Point mutations in a nucleoside transporter gene from Leishmania donovani confer drug resistance and alter substrate selectivity

Leishmania parasites lack a purine biosynthetic pathway and depend on surface nucleoside and nucleobase transporters to provide them with host purines. Leishmania donovani possess two closely related genes that encode high affinity adenosine-pyrimidine nucleoside transporters LdNT1.1 and LdNT1.2 and that transport the toxic adenosine analog tubercidin in addition to the natural substrates. In this study, we have characterized a drug-resistant clonal mutant of L. donovani (TUBA5) that is deficient in LdNT1 transport and consequently resistant to tubercidin. In TUBA5 cells, the LdNT1.2 genes had the same sequence as wild-type cells. However, because LdNT1.2 mRNA is not detectable in either wild-type or TUBA5 promastigotes, LdNT1.2 does not contribute to nucleoside transport in this stage of the life cycle. In contrast, the TUBA5 cells were compound heterozygotes at the LdNT1.1 locus containing two mutant alleles that encompassed distinct point mutations, each of which impaired transport function. One of the mutant LdNT1.1 alleles encoded a G183D substitution in predicted TM 5, and the other allele contained a C337Y change in predicted TM 7. Whereas G183D and C337Y mutants had only slightly elevated adenosine Km values, the severe impairment in transport resulted from drastically (≈20-fold) reduced Vmax values. Because these transporters were correctly targeted to the plasma membrane, the reduction in Vmax apparently resulted from a defect in translocation. Strikingly, G183 was essential for pyrimidine nucleoside but not adenosine transport. A mutant transporter with a G183A substitution had an altered substrate specificity, exhibiting robust adenosine transport but undetectable uridine uptake. These results suggest that TM 5 is likely to form part of the nucleoside translocation pathway in LdNT1.1

Glutamate antagonists limit tumor growth

Neuronal progenitors and tumor cells possess propensity to proliferate and to migrate. Glutamate regulates proliferation and migration of neurons during development, but it is not known whether it influences proliferation and migration of tumor cells. We demonstrate that glutamate antagonists inhibit proliferation of human tumor cells. Colon adenocarcinoma, astrocytoma, and breast and lung carcinoma cells were most sensitive to the antiproliferative effect of the N-methyl-d-aspartate antagonist dizocilpine, whereas breast and lung carcinoma, colon adenocarcinoma, and neuroblastoma cells responded most favorably to the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate antagonist GYKI52466. The antiproliferative effect of glutamate antagonists was Ca2+ dependent and resulted from decreased cell division and increased cell death. Morphological alterations induced by glutamate antagonists in tumor cells consisted of reduced membrane ruffling and pseudopodial protrusions. Furthermore, glutamate antagonists decreased motility and invasive growth of tumor cells. These findings suggest anticancer potential of glutamate antagonists.

The copper transporter CTR1 provides an essential function in mammalian embryonic development

Copper serves as an essential cofactor for a variety of proteins in all living organisms. Previously, we described a human gene (CTR1;SLC31A1) that encodes a high-affinity copper-uptake protein and hypothesized that this protein is required for copper delivery to mammalian cells. Here, we test this hypothesis by inactivating the Ctr1 gene in mice by targeted mutagenesis. We observe early embryonic lethality in homozygous mutant embryos and a deficiency in copper uptake in the brains of heterozygous animals. Ctr1−/− embryos can be recovered at E8.5 but are severely developmentally retarded and morphologically abnormal. Histological analysis reveals discontinuities and variable thickness in the basement membrane of the embryonic region and an imperfect Reichert's membrane, features that are likely due to lack of activity in the collagen cross-linking cupro-enzyme lysyl oxidase. A collapsed embryonic cavity, the absence of an allantois, retarded mesodermal migration, and increased cell death are also apparent. In the brains of heterozygous adult mice, which at 16 months are phenotypically normal, copper is reduced to approximately half compared with control littermates, implicating CTR1 as the required port for copper entry into at least this organ. A study of the spatial and temporal expression pattern of Ctr1 during mouse development and adulthood further shows that CTR1 is ubiquitously transcribed with highest expression observed in the specialized epithelia of the choroid plexus and renal tubules and in connective tissues of the eye, ovary, and testes. We conclude that CTR1 is the primary avenue for copper uptake in mammalian cells.

