Cloning of the human thiamine transporter, a member of the folate transporter family.

We have isolated a cDNA from human placenta, which, when expressed heterologously in mammalian cells, mediates the transport of the water-soluble vitamin thiamine. The cDNA codes for a protein of 497 amino acids containing 12 putative transmembrane domains. Northern blot analysis indicates that this transporter is widely expressed in human tissues. When expressed in HeLa cells, the cDNA induces the transport of thiamine (K(t) = 2.5 +/- 0.6 microM) in a Na(+)-independent manner. The cDNA-mediated transport of thiamine is stimulated by an outwardly directed H(+) gradient. Substrate specificity assays indicate that the transporter is specific to thiamine. Even though thiamine is an organic cation, the cDNA-induced thiamine transport is not inhibited by other organic cations. Similarly, thiamine is not a substrate for the known members of mammalian organic cation transporter family. The thiamine transporter gene, located on human chromosome 1q24, consists of 6 exons and is most likely the gene defective in the metabolic disorder, thiamine-responsive megaloblastic anemia. At the level of amino acid sequence, the thiamine transporter is most closely related to the reduced-folate transporter and thus represents the second member of the folate transporter family.     

A Na+/Cl- -coupled GABA transporter, GAT-1, from Caenorhabditis elegans: structural and functional features, specific expression in GABA-ergic neurons, and involvement in muscle function

GABA functions as an inhibitory neurotransmitter in body muscles and as an excitatory neurotransmitter in enteric muscles in Caenorhabditis elegans. Whereas many of the components of the GABA-ergic neurotransmission in this organism have been identified at the molecular and functional levels, no transporter specific for this neurotransmitter has been identified to date. Here we report on the cloning and functional characterization of a GABA transporter from C. elegans (ceGAT-1) and on the functional relevance of the transporter to the biology of body muscles and enteric muscles. ceGAT-1 is coded by snf-11 gene, a member of the sodium-dependent neurotransmitter symporter gene family in C. elegans. The cloned ceGAT-1 functions as a Na(+)/Cl(-)-coupled high-affinity transporter selective for GABA with a K(t) of approximately 15 microm. The Na(+):Cl(-):GABA stoichiometry for ceGAT-1-mediated transport process is 2:1:1. The transport process is electrogenic as evidenced from GABA-induced inward currents in Xenopus laevis oocytes that express ceGAT-1 heterologously. The transporter is expressed exclusively in GABA-ergic neurons and in two other additional neurons. We also investigated the functional relevance of ceGAT-1 to the biology of body muscles and enteric muscles by ceGAT-1-specific RNA interference (RNAi) in rrf-3 mutant, a strain of C. elegans in which neurons are not refractory to RNAi as in the wild type strain. The down-regulation of ceGAT-1 by RNAi leads to an interesting phenotype associated with altered function of body muscles (as evident from changes in thrashing frequency) and enteric muscles (as evident from the rates of defecation failure) and also with altered sensitivity to aldicarb-induced paralysis. These findings provide unequivocal evidence for a modulatory role of GABA and ceGAT-1 in the biology of cholinergic neurons and in the function of body muscles and enteric muscles in this organism.     

Cloning of an amino acid transporter with functional characteristics and tissue expression pattern identical to that of system A

We report here on the cloning and functional characterization of the protein responsible for the system A amino acid transport activity that is known to be expressed in most mammalian tissues. This transporter, designated ATA2 for amino acid transporter A2, was cloned from rat skeletal muscle. It is distinct from the neuron-specific glutamine transporter (GlnT/ATA1). Rat ATA2 consists of 504 amino acids and bears significant homology to GlnT/ATA1 and system N (SN1). ATA2-specific mRNA is ubiquitously expressed in rat tissues. When expressed in mammalian cells, ATA2 mediates Na(+)-dependent transport of alpha-(methylamino)isobutyric acid, a specific model substrate for system A. The transporter is specific for neutral amino acids. It is pH-sensitive and Li(+)-intolerant. The Na(+):amino acid stoichiometry is 1:1. When expressed in Xenopus laevis oocytes, transport of neutral amino acids via ATA2 is associated with inward currents. The substrate-induced current is Na(+)-dependent and pH-sensitive. The amino acid transport system A is particularly known for its adaptive and hormonal regulation, and therefore the successful cloning of the protein responsible for this transport activity represents a significant step toward understanding the function and expression of this transporter in various physiological and pathological states.     

