Structure,function and regulation of the tricarboxylate transport protein from rat liver mitochondria

Recent progress is summarized on the structure, function, and regulation of the tricarboxylate (i.e., citrate) transport protein (CTP) from the rat liver mitochondrial inner membrane. The transporter has been purified and its reconstituted function characterized. A cDNA clone encoding the CTP has been isolated and sequenced, thus enabling a deduction of the complete amino acid sequence of this 32.6 kDa transport protein. Dot matrix analysis and sequence alignment indicate that based on structural considerations the CTP can be assigned to the mitochondrial carrier family. Hydropathy analysis of the transporter sequence indicates six putative membrane-spanning -helices and has permitted the development of an initial model for the topography of the CTP within the inner membrane. The questions as to whether more than one gene encodes the CTP and whether more than one isoform is expressed remain unanswered at this time. Studies documenting a diabetes-induced alteration in the function of several mitochondrial anion transporters, which can be reversed by treatment with insulin, provide a physiologically/pathologically relevant experimental system for studying the molecular mechanism(s) by which mitochondrial transporters are regulated. Potential future research directions are discussed.     

Amino acid neurotransmitter transporters: Structure,function, and molecular diversity

Many biologically active compounds including neurotransmitters, metabolic precursors, and certain drugs are accumulated intracellularly by transporters that are coupled to the transmembrane Na⁺ gradient. Amino acid neurotransmitter transporters play a key role in the regulation of extracellular amino acid concentrations and termination of neurotransmission in the CNS

1 Abbreviations: CNS, central nervous system; GABA, γ-aminobutyric acid; cDNA, complementary deoxyribonucleic acid; mRNA, messenger ribonucleic acid; NMDA, N-methyl-D-aspartate; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; DAG, diacyl glycerol; R59022, DAG kinase inhibitor; AA, arachidonic acid; ACHC, cis-3-aminocyclohexanecarboxylic acid; GAT-A, ACHC-sensitive GABA transporter; GAT-B, β-alanine-sensitive GABA transporter; GLY-1 and GLYT-1, glycine transporters; PROT-1, proline transporter; BGT-1, betaine transporter.

. Transporters for the major amino acid neurotransmitters glutamate, GABA, and glycine are found in both neurons and glial cells. Recent work has resulted in the identification of cDNAs encoding several amino acid neurotransmitter transport proteins, all of which belong to the Na⁺-and Cl^?-dependent transporter gene family. The diversity of this family suggests a degree of transporter heterogeneity that is greater than that indicated by biochemical and pharmacological studies.     

PC12 variants deficient in norepinephrine transporter mRNA have wild type activities of several other related transporters

Wild type PC12 pheochromocytoma cells express a Na⁺-dependent norepinephrine transporter that operates in the uptake of catecholamines. In addition to the previously described Na⁺-dependent system A for the uptake of -amino-isobutyric acid and system Gly for glycine, we have identified two other Na⁺-dependent transporter systems for amino acid uptake in these cells: 1) system for -alanine and taurine; and 2) a system for creatine. Uptake of -amino-isobutyric acid, glycine, -alanine, and creatine is not affected in some PC12 variants that were previously shown to be deficient in catecholamine uptake and to have decreased levels of norepinephrine transporter mRNA. We have isolated two PC12 cDNA clones that are essentially identical in sequence to recently reported cDNAs for rat brain taurine and creatine transporters, respectively, and a third cDNA that appears to code for a novel transporter. mRNAs for these three transporters are present at wild type levels in those variants that express no or little norepinephrine transporter mRNA. These results support the notion that the expression of catecholamine reuptake transporters may be particularly susceptible to down-regulation.     

Phosphate transport in mitochondria: Past accomplishments,present problems,and future challenges

The requirement of inorganic phosphate (P_i) for oxidative phosphorylation in eukaryotic cells is fulfilled through specific P_i transport systems. The mitochondrial proton/phosphate symporter (P_ic) is a membrane-embedded protein which translocates P_i from the cytosol into the mitochondrial matrix. P_ic is responsible for the very rapid transport of most of the P_i used in ATP synthesis. During the past five years there have been advances on several fronts. Genomic and cDNA clones for yeast, bovine, rat, and human P_ic have been isolated and sequenced. Functional expression of yeast P_ic in yeast strains deficient in P_i transport and expression inEscherichia coli of a chimera protein involving P_ic and ATP synthase subunit have been accomplished. P_ic, in contrast to other members of the family of transporters involved in energy metabolism, was demonstrated to have a presequence, which optimizes the import of the precursor protein into mitochondria. Six transmembrane segments appear to be a structural feature shared between P_ic and other mitochondrial anion carriers, and recent-site directed mutagenesis studies implicate structure-functional relationships to bacteriorhodopsin. These recent advances on P_ic will be assessed in light of a more global interpretation of transport mechanism across the inner mitochondrial membrane.     