Essential role for mammalian copper transporter Ctr1 in copper homeostasis and embryonic development

The trace metal copper (Cu) plays an essential role in biology as a cofactor for many enzymes that include Cu, Zn superoxide dismutase, cytochrome oxidase, ceruloplasmin, lysyl oxidase, and dopamine β-hydroxylase. Consequently, Cu transport at the cell surface and the delivery of Cu to intracellular compartments are critical events for a wide variety of biological processes. The components that orchestrate intracellular Cu trafficking and their roles in Cu homeostasis have been elucidated by the studies of model microorganisms and by the characterizations of molecular basis of Cu-related genetic diseases, including Menkes disease and Wilson disease. However, little is known about the mechanisms for Cu uptake at the plasma membrane and the consequences of defects in this process in mammals. Here, we show that the mouse Ctr1 gene encodes a component of the Cu transport machinery and that mice heterozygous for Ctr1 exhibit tissue-specific defects in copper accumulation and in the activities of copper-dependent enzymes. Mice completely deficient for Ctr1 exhibit profound growth and developmental defects and die in utero in mid-gestation. These results demonstrate a crucial role for Cu acquisition through the Ctr1 transporter for mammalian Cu homeostasis and embryonic development.

Identification and characterization of a lysosomal transporter for small neutral amino acids

In eukaryotic cells, lysosomes represent a major site for macromolecule degradation. Hydrolysis products are eventually exported from this acidic organelle into the cytosol through specific transporters. Impairment of this process at either the hydrolysis or the efflux step is responsible of several lysosomal storage diseases. However, most lysosomal transporters, although biochemically characterized, remain unknown at the molecular level. In this study, we report the molecular and functional characterization of a lysosomal amino acid transporter (LYAAT-1), remotely related to a family of H+-coupled plasma membrane and synaptic vesicle amino acid transporters. LYAAT-1 is expressed in most rat tissues, with highest levels in the brain where it is present in neurons. Upon overexpression in COS-7 cells, the recombinant protein mediates the accumulation of neutral amino acids, such as γ-aminobutyric acid, l-alanine, and l-proline, through an H+/amino acid symport. Confocal microscopy on brain sections revealed that this transporter colocalizes with cathepsin D, an established lysosomal marker. LYAAT-1 thus appears as a lysosomal transporter that actively exports neutral amino acids from lysosomes by chemiosmotic coupling to the H+-ATPase of these organelles. Homology searching in eukaryotic genomes suggests that LYAAT-1 defines a subgroup of lysosomal transporters in the amino acid/auxin permease family.

Use of chimeric proteins to investigate the role of transporter associated with antigen processing (TAP) structural domains in peptide binding and translocation

The transporter associated with antigen processing (TAP) comprises two subunits, TAP1 and TAP2, each containing a hydrophobic membrane-spanning region (MSR) and a nucleotide binding domain (NBD). The TAP1/TAP2 complex is required for peptide translocation across the endoplasmic reticulum membrane. To understand the role of each structural unit of the TAP1/TAP2 complex, we generated two chimeras containing TAP1 MSR and TAP2 NBD (T1MT2C) or TAP2 MSR and TAP1 NBD (T2MT1C). We show that TAP1/T2MT1C, TAP2/T1MT2C, and T1MT2C/T2MT1C complexes bind peptide with an affinity comparable to wild-type complexes. By contrast, TAP1/T1MT2C and TAP2/T2MT1C complexes, although observed, are impaired for peptide binding. Thus, the MSRs of both TAP1 and TAP2 are required for binding peptide. However, neither NBD contains unique determinants required for peptide binding. The NBD-switched complexes, T1MT2C/T2MT1C, TAP1/T2MT1C, and TAP2/T1MT2C, all translocate peptides, but with progressively reduced efficiencies relative to the TAP1/TAP2 complex. These results indicate that both nucleotide binding sites are catalytically active and support an alternating catalytic sites model for the TAP transport cycle, similar to that proposed for P-glycoprotein. The enhanced translocation efficiency of TAP1/T2MT1C relative to TAP2/T1MT2C complexes correlates with enhanced binding of the TAP1 NBD-containing constructs to ATP-agarose beads. Preferential ATP interaction with TAP1, if occurring in vivo, might polarize the transport cycle such that ATP binding to TAP1 initiates the cycle. However, our observations that TAP complexes containing two identical TAP NBDs can mediate translocation indicate that distinct properties of the nucleotide binding site per se are not essential for the TAP catalytic cycle.