Functional expression of a high affinity mammalian hepatic choline/organic cation transporter

Uptake by the liver of the organic cation and essential nutrient choline is required for the hepatic synthesis of phosphatidylcholine. Uptake of other organic cations is also important for the metabolism and secretion of numerous endobiotics and drugs. Although a high affinity mammalian hepatic choline transporter has been kinetically defined, it has not been previously identified. We have developed stable transfectants of BALB/3T3 cells, using a murine member of the organic cation transporter gene family (mOct1/Slc22a1), and used these cells to characterize the transport of the organic cation choline and model organic cation tetraethylammonium (TEA). Functional expression of mOct1/Slc22a1 in BALB/3T3 cells confers the saturable, temperature-dependent uptake of choline with a K(m) of 42 micrometer, and uptake of TEA with a K(m) of 43 micrometer. We subsequently used our cell culture uptake system to kinetically define in HepG2 cells a high affinity choline uptake process, which transports choline with a K(m) similar to that of mOct1/Slc22a1 protein. We also demonstrated that organic cation transport by mOct1/Slc22a1 is inhibited by several organic cations, and that the gene is expressed in the perinatal period, at a time when phosphatidylcholine synthesis increases.We conclude that mOct1/Slc22a1 encodes a high affinity mammalian hepatic choline/organic cation transporter. This transporter may be important for hepatic phosphatidylcholine synthesis, and for the metabolism and secretion of many organic cationic drugs.     

Transport of choline and its relationship to the expression of the organic cation transporters in a rat brain microvessel endothelial cell line (RBE4)

The present study was undertaken to elucidate the functional characteristics of choline uptake and deduce the relationship between choline uptake and the expression of organic cation transporters in the rat brain microvessel endothelial cell line RBE4. Confluent RBE4 cells were found to express a high affinity choline uptake system. The system is Na(+)-independent and shows a Michaelis-Menten constant of approx. 20 microM for choline. The choline analogue hemicholinium-3 inhibits choline uptake in these cells with an inhibition constant of approx. 50 microM. The uptake system is also susceptible for inhibition by various organic cations, including 1-methyl-4-phenylpyridinium, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, clonidine, procainamide, and tetramethylammonium. The prototypical organic cation tetraethylammonium shows very little affinity for the choline uptake system in these cells. The inhibition of choline uptake by hemicholinium-3 is competitive. Northern analysis and RT-PCR show that these cells do not express the organic cation transporters OCT2 and OCT3. These cells do express, however, low levels of OCT1, but the functional characteristics of choline uptake in these cells are very different from the known properties of choline uptake via OCT1. The Na(+)-coupled high affinity choline transporter CHT1 is not expressed in these cells as evidenced by RT-PCR. This corroborates the Na(+)-independent nature of choline uptake in these cells. It is concluded that RBE4 cells express an organic cation transporter that is responsible for choline uptake in these cells and that this transporter is not identical to any of the organic cation transporters thus far identified at the molecular level in mammalian cells.     

F1Aalpha, a death receptor-binding protein homologous to the Caenorhabditis elegans sex-determining protein, FEM-1, is a caspase substrate that mediates apoptosis.