Hexose transporters of tomato: molecular cloning,expression analysis and functional characterization

A full-length (LeHT2) and two partial (LeHT1 and LeHT3) cDNA clones, encoding hexose transporters, were isolated from tomato (Lycopersicon esculentum) fruit and flower cDNA libraries. Southern blot analysis confirmed the presence of a gene family of hexose transporters in tomato consisting of at least three members. The full-length cDNA (LeHT2) encodes a protein of 523 amino acids, with a calculated molecular mass of 57.6 kDa. The predicted protein has 12 putative membrane-spanning domains and belongs to the Major Facilitator Superfamily of membrane carriers. The three clones encode polypeptides that are homologous to other plant monosaccharide transporters and contain conserved amino acid motifs characteristic of this superfamily. Expression of the three genes in different organs of tomato was investigated by quantitative PCR. LeHT1 and LeHT3 are expressed predominantly in sink tissues, with both genes showing highest expression in young fruit and root tips. LeHT2 is expressed at relatively high levels in source leaves and certain sink tissues such as flowers. LeHT2 was functionally expressed in a hexose transport-deficient mutant (RE700A) of Saccharomyces cerevisiae. LeHT2-dependent transport of glucose in RE700A exhibited properties consistent with the operation of an energy-coupled transporter and probably a H⁺/hexose symporter. The K _m of the symporter for glucose is 45 M.     

The S7 ribosomal protein gene is truncated and overlaps a cytochrome c biogenesis gene in pea mitochondria

The pea mitochondrial genome contains a truncated rps7 gene lacking ca. 40 codons at its 5 terminus. This single-copy sequence is immediately downstream of and slightly overlapping an actively transcribed and edited reading frame of 744 bp (designated ccb248) homologous to the bacterial helC gene which encodes a subunit of the ABC-type heme transporter involved in cytochrome c biogenesis. This region of mitochondrial DNA appears recombinogenic, and the carboxy-termini of helC-type proteins are predicted to vary in sequence and length among plants. Sequences corresponding to the 5 coding region of rps7 were not detected elsewhere in the pea mitochondrial genome using wheat rps7 probes, and only a very short internal rps7 segment was observed in soybean mitochondrial DNA. The presence of rps7-homologous sequences in the nuclear genomes of pea and soybean is consistent with the recent transfer of a functional mitochondrial rps7 gene to the nucleus in certain plant lineages.     

Energy-linked anion transport. Cloning, sequencing, and characterization of a full length cDNA encoding the rat liver mitochondrial proton/phosphate symporter

A full length cDNA clone encoding the precursor of the rat liver mitochondrial phosphate transporter (H+/Pi symporter) has been isolated from a cDNA library using a bovine heart partial length phosphate transporter clone as a hybridization probe. The entire clone is 1263 base pairs in length with 5'- and 3'-untranslated regions of 16 and 168 base pairs, respectively. The open reading frame encodes for the mature protein (312 amino acids) preceded by a presequence of 44 amino acids enriched in basic residues. The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the first 17 amino-terminal amino acids of the pure phosphate transporter protein. The rat liver phosphate transporter differs from the bovine heart transporter in 32 amino acids (i.e. approximately 10%). It contains a region from amino acid 139 to 159 which is 37% identical with the beta-subunit of the liver mitochondrial ATP synthase. Amino acid sequence comparisons of the Pi transporter with Pi binding proteins, other H+-linked symporters, and the human glucose transporter did not reveal significant sequence homology. Analysis of genomic DNA from both rat and S. cerevisiae by Southern blots using the rat liver mitochondrial Pi carrier cDNA as a probe revealed remarkably similar restriction patterns, a finding consistent with the presence in lower and higher eukaryotes of homologous Pi carrier proteins. This is the first report of the isolation, sequencing, and characterization of a full length cDNA coding for a protein involved in energy-coupled Pi transport.     