Distinct functions and cooperative interaction of the subunits of the transporter associated with antigen processing (TAP)

The ATP-binding cassette (ABC) transporter TAP translocates peptides from the cytosol to awaiting MHC class I molecules in the endoplasmic reticulum. TAP is made up of the TAP1 and TAP2 polypeptides, which each possess a nucleotide binding domain (NBD). However, the role of ATP in peptide binding and translocation is poorly understood. We present biochemical and functional evidence that the NBDs of TAP1 and TAP2 are non-equivalent. Photolabeling experiments with 8-azido-ATP demonstrate a cooperative interaction between the two NBDs that can be stimulated by peptide. The substitution of key lysine residues in the Walker A motifs of TAP1 and TAP2 suggests that TAP1-mediated ATP hydrolysis is not essential for peptide translocation but that TAP2-mediated ATP hydrolysis is critical, not only for translocation, but for peptide binding.

Rapid Up-Regulation of HKT1, a High-Affinity Potassium Transporter Gene, in Roots of Barley and Wheat following Withdrawal of Potassium1

High-affinity K+ uptake in plant roots is rapidly up-regulated when K+ is withheld and down-regulated when K+ is resupplied. These processes make important contributions to plant K+ homeostasis. A cDNA coding for a high-affinity K+ transporter, HKT1, was earlier cloned from wheat (Triticum aestivum L.) roots and functionally characterized. We demonstrate here that in both barley (Hordeum vulgare L.) and wheat roots, a rapid and large up-regulation of HKT1 mRNA levels resulted when K+ was withdrawn from growth media. This effect was specific for K+; withholding N caused a modest reduction of HKT1 mRNA levels. Up-regulation of HKT1 transcript levels in barley roots occurred within 4 h of removing K+, which corresponds to the documented increase of high-affinity K+ uptake in roots following removal of K+. Increased expression of HKT1 mRNA was evident before a decline in total root K+ concentration could be detected. Resupply of 1 mm K+ was sufficient to strongly reduce HKT1 transcript levels. In wheat root cortical cells, both membrane depolarizations in response to 100 μm K+, Cs+, and Rb+, and high-affinity K+ uptake were enhanced by K+ deprivation. Thus, in both plant systems the observed physiological changes associated with manipulating external K+ supply were correlated with levels of HKT1 mRNA expression. Implications of these findings for K+ sensing and regulation of the HKT1 mRNA levels in plant roots are discussed.

Characterization of Two Glutamate Decarboxylase cDNA Clones from Arabidopsis

Two distinct cDNA clones encoding for the glutamate decarboxylase (GAD) isoenzymes GAD1 and GAD2 from Arabidopsis (L.) Heynh. were characterized. The open reading frames for GAD1 and GAD2 were expressed in Escherichia coli and the recombinant proteins were purified by affinity chromatography. Analysis of the recombinant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis suggest that GAD1 and GAD2 encode for 58- and 56-kD peptides, respectively. The enzymatic activities of the pure recombinant GAD1 and GAD2 proteins were stimulated 35- and 13-fold, respectively, by Ca2+/calmodulin but not by Ca2+ or calmodulin alone. Southern-blot analysis of genomic DNA suggests that there is only one copy of each gene in Arabidopsis. The GAD1 transcript and a corresponding 58-kD peptide were detected in roots only. Conversely, the GAD2 transcript and a corresponding 56-kD peptide were detected in all organs tested. The specific activity, GAD2 transcript, and 56-kD peptide increased in leaves of plants treated with 10 mm NH4Cl, 5 mm NH4NO3, 5 mm glutamic acid, or 5 mm glutamine as the sole nitrogen source compared with samples from plants treated with 10 mm KNO3. The results from these experiments suggest that in leaves GAD activity is partially controlled by gene expression or RNA stability. Results from preliminary analyses of different tissues imply that these tendencies were not the same in flower stalks and flowers, suggesting that other factors may control GAD activity in these organs. The results from this investigation demonstrate that GAD activity in leaves is altered by different nitrogen treatments, suggesting that GAD2 may play a unique role in nitrogen metabolism.