Apoptosis is an evolutionarily conserved process that is critical for tissue homeostasis and development including sex determination in essentially all multicellular organisms. Here, we report the cloning of an ankyrin repeat-containing protein, termed F1Aalpha, in a yeast two-hybrid screen using the cytoplasmic domain of Fas (CD95/APO-1) as bait. Amino acid sequence analysis indicates that F1Aalpha has extensive homology to the sex-determining protein FEM-1 of the Caenorhabditis elegans, which is required for the development of all aspects of the male phenotype. F1Aalpha associates with the cytoplasmic domains of Fas and tumor necrosis factor receptor 1, two prototype members of the "death receptor" family. The F1Aalpha protein also oligomerizes. Overexpression of F1Aalpha induces apoptosis in mammalian cells, and co-expression of Bcl-XL or the dominant negative mutants of either FADD or caspase-9 blocks this effect. Deletion analysis revealed the center region of F1Aalpha, including a cluster of five ankyrin repeats to be necessary and sufficient for maximum apoptotic activity, and the N-terminal region appears to regulate negatively this activity. Furthermore, F1Aalpha is cleaved by a caspase-3-like protease at Asp(342), and the cleavage-resistant mutant is unable to induce apoptosis upon overexpression. F1Aalpha is therefore a member of a growing family of death receptor-associated proteins that mediates apoptosis.     

Divergence in enzyme regulation between Caenorhabditis elegans and human tyrosine hydroxylase, the key enzyme in the synthesis of dopamine

TH (tyrosine hydroxylase) is the rate-limiting enzyme in the synthesis of catecholamines. The cat-2 gene of the nematode Caenorhabditis elegans is expressed in mechanosensory dopaminergic neurons and has been proposed to encode a putative TH. In the present paper, we report the cloning of C. elegans full-length cat-2 cDNA and a detailed biochemical characterization of the encoded CAT-2 protein. Similar to other THs, C. elegans CAT-2 is composed of an N-terminal regulatory domain followed by a catalytic domain and a C-terminal oligomerization domain and shows high substrate specificity for L-tyrosine. Like hTH (human TH), CAT-2 is tetrameric and is phosphorylated at Ser35 (equivalent to Ser40 in hTH) by PKA (cAMP-dependent protein kinase). However, CAT-2 is devoid of characteristic regulatory mechanisms present in hTH, such as negative co-operativity for the cofactor, substrate inhibition or feedback inhibition exerted by catecholamines, end-products of the pathway. Thus TH activity in C. elegans displays a weaker regulation in comparison with the human orthologue, resembling a constitutively active enzyme. Overall, our data suggest that the intricate regulation characteristic of mammalian TH might have evolved from more simple models to adjust to the increasing complexity of the higher eukaryotes neuroendocrine systems.     

Structural and functional characteristics of two sodium-coupled dicarboxylate transporters (ceNaDC1 and ceNaDC2) from Caenorhabditis elegans and their relevance to life span

We have cloned and functionally characterized two Na(+)-coupled dicarboxylate transporters, namely ceNaDC1 and ceNaDC2, from Caenorhabditis elegans. These two transporters show significant sequence homology with the product of the Indy gene identified in Drosophila melanogaster and with the Na(+)-coupled dicarboxylate transporters NaDC1 and NaDC3 identified in mammals. In a mammalian cell heterologous expression system, the cloned ceNaDC1 and ceNaDC2 mediate Na(+)-coupled transport of various dicarboxylates. With succinate as the substrate, ceNaDC1 exhibits much lower affinity compared with ceNaDC2. Thus, ceNaDC1 and ceNaDC2 correspond at the functional level to the mammalian NaDC1 and NaDC3, respectively. The nadc1 and nadc2 genes are not expressed at the embryonic stage, but the expression is detectable all through the early larva stage to the adult stage. Tissue-specific expression pattern studies using a reporter gene fusion approach in transgenic C. elegans show that both genes are coexpressed in the intestinal tract, an organ responsible for not only the digestion and absorption of nutrients but also for the storage of energy in this organism. Independent knockdown of the function of these two transporters in C. elegans using the strategy of RNA interference suggests that NaDC1 is not associated with the regulation of average life span in this organism, whereas the knockdown of NaDC2 function leads to a significant increase in the average life span. Disruption of the function of the high affinity Na(+)-coupled dicarboxylate transporter NaDC2 in C. elegans may lead to decreased availability of dicarboxylates for cellular production of metabolic energy, thus creating a biological state similar to that of caloric restriction, and consequently leading to life span extension.     

