not affect symporter induction by IBMX. Changes in symporter activity were accompanied by changes in levels of the 75 kD symporter subunit detected by Western blot. Cultures exposed to HMBA exhibited reduced levels of ornithine decarboxylase activity. Our results suggest that induction of symporter expression by HMBA may be mediated in part by its effects on polymine metabolism, and point to parallel roles of polyamines and cyclic AMP in regulating the expression of this physiologically important renal transport system. © 1993 Wiley-Liss, Inc.     

Functional integration of the HUP1 hexose symporter gene into the genome of C. reinhardtii: Impacts on biological H(2) production

Phototrophic organisms use photosynthesis to convert solar energy into chemical energy. In nature, the chemical energy is stored in a diverse range of biopolymers. These sunlight-derived, energy-rich biopolymers can be converted into environmentally clean and CO(2) neutral fuels. A select group of photosynthetic microorganisms have developed the ability to extract and divert protons and electrons derived from water to chloroplast hydrogenase(s) to produce molecular H(2) fuel. Here, we describe the development and characterization of C. reinhardtii strains, derived from the high H(2) production mutant Stm6, into which the HUP1 (hexose uptake protein) hexose symporter from Chlorella kessleri was introduced. The isolated cell lines can use externally supplied glucose for heterotrophic growth in the dark. More importantly, external glucose supply (1mM) was shown to increase the H(2) production capacity in strain Stm6Glc4 to approximately 150% of that of the high-H(2) producing strain, Stm6. This establishes the foundations for a new fuel production process in which H(2)O and glucose can simultaneously be used for H(2) production. It also opens new perspectives on future strategies for improving bio-H(2) production efficiency under natural day/night regimes and for using sugar waste material for energy production in green algae as photosynthetic catalysts.     

人钠/碘同向转运体基因cDNA的克隆与序列测定

为研究人钠／碘同向转运体(hNIS)的生物学性能和用于肿瘤放射性碘治疗的可能性,运用反转录聚合酶链反应(RT—PCR)从人甲状腺组织总RNA中扩增出hNIS基因cDNA序列,将其克隆至pUCm-T载体中。序列分析证实克隆片段与献报道的hNIS基因cDNA序列完全一致,说明已成功克隆到hNIS基因cDNA。     

Expression of the thyroid sodium/iodide symporter in Xenopus laevis oocytes

Poly(A+) RNA isolated from FRTL-5 cells (a continuous line of cultured and fully functional rat thyroid cells (Ambesi-Impiombato, F. S., Parks, L. A. M., and Coons, H. G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 3455-3459] was injected into Xenopus laevis oocytes, and the expression of the Na+/I- symporter in the plasma membrane was assayed by measuring the Na+-dependent ClO4--sensitive uptake of 125I. Expression of the Na+/I- symporter was detected as a 7-fold average increase in transport over background, 5-6 days after injection. Poly(A+) RNA was subsequently fractionated by sucrose gradient centrifugation, and fractions were assayed for their ability to induce I- transport activity. The poly(A+) RNA encoding the Na+/I- symporter was found in a fraction containing messages of 2.8-4.0 kilobases in length.