Characterization of a Saccharomyces cerevisiae gene that encodes a mitochondrial phosphate transporter-like protein

The mitochondrial phosphate transporter of Saccharomyces cerevisiae, encoded by MIR1 (YJR077C) gene, shows divergence among the transporters in various eukaryotes. We have characterized another gene, YER053C, that appeared to encode an orthologous mitochondrial phosphate transporter of yeast. The predicted amino acid sequence of the YER053C protein is much more similar to that of mitochondrial phosphate transporters of other species than that of MIR1. RNA gel blot analysis indicated that, like the MIR1 promoter, the YER053C promoter is functional and that its activity varies according to aeration. An MIR1 gene null mutant did not grow on glycerol medium, whereas a YER053C null mutant grew well on the medium, suggesting that the YER053C gene is not essential for the mitochondrial function. YER053C also did not support the growth of the MIR1 null mutant on glycerol. The MIR1 and YER053C proteins were expressed in Escherichia coli and then reconstituted into liposomes. Unlike the proteoliposomes of MIR1, those of YER053C did not exhibit significant phosphate transport activity. Unexpectedly, it was shown that YER053C is localized in vacuoles, not mitochondria, by immunological electron microscopy. These results suggest that, during evolution, yeast lost the function and/or mitochondrial targeting of YER053C and then recruited an atypical MIR1 as the only transporter.     

大豆磷、硫转运蛋白研究进展

磷、硫转运蛋白是大豆（Glycine max（L.）Merr.）体内磷、硫转运的重要载体,参与调节磷和硫酸盐的吸收与转运,对提高大豆的磷、硫利用效率至关重要。大豆磷转运蛋白可划分为Pht1、Pht2、Pht3、Pho1和Pho2 5大家族,目前对Pht1的研究最为深入。大豆14个Pht1家族可分为3个亚家族,他们对磷吸收和转运具有重要作用。大豆硫转运蛋白基因GmSULTR1;2b可在大豆根中特异性表达并被低硫胁迫诱导。本文基于大豆磷、硫的营养吸收、转运与利用过程中的相关性,对Pht1家族以及GmSULTR1;2b基因在大豆中的研究进展进行了综述,并对近年来大豆磷、硫转运蛋白的研究进展及未来的研究方向进行了展望。     

Uptake of Selenite by Saccharomyces cerevisiae Involves the High and Low Affinity Orthophosphate Transporters

Although the general cytotoxicity of selenite is well established, the mechanism by which this compound crosses cellular membranes is still unknown. Here, we show that in Saccharomyces cerevisiae, the transport system used opportunistically by selenite depends on the phosphate concentration in the growth medium. Both the high and low affinity phosphate transporters are involved in selenite uptake. When cells are grown at low P_i concentrations, the high affinity phosphate transporter Pho84p is the major contributor to selenite uptake. When phosphate is abundant, selenite is internalized through the low affinity P_i transporters (Pho87p, Pho90p, and Pho91p). Accordingly, inactivation of the high affinity phosphate transporter Pho84p results in increased resistance to selenite and reduced uptake in low P_i medium, whereas deletion of SPL2, a negative regulator of low affinity phosphate uptake, results in exacerbated sensitivity to selenite. Measurements of the kinetic parameters for selenite and phosphate uptake demonstrate that there is a competition between phosphate and selenite ions for both P_i transport systems. In addition, our results indicate that Pho84p is very selective for phosphate as compared with selenite, whereas the low affinity transporters discriminate less efficiently between the two ions. The properties of phosphate and selenite transport enable us to propose an explanation to the paradoxical increase of selenite toxicity when phosphate concentration in the growth medium is raised above 1 mm.     

The role of membrane transport in metabolic engineering of plant primary metabolism

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Highlights► The plastidial pyruvate transporter in plants is driven by sodium symport. ► The mitochondrial pyruvate transporter MPC has been identified. ► All transporters needed for a synthetic PEP-CK/C₄ photosynthetic cycle are now known. ► The SWEET proteins define a new class of sugar efflux carriers in plants.     

Phosphate transport and sensing in Saccharomyces cerevisiae.