Functional characterization of Caenorhabditis elegans heteromeric amino acid transporters

Mammalian heteromeric amino acid transporters (HATs) are composed of a multi-transmembrane spanning catalytic protein covalently associated with a type II glycoprotein (e.g. 4F2hc, rBAT) through a disulfide bond. Caenorhabditis elegans has nine genes encoding close homologues of the HAT catalytic proteins. Three of these genes (designated AAT-1 to AAT-3) have a much higher degree of similarity to the mammalian homologues than the other six, including the presence of a cysteine residue at the position known to form a disulfide bridge to the glycoprotein partner in mammalian HATs. C. elegans also has two genes encoding homologues of the heteromeric amino acid transporter type II glycoprotein subunits (designated ATG-1 and ATG-2). Both ATG, and/or AAT-1, -2, -3 proteins were expressed in Xenopus oocytes and tested for amino acid transport function. This screen revealed that AAT-1 and AAT-3 facilitate amino acid transport when expressed together with ATG-2 but not with ATG-1 or the mammalian type II glycoproteins 4F2hc and rBAT. AAT-1 and AAT-3 covalently bind to both C. elegans ATG glycoproteins, but only the pairs with ATG-2 traffic to the oocyte surface. Both of these functional, surface-expressed C. elegans HATs transport most neutral amino acids and display the highest transport rate for l-Ala and l-Ser (apparent K(m) 100 microm range). Similar to their mammalian counterparts, the C. elegans HATs function as (near) obligatory amino acid exchangers. Taken together, this study demonstrates that the heteromeric structure and the amino acid exchange function of HATs have been conserved throughout the evolution of nematodes to mammals.     

The Caenorhabditis elegans sex determination protein FEM-1 is a CED-3 substrate that associates with CED-4 and mediates apoptosis in mammalian cells

Sex-specific elimination of cells by apoptosis plays a role in sex determination in Caenorhabditis elegans. Recently, a mammalian pro-apoptotic protein named F1Aalpha has been identified. F1Aalpha shares extensive homology throughout the entire protein with the C. elegans protein, FEM-1, which is essential for achieving all aspects of the male phenotype in the nematode. In this report, the role of FEM-1 in apoptosis was investigated. Overexpression of FEM-1 induces caspase-dependent apoptosis in mammalian cells. FEM-1 is cleaved in vitro by the C. elegans caspase, CED-3, generating an N-terminal cleavage product that corresponds to the minimal effector domain for apoptosis. Furthermore, CED-4 associates with FEM-1 in vitro and in vivo in mammalian cells and potentiates FEM-1-mediated apoptosis. Similarly, Apaf-1, the mammalian homologue of CED-4 was found to associate with F1Aalpha. These data suggest that FEM-1 and F1Aalpha may mediate apoptosis by communicating directly with the core machinery of apoptosis.     

Nramp2 expression is associated with pH-dependent iron uptake across the apical membrane of human intestinal Caco-2 cells

The absorption of dietary non-heme iron by intestinal enterocytes is crucial to the maintenance of body iron homeostasis. This process must be tightly regulated since there are no distinct mechanisms for the excretion of excess iron from the body. An insight into the cellular mechanisms has recently been provided by expression cloning of a divalent cation transporter (DCT1) from rat duodenum and positional cloning of its human homologue, Nramp2. Here we demonstrate that Nramp2 is expressed in the apical membrane of the human intestinal epithelial cell line, Caco 2 TC7, and is associated with functional iron transport in these cells with a substrate preference for iron over other divalent cations. Iron transport occurs by a proton-dependent mechanism, exhibiting a concurrent intracellular acidification. Taken together, these data suggest that the expression of the Nramp2 transporter in human enterocytes may play an important role in intestinal iron absorption.     

