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ABSTRACT: The vesicular nucleotide transporter (VNUT) is a secretory vesicle protein that is responsible for the vesicular storage and subsequent exocytosis of ATP (Sawada, K., Echigo, N., Juge, N., Miyaji, T., Otsuka, M., Omote, H., and Moriyama, Y. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 5683-5686). Because VNUT actively transports ATP in a membrane potential (Δψ)-dependent manner irrespective of divalent cations such as Mg(2+) and Ca(2+), VNUT recognizes free ATP as a transport substrate. However, whether or not VNUT transports chelating complexes with divalent cations remains unknown. Here, we show that proteoliposomes containing purified VNUT actively took up Mg(2+) when ATP was present, as detected by atomic absorption spectroscopy. The VNUT-containing proteoliposomes also took up radioactive Ca(2+) upon imposing Δψ (positive-inside) but not ΔpH. The Δψ-driven Ca(2+) uptake required ATP and a millimolar concentration of Cl(-), which was inhibited by Evans blue, a specific inhibitor of SLC17-type transporters. VNUT in which Arg-119 was specifically mutated to alanine, the counterpart of the essential amino acid residue of the SLC17 family, lost the ability to take up both ATP and Ca(2+). Ca(2+) uptake was also inhibited in the presence of various divalent cations such as Mg(2+). Kinetic analysis indicated that Ca(2+) or Mg(2+) did not affect the apparent affinity for ATP. RNAi of the VNUT gene in PC12 cells decreased the vesicular Mg(2+) concentration to 67.7%. These results indicate that VNUT transports both nucleotides and divalent cations probably as chelating complexes and suggest that VNUT functions as a divalent cation importer in secretory vesicles under physiological conditions.
Journal of Biological Chemistry 11/2011; 286(50):42881-7. · 4.77 Impact Factor
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ABSTRACT: ATP is known to be coreleased with glutamate at certain central synapses. However, the nature of its release is controversial. Here, we demonstrate that ATP release from cultured rat hippocampal neurons is sensitive to RNAi-mediated knockdown of the recently identified vesicular nucleotide transporter (VNUT or SLC17A9). In the intact brain, light microscopy showed particularly strong VNUT immunoreactivity in the cerebellar cortex, the olfactory bulb, and the hippocampus. Using immunoelectron microscopy, we found VNUT immunoreactivity colocalized with synaptic vesicles in excitatory and inhibitory terminals in the hippocampal formation. Moreover, VNUT immunolabeling, unlike that of the vesicular glutamate transporter VGLUT1, was enriched in preterminal axons and present in postsynaptic dendritic spines. Immunoisolation of synaptic vesicles indicated presence of VNUT in a subset of VGLUT1-containing vesicles. Thus, we conclude that VNUT mediates transport of ATP into synaptic vesicles of hippocampal neurons, thereby conferring a purinergic phenotype to these cells.
Cerebral Cortex 08/2011; 22(5):1203-14. · 6.54 Impact Factor
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ABSTRACT: Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. Large amounts of purified proteins are required for activity and crystallization attempts. Thus, there is an unmet need for a heterologous membrane protein overexpression system for purification, crystallization, and activity determination. We developed a combinatorial method for overexpressing and purifying membrane proteins using Escherichia coli. This method utilizes short hydrophilic bacterial proteins, YaiN and YbeL, fused to the ends of the membrane proteins to serve as facilitating factors for expression and purification. Fourteen prokaryotic and mammalian membrane proteins were expressed using this system. Moderate to high expression was obtained for most proteins, and detergent solubilization combined with a short purification process produced stable, monodispersed membrane proteins. Five of the mammalian membrane proteins, overexpressed using our system, were reconstituted into liposomes and exhibited transport activity comparable with the native transporters.
Journal of Biological Chemistry 07/2010; 285(31):23548-56. · 4.77 Impact Factor
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ABSTRACT: Alkaloids play a key role in plant defense mechanisms against pathogens and herbivores, but the plants themselves need to cope with their toxicity as well. The major alkaloid of the Nicotiana species, nicotine, is translocated via xylem transport from the root tissues where it is biosynthesized to the accumulation sites, the vacuoles of leaves. To unravel the molecular mechanisms behind this membrane transport, we characterized one transporter, the tobacco (Nicotiana tabacum) jasmonate-inducible alkaloid transporter 1 (Nt-JAT1), whose expression was coregulated with that of nicotine biosynthetic genes in methyl jasmonate-treated tobacco cells. Nt-JAT1, belonging to the family of multidrug and toxic compound extrusion transporters, was expressed in roots, stems, and leaves, and localized in the tonoplast of leaf cells. When produced in yeast cells, Nt-JAT1 occurred mainly in the plasma membrane and showed nicotine efflux activity. Biochemical analysis with proteoliposomes reconstituted with purified Nt-JAT1 and bacterial F(0)F(1)-ATPase revealed that Nt-JAT1 functioned as a proton antiporter and recognized endogenous tobacco alkaloids, such as nicotine and anabasine, and other alkaloids, such as hyoscyamine and berberine, but not flavonoids. These findings strongly suggest that Nt-JAT1 plays an important role in the nicotine translocation by acting as a secondary transporter responsible for unloading of alkaloids in the aerial parts and deposition in the vacuoles.
Proceedings of the National Academy of Sciences 02/2009; 106(7):2447-52. · 9.68 Impact Factor
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ABSTRACT: ATP is a major chemical transmitter in purinergic signal transmission. Before secretion, ATP is stored in secretory vesicles found in purinergic cells. Although the presence of active transport mechanisms for ATP has been postulated for a long time, the proteins responsible for its vesicular accumulation remains unknown. The transporter encoded by the human and mouse SLC17A9 gene, a novel member of an anion transporter family, was predominantly expressed in the brain and adrenal gland. The mouse and bovine counterparts were associated with adrenal chromaffin granules. Proteoliposomes containing purified transporter actively took up ATP, ADP, and GTP by using membrane potential as the driving force. The uptake properties of the reconstituted transporter were similar to that of the ATP uptake by synaptic vesicles and chromaffin granules. Suppression of endogenous SLC17A9 expression in PC12 cells decreased exocytosis of ATP. These findings strongly suggest that SLC17A9 protein is a vesicular nucleotide transporter and should lead to the elucidation of the molecular mechanism of ATP secretion in purinergic signal transmission.
Proceedings of the National Academy of Sciences 05/2008; 105(15):5683-6. · 9.68 Impact Factor