Cellular metabolism depends on the appropriate concentration of intracellular inorganic phosphate; however, little is known about how phosphate concentrations are sensed. The similarity of Pho84p, a high-affinity phosphate transporter in Saccharomyces cerevisiae, to the glucose sensors Snf3p and Rgt2p has led to the hypothesis that Pho84p is an inorganic phosphate sensor. Furthermore, pho84Delta strains have defects in phosphate signaling; they constitutively express PHO5, a phosphate starvation-inducible gene. We began these studies to determine the role of phosphate transporters in signaling phosphate starvation. Previous experiments demonstrated a defect in phosphate uptake in phosphate-starved pho84Delta cells; however, the pho84Delta strain expresses PHO5 constitutively when grown in phosphate-replete media. We determined that pho84Delta cells have a significant defect in phosphate uptake even when grown in high phosphate media. Overexpression of unrelated phosphate transporters or a glycerophosphoinositol transporter in the pho84Delta strain suppresses the PHO5 constitutive phenotype. These data suggest that PHO84 is not required for sensing phosphate. We further characterized putative phosphate transporters, identifying two new phosphate transporters, PHO90 and PHO91. A synthetic lethal phenotype was observed when five phosphate transporters were inactivated, and the contribution of each transporter to uptake in high phosphate conditions was determined. Finally, a PHO84-dependent compensation response was identified; the abundance of Pho84p at the plasma membrane increases in cells that are defective in other phosphate transporters.     

Arabidopsis thaliana Atvsp is homologous to soybean VspA and VspB,genes encoding vegetative storage protein acid phosphatases,and is regulated similarly by methyl jasmonate,wounding, sugars,light and phosphate

The soybean vegetative storage proteins, VSP and VSP, are acid phosphatases that accumulate to very high levels in hypocotyls, young leaves and flowers and pods. The genes encoding the soybean VSP are activated by jasmonate, wounding, sugars and light and down regulated by phosphate and auxin. In this study, expression of an Arabidopsis thaliana gene (Atvsp) encoding a protein homologous to soybean Vsp and Vsp, was examined and compared to expression of the soybean Vsp genes. Atvsp mRNA was present at high levels in flowers and buds and at low levels in roots, stems, leaves and siliques. Expression of Atvsp in leaves could be induced by wounding or by treatment of illuminated plants with methyl jasmonate and sucrose. Roots of plants with wounded leaves also accumulated Atvsp mRNA indicating that this gene can be regulated by a transmissible wound signal. Phosphate partially inhibited expression of Atvsp. Arabidopsis proteins of 29 and 30 kDa crossreacted with antibodies against soybean VSP. These proteins were very abundant in flowers and the proteins accumulated in leaves and roots of plants treated with methyl jasmonate. The level of these proteins in flowers was similar to the levels of soybean VSP in young soybean leaves. Overall, these data indicate that Arabidopsis Atvsp and soybean VspA/B genes are regulated similarly and that in both plants, the gene products can accumulate to high levels. This suggests that genes homologous to VspA/B may be of greater general significance than previously recognized.     

Effect of the electrochemical proton gradient and anions on the ATPase activity of soybean submitochondrial particles

Submitochondrial particles from soybean (Glycine max L. cv Jupiter) hypocotyls with an ATPase activity of 0.3 to 1.0 micromole per minute per milligram were prepared by sonication with Mg-ATP. The particles catalyzed ATP synthesis with NADH and succinate; the ratios of ATP/O with these substrates were 1.0 and 0.1, respectively. As monitored by oxonol-VI, the particles built up and maintained a membrane potential that was higher with NADH than with succinate or Mg-ATP. The ATPase activity of the particles increased two to threefold by preincubation with 50 millimolar phosphate at a temperature of 38°C. The increase in ATPase activity became higher (five to sixfold) when particles were preincubated with Mg-ATP plus phosphate. Under the latter conditions, collapse of μH by carbonyl cyanide p-trifluoromethoxyphenylhydrazone prevented the activation. An increase in ATPase activity of the particles was also observed with NADH and succinate, although activation was lower with succinate. With these substrates, phosphate did not increase ATPase activation. When particles were preincubated with Mg-ATP, anions that stimulate ATP hydrolysis (malate, malonate, and bicarbonate) had an activating effect similar to that of phosphate. The data suggest that the soybean mitochondrial ATPase can be activated by μH but that this activation is increased by the binding of certain anions to a conformation of the enzyme that appears during hydrolytic cycles.     