Molecular cloning and characterization of a novel mammalian endo-apyrase (LALP1)

Here we describe the cloning, localization, and characterization of a novel mammalian endo-apyrase (LALP1) in human and mouse. The predicted human LALP1 gene encodes a 604-amino acid protein, whereas the mouse Lalp1 gene encodes a 606-amino acid protein. The human and mouse genes have 88% amino acid sequence identity. These genes share considerable homologies with hLALP70, a recently discovered mammalian lysosomal endo-apyrase. The human LALP1 gene resides on chromosome 10q23-q24 and contains 12 exons and 11 introns covering a genomic region of approximately 46 kilobase pairs. The subcellular localization and enzymatic activity of LALP1 indicated that LALP1 is indeed an endo-apyrase with substrate preference for nucleoside triphosphates UTP, GTP, and CTP.     

A novel neutral amino acid transporter from the hyperthermophilic archaeon Thermococcus sp. KS-1

A novel gene encoding a small neutral amino acid transporter was cloned from the genome of the hyperthermophilic archaeon Thermococcus sp. KS-1 by functional cloning using Escherichia coli strain AK430, which is defective in transporting glycine and D-alanine. The cloned gene, snatA, encoded a protein of 216 amino acid residues, SnatA, and was predicted to be a membrane protein with six membrane-spanning segments. E. coli AK430 cells transformed with snatA transported glycine with an apparent K(t) value of 24 micro M, which was one order of magnitude higher than that of other known glycine/alanine transporters, including cycA of E. coli and acp of thermophilic bacterium PS3. Competition studies revealed that SnatA transported various L-type neutral amino acids, but its substrate specificity was different from that of CycA or ACP. The glycine transport was inhibited by a protonophore, FCCP, or valinomycin plus nigericin, indicating that the process is dependent on an electrochemical potential of H(+). Homology searches revealed no homology with any transporters known to date. However, several hypothetical genes in prokaryote cells enrolled in the gene bank showed significantly high homology scores, indicating that snatA and its homologues form a family of prokaryotes. To our knowledge, this is the first report on the cloning of a gene of an amino acid transporter from a hyperthermophilic archaeon.     

Cloning and characterization of the rabbit POU5F1 gene.

The product of the POUSF1 gene, Oct4, plays an important role both in embryonic development and in the self-renewal and differentiation of totipotent cells. To understand the function of Oct4 in rabbit ES cells, we cloned and sequenced the rabbit POU5F1 gene, as well as the cDNA encoded by the gene. The Oct4 cDNA contains a 1083 bp ORF encoding a 360 aa protein and a 241 bp 3' UTR sequence. Oct4 mRNA was expressed at a high level in rabbit ES cells and was barely detectable in the adult spleen, kidney, brain and muscle tissues. The POU5F1 gene is approximately 6 kb in length and includes five exons and four introns. Gene organization is similar to that of the mouse, human and bovine orthologs. Sequencing of the gene revealed an 82% (mouse), 90% (human) and 89% (bovine) overall identity at the protein level. The rabbit POUSF1 gene was mapped to chromosome 12q1.1 by PCR amplification of DNA from two putative POU5F1-containing BAC clones, which were previously mapped to chromosome 12q1.1. The cloning of the rabbit POU5F1 gene will facilitate studies on its roles in rabbit embryogenesis and ES cells.     

Monosaccharide transporters in plants: structure, function and physiology

Monosaccharide transport across the plant plasma membrane plays an important role both in lower and higher plants. Algae can switch between phototrophic and heterotrophic growth and utilize organic compounds, such as monosaccharides as additional or sole carbon sources. Higher plants represent complex mosaics of phototrophic and heterotrophic cells and tissues and depend on the activity of numerous transporters for the correct partitioning of assimilated carbon between their different organs. The cloning of monosaccharide transporter genes and cDNAs identified closely related integral membrane proteins with 12 transmembrane helices exhibiting significant homology to monosaccharide transporters from yeast, bacteria and mammals. Structural analyses performed with several members of this transporter superfamily identified protein domains or even specific amino acid residues putatively involved in substrate binding and specificity. Expression of plant monosaccharide transporter cDNAs in yeast cells and frog oocytes allowed the characterization of substrate specificities and kinetic parameters. Immunohistochemical studies, in situ hybridization analyses and studies performed with transgenic plants expressing reporter genes under the control of promoters from specific monosaccharide transporter genes allowed the localization of the transport proteins or revealed the sites of gene expression. Higher plants possess large families of monosaccharide transporter genes and each of the encoded proteins seems to have a specific function often confined to a limited number of cells and regulated both developmentally and by environmental stimuli.     