Tomato Phosphate Transporter Genes Are Differentially Regulated in Plant Tissues by Phosphorus1

Phosphorus is a major nutrient acquired by roots via high-affinity inorganic phosphate (Pi) transporters. In this paper, we describe the tissue-specific regulation of tomato (Lycopersicon esculentum L.) Pi-transporter genes by Pi. The encoded peptides of the LePT1 and LePT2 genes belong to a family of 12 membrane-spanning domain proteins and show a high degree of sequence identity to known high-affinity Pi transporters. Both genes are highly expressed in roots, although there is some expression of LePT1 in leaves. Their expression is markedly induced by Pi starvation but not by starvation of nitrogen, potassium, or iron. The transcripts are primarily localized in root epidermis under Pi starvation. Accumulation of LePT1 message was also observed in palisade parenchyma cells of Pi-starved leaves. Our data suggest that the epidermally localized Pi transporters may play a significant role in acquiring the nutrient under natural conditions. Divided root-system studies support the hypothesis that signal(s) for the Pi-starvation response may arise internally because of the changes in cellular concentration of phosphorus.

A Saccharomyces cerevisiae homolog of the human adrenoleukodystrophy transporter is a heterodimer of two half ATP-binding cassette transporters.

The adrenoleukodystrophy protein (ALDP) and the 70-kDa peroxisomal membrane protein (PMP70) are half ATP-binding cassette (ABC) transporters in the human peroxisome membrane. ALDP and PMP70 share sequence homology and both are implicated in genetic diseases. PXA1 and YKL741 are Saccharomyces cerevisiae genes that encode homologs of ALDP and PMP70. Pxa1p, a putative ortholog of ALDP, is involved in peroxisomal beta-oxidation of fatty acids while YKL741 is an open reading frame found by the yeast genome sequencing project. Here we designate YKL741 as PXA2 and show that its protein product, Pxa2p, like Pxa1p, is associated with peroxisomes but not required for their assembly. Yeast strains carrying gene disruption of PXA1, PXA2, or both have similar and, in the case of the latter, nonadditive phenotypes. We also find that the stability of Pxa1p, but not Pxa2p, is markedly reduced in the absence of the other. Finally, we find that Pxa1p and Pxa2p coimmuno-precipitate. These genetic and physical data suggest that Pxa1p and Pxa2p heterodimerize to form a complete peroxisomal ABC transporter involved in fatty acid beta-oxidation. This result predicts the presence of similar heterodimeric ABC transporters in the mammalian peroxisome membrane.

Multidrug resistance mediated by a bacterial homolog of the human multidrug transporter MDR1.

Resistance of Lactococcus lactis to cytotoxic compounds shares features with the multidrug resistance phenotype of mammalian tumor cells. Here, we report the gene cloning and functional characterization in Escherichia coli of LmrA, a lactococcal structural and functional homolog of the human multidrug resistance P-glycoprotein MDR1. LmrA is a 590-aa polypeptide that has a putative topology of six alpha-helical transmembrane segments in the N-terminal hydrophobic domain, followed by a hydrophilic domain containing the ATP-binding site. LmrA is similar to each of the two halves of MDR1 and may function as a homodimer. The sequence conservation between LmrA and MDR1 includes particular regions in the transmembrane domains and connecting loops, which, in MDR1 and the MDR1 homologs in other mammalian species, have been implicated as determinants of drug recognition and binding. LmrA and MDR1 extrude a similar spectrum of amphiphilic cationic compounds, and the activity of both systems is reversed by reserpine and verapamil. As LmrA can be functionally expressed in E. coli, it offers a useful prokaryotic model for future studies on the molecular mechanism of MDR1-like multidrug transporters.