Ectomycorrhizal symbiosis enhances tolerance to low phosphorous through expression of phosphate transporter genes in masson pine (<Emphasis Type="Italic">Pinus massoniana</Emphasis>)

Ectomycorrhizal symbiosis promotes the growth of masson pine (Pinus massoniana) in low-phosphorus (low-P) conditions; however, the mechanism underlying this phenomenon has not yet been fully described. Here, we cloned four members of the Pht1 phosphate transporter protein family (GenBank accession: AMR43649.1 to AMR43652.1) encoding phosphate transporters in masson pine (PmPTs) by rapid amplification of cDNA ends (RACE) and characterized them in Boletus edulis and Pisolithus tinctorius colonized plants under low-P stress. PmPT1 to PmPT4 encoded 548, 548, 535, 535 amino acid polypeptides, respectively, containing the typical domain of the Pi:H⁺ symporter (PHS-transporter). Homology multiple sequence alignment indicated that these PmPTs were highly similar to phosphate transporters from other species. The polypeptides were characterized by high hydropathicity and contained 12 putative intra-membrane regions and 1 cytoplasmic loop. The temporal and spatial expression profiles showed higher expression of these PmPTs in ectomycorrhiza (ECM)-inoculated plants compared to non-inoculated ones. In addition, expressions of these PmPT members shared a similar pattern and might be intensively activated by low-P stress or inhibited under P excess. Interestingly, the ECM-colonized plants accumulated more phosphate compared to non-ECM-colonized specimens when exposed to low-P. Therefore, enhanced low-P tolerance in ECM-inoculated masson pine to low-P stress was at least partially dependent on the up-regulation of phosphate transporter genes, reflecting that the intimate interaction between plants and ECM fungi resulted in the improvement of P nutrition.     

Molecular cloning and characterization of a tobacco leaf cDNA encoding a phosphate transporter

Phosphate acquired by roots is translocated to and utilized by the upper part of the plant, where the phosphate transport in the cell is also important in the phosphate metabolism. In order to study the role of the phosphate transporter in the regulation of the phosphate movement across the membranes in leaf cells, we isolated and characterized a 2,059 bp tobacco leaf cDNA clone, NtPT1. The 537 amino acid sequences, deduced from NtPT1, exhibited 93 and 91% identites to one of the high affinity phosphate transporters constitutively expressed in potato and tomato roots, respectively. The NtPT1 contains 12 membrane-spanning domain with a central hydrophilic region. The expression of NtPT1 in the yeast high affinity phosphate transporter mutant strain, NS219, complemented the mutant and promoted cell growth significantly. These results strongly suggest that NtPT1 encodes a functional phosphate transporter and that one of the high affinity phosphate transporters expressed in roots is also expressed in leaves. Southern analysis indicated that tobacco phosphate transporter genes are low copy number genes and members of a small multi-gene family.     

Mitochondrial phosphate transport. Import of the H+/Pi symporter and role of the presequence

The rat liver mitochondrial phosphate transporter contains a 44-amino acid presequence. The role of this presequence is not clear since the ADP/ATP carrier and the brown fat uncoupling protein, related members of a family of inner membrane anion transporters, lack a presequence and contain targeting information within the mature protein. Here, we present evidence that the rat liver mitochondrial phosphate transporter can be synthesized in vitro, imported into mitochondria, and processed to a protein of Mr 33,000. Import requires the membrane potential and external nucleotide triphosphate. The presequence inserts into the outer mitochondrial membrane, and import proceeds via a process similar to other proteins destined for the inner membrane or matrix. A mutant phosphate transporter lacking 35 amino acids at the NH2 terminus of the presequence has little capacity for mitochondrial import. The rat liver phosphate transporter is also imported and processed by rat kidney mitochondria and by mitochondria from the yeast Saccharomyces cerevisiae. A site-directed mutation of the N-ethyl-maleimide reactive cysteine 41 does not affect import or processing. The results presented show that optimal import of the mitochondrial phosphate transporter, unlike the ADP/ATP carrier and the brown fat uncoupling protein, is dependent on a presequence. As these carriers are believed to have evolved from a single gene, it seems likely that the H+/Pi carrier, known to be present in prokaryotes, appeared first and that subsequent evolutionary events leading to the other anion carriers eliminated the presequence.