Cloning and functional characterization of a cocaine-sensitive dopamine transporter.

We report the cloning of a rat cDNA encoding a functional dopamine transporter. This cDNA, derived from an intron-containing gene, encodes a protein of 620 amino acids. Hydropathicity analysis of the protein sequence suggests the presence of 12 putative transmembrane domains. The protein displays considerable identity with transporters for noradrenaline and GABA (64 and 30%, respectively). Transient expression of the cDNA in COS7 cells directs the expression of dopamine uptake activity with appropriate pharmacology and in a sodium-dependent fashion. In situ hybridization reveals that the mRNA for this transporter is expressed in the substantia nigra and ventral tegmental area, regions that contain dopaminergic cell bodies.     

The human OCTN1 (SLC22A4) reconstituted in liposomes catalyzes acetylcholine transport which is defective in the mutant L503F associated to the Crohn's disease

The organic cation transporter (OCTN1) plays key roles in transport of selected organic cations, but understanding of its biological functions remains limited by restricted knowledge of its substrate targets. Here we show capacity of human OCTN1-reconstituted proteoliposomes to mediate uptake and efflux of [(3)H]acetylcholine, the Km of transport being 1.0mM with V(max) of 160nmol?mg(-1)protein?min(-1). OCTN1-mediated transport of this neurotransmitter was time-dependent and was stimulated by intraliposomal ATP. The transporter operates as uniporter but translocates acetylcholine in both directions. [(3)H]acetylcholine uptake was competitively inhibited by tetraethylammonium, γ-butyrobetaine and acetylcarnitine, and was also inhibited by various polyamines. Decreasing intraliposomal ATP concentrations increased OCTN Km for acetylcholine, but V(max) was unaffected. Evaluation of the acetylcholine transporter properties of a variant form of OCTN1, the Crohn's disease-associated 503F variant, revealed time course, Km and V(max) for acetylcholine uptake to be comparable to that of wild-type OCTN1. Km for acetylcholine efflux was also comparable for both OCTN1 species, but V(max) of OCTN1 503F-mediated acetylcholine efflux (1.9nmol?mg(-1)protein?min(-1)) was significantly lower than that of wild-type OCTN1 (14nmol?mg(-1)protein?min(-1)). These data identify a new transport role for OCTN1 and raise the possibility that its involvement in the non-neuronal acetylcholine system may be relevant to the pathogenesis of Crohn's disease.     

Evolution of the Na-P(i) cotransport systems

Membrane transport systems for P(i) transport are key elements in maintaining homeostasis of P(i) in organisms as diverse as bacteria and human. Two Na-P(i) cotransporter families with well-described functional properties in vertebrates, namely NaPi-II and NaPi-III, show conserved structural features with prokaryotic origin. A clear vertical relationship can be established among the mammalian protein family NaPi-III, a homologous system in C. elegans, the yeast system Pho89, and the bacterial P(i) transporter Pit. An alternative lineage connects the mammalian NaPi-II-related transporters with homologous proteins from Caenorhabditis elegans and Vibrio cholerae. The present review focuses on the molecular evolution of the NaPi-II protein family. Preliminary results indicate that the NaPi-II homologue cloned from V. cholerae is indeed a functional P(i) transporter when expressed in Xenopus oocytes. The closely related NaPi-II isoforms NaPi-IIa and NaPi-IIb are responsible for regulated epithelial Na-dependent P(i) transport in all vertebrates. Most species express two different NaPi-II proteins with the exception of the flounder and Xenopus laevis, which rely on only a single isoform. Using an RT-PCR-based approach with degenerate primers, we were able to identify NaPi-II-related mRNAs in a variety of vertebrates from different families. We hypothesize that the original NaPi-IIb-related gene was duplicated early in vertebrate development. The appearance of NaPi-IIa correlates with the development of the mammalian nephron.