Relationship between psychostimulant-induced "high" and dopamine transporter occupancy.

The ability of cocaine to inhibit the dopamine transporter (DAT) appears to be crucial for its reinforcing properties. The potential use of drugs that produce long-lasting inhibition of the DAT as a mean of preventing the "high" and reducing drug-seeking behavior has become a major strategy in medication development. However, neither the relation between the high and DAT inhibition nor the ability to block the high by prior DAT blockade have ever been demonstrated. To evaluate if DAT could prevent the high induced by methylphenidate (MP), a drug which like cocaine inhibits the DAT, we compared the responses in eight non-drug-abusing subjects between the first and the second of two MP doses (0.375 mg/kg, i.v.) given 60 min apart. At 60 min the high from MP has returned to baseline, but 75-80% of the drug remains in brain. Positron-emission tomography and [11C]d-threo-MP were used to estimate DAT occupancies at different times after MP. DAT inhibition by MP did not block or attenuate the high from a second dose of MP given 60 min later, despite a 80% residual transporter occupancy from the first dose. Furthermore some subjects did not perceive a high after single or repeated administration despite significant DAT blockade. These results indicate that DAT occupancy is not sufficient to account for the high, and that for DAT inhibitors to be therapeutically effective, occupancies > 80% may be required.

Evidence for an insulin receptor substrate 1 independent insulin signaling pathway that mediates insulin-responsive glucose transporter (GLUT4) translocation.

Interaction of the activated insulin receptor (IR) with its substrate, insulin receptor substrate 1 (IRS-1), via the phosphotyrosine binding domain of IRS-1 and the NPXY motif centered at phosphotyrosine 960 of the IR, is important for IRS-1 phosphorylation. We investigated the role of this interaction in the insulin signaling pathway that stimulates glucose transport. Utilizing microinjection of competitive inhibitory reagents in 3T3-L1 adipocytes, we have found that disruption of the IR/IRS-1 interaction has no effect upon translocation of the insulin-responsive glucose transporter (GLUT4). The activity of these reagents was demonstrated by their ability to block insulin stimulation of two distinct insulin bioeffects, membrane ruffling and mitogenesis, in 3T3-L1 adipocytes and insulin-responsive rat 1 fibroblasts. These data suggest that phosphorylated IRS-1 is not an essential component of the metabolic insulin signaling pathway that leads to GLUT4 translocation, yet it appears to be required for other insulin bioeffects.

Expression cloning of the Golgi CMP-sialic acid transporter.

Translocation of nucleotide sugars across the membrane of the Golgi apparatus is a prerequisite for the synthesis of complex carbohydrate structures. While specific transport systems for different nucleotide sugars have been identified biochemically in isolated microsomes and Golgi vesicles, none of these transport proteins has been characterized at the molecular level. Chinese hamster ovary (CHO) mutants of the complementation group Lec2 exhibit a strong reduction in sialylation of glycoproteins and glycolipids due to a defect in the CMP-sialic acid transport system. By complementation cloning in the mutant 6B2, belonging to the Lec2 complementation group, we were able to isolate a cDNA encoding the putative murine Golgi CMP-sialic acid transporter. The cloned cDNA encodes a highly hydrophobic, multiple membrane spanning protein of 36.4 kDa, with structural similarity to the recently cloned ammonium transporters. Transfection of a hemagglutinin-tagged fusion protein into the mutant 6B2 led to Golgi localization of the hemagglutinin epitope. Our results, together with the observation that the cloned gene shares structural similarities to other recently cloned transporter proteins, strongly suggest that the isolated cDNA encodes the CMP-sialic acid transporter.