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Identification of a Vesicular Nucleotide Transporter

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Identification of a Vesicular Nucleotide Transporter Identification of a vesicular nucleotide transporter Keisuke Sawada*, Noriko Echigo*, Narinobu Juge*, Takaaki Miyaji*, Masato Otsuka†, Hiroshi Omote*, Akitsugu Yamamoto‡, and Yoshinori Moriyama*†§ *Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, and †Advanced Science Research Center, Okayama University, Okayama 700-8530, Japan; and ‡Department of Cell Biology, Nagahama Institute of Technology, Nagahama 526-0829, Japan Edited by Nathan Nelson, Tel Aviv University, Tel Aviv, Israel, and accepted by the Editorial Board February 11, 2008 (received for review January 7, 2008) ATP is a major chemical transmitter in purinergic signal transmis- A NPT4 sion. Before secretion, ATP is stored in secretory vesicles found in NPT1 SLC17A3 NPT homolog purinergic cells. Although the presence of active transport mech- SLC17A1 SLC17A4 anisms for ATP has been postulated for a long time, the proteins responsible for its vesicular accumulation remains unknown. The NPT3 transporter encoded by the human and mouse SLC17A9 gene, a SLC17A2 novel member of an anion transporter family, was predominantly VNUT expressed in the brain and adrenal gland. The mouse and bovine SLC17A9 counterparts were associated with adrenal chromaffin granules. Sialin SLC17A5 Proteoliposomes containing purified transporter actively took up VGLUT1 SLC17A7 ATP, ADP, and GTP by using membrane potential as the driving VGLUT3 VGLUT2 force. The uptake properties of the reconstituted transporter were SLC17A8 SLC17A6 similar to that of the ATP uptake by synaptic vesicles and chro- maffin granules. Suppression of endogenous SLC17A9 expression B P G W N FD T A T G in PC12 cells decreased exocytosis of ATP. These findings strongly F K SS T S D K L A W Y E K SS E W Q E H H E G E G T F M T W M S A A L A L Q F F W H C L C S G L L S T I G E Y L suggest that SLC17A9 protein is a vesicular nucleotide transporter V I L M F L L P F L S G G F C T V P T S I W N C V L N L T F G F S V V C L I S V V Y L L V L F V V MP S R S A F I P A A G A S F A I G S F F W F S V I and should lead to the elucidation of the molecular mechanism of S F T L T G S L A V I A R W I M L C V G C G S G L G A A V S S A S L Y L T L S A I A L N Y G L G T Q L I S G G L L C W Q L S P L L A G ATP secretion in purinergic signal transmission. C L A G Q F L A G Q T L G T T V S A V N L Q S Y G W V S L T A C LL V A F A V A L G F G V T S P G W V W F M N F F T G V A V Y A S G L G L L I V P G H L W T G L T S Y R K F Q S G F V A H I L S Y Y R F L M G A F Q L V L T L L S L I C G G L C K S F L R D K S S Q ͉ ͉ ͉ D S A R ATP chromaffin granule purinergic signaling NCM E R E Q A V H R V P A I G K R E G W N A T P G K Q L V Q L R V E L S P I I L L D T Q D R D R S A V N I storage and exocytosis ͉ synaptic vesicle T A E L R V E I L P Q A S E R G R D H R Q V S Y L T R S S H SS Q D R esicular storage and subsequent exocytosis of neurotrans- Fig. 1. Phylogenetic tree of SLC17 family (A) and putative secondary struc- Vmitters is essential for chemical transmission in neurons and ture of SLC17A9 protein (B). endocrine cells. Thus far four distinct classes of transporters are known to participate in the uptake of neurotransmitters into neuronal synaptic vesicles and secretory granules in endocrine NPT3, NPT4, and Naϩ/PO42Ϫcotransporter homologue), Naϩ cells. These are vesicular monoamine transporters, vesicular and inorganic phosphate cotransporters, (ii) SLC17A5 (sialin), acetylcholine transporters, vesicular inhibitory amino acid trans- a lysosomal Hϩ/sialic acid cotransporter, and (iii) SLC17A6–8, porters, and vesicular glutamate transporters (VGLUTs) (1–7). VGLUT (5). We found that a fourth subfamily, tentatively These vesicular transporters mediate the active accumulation of designated SLC17A9, was identified in a genome data bank their respective neurotransmitters through an electrochemical screening (Fig. 1A). SLC17A9 was located on chromosome 20 gradient of protons across the membrane generated by vacuolar and contained 14 exons and 13 introns [supporting information proton-ATPase. ATP is stored in secretory vesicles and subse- (SI) Fig. S1]. The SLC17A9 protein was 430 aa residues long with quently exocytosed, which leads to the various purinergic re- 12 putative transmembrane helices with Ϸ23–29% identity and sponses, such as central control of autonomic functions, pain and 41–48% similarity to that of other SLC17 members (Fig. 1B and mechanosensory transduction, neural-glial interactions, control Fig. S2). Orthologues of other mammalian species have been of vessel tone and angiogenesis, and platelet aggregation through identified. Although amino acid sequences of the amino- purinoceptors, thus establishing its role as a chemical transmitter terminal region in mammals exhibited species-specific variation, (8–12). Because the concentration of nucleotides in the vesicles Ϸ the remaining regions showed the relatively well conserved BIOCHEMISTRY was maintained at 0.1–1 M, an active transport mechanisms to features (83% identity) (Fig. S3). accumulate nucleotides has been postulated (8–18). Although We cloned cDNA-encoded SLC17A9 (Fig. S2). Northern blot evidence increasingly supports the presence of a vesicular ATP analysis indicated that the SLC17A9 and mouse counterpart transporters in secretory vesicles such as synaptic vesicles and (mSLC17A9) were widely expressed in various organs, but adrenal chromaffin granules (13–19), the protein responsible for predominantly in the adrenal gland, brain, and thyroid gland the ATP accumulation has not yet been identified. Here, we report the expression and function of human and mouse SLC17A9, an isoform of the SLC17 phosphate trans- Author contributions: K.S., N.E., and N.J. contributed equally to this work; H.O. and Y.M. porter family. We present evidence that the SLC17A9 protein designed research; K.S., N.E., N.J., T.M., M.O., and A.Y. performed research; H.O. and Y.M. acts as a vesicular nucleotide transporter (VNUT) and that it analyzed data; and H.O. and Y.M. wrote the paper. plays an essential role in vesicular storage of ATP in the The authors declare no conflict of interest. ATP-secreting cells. This article is a PNAS Direct Submission. N.N. is a guest editor invited by the Editorial Board. §To whom correspondence should be addressed. E-mail: [email protected]. Results and Discussion ac.jp. Gene Organization and Expression of SLC17A9. SLC17 is a type I This article contains supporting information online at www.pnas.org/cgi/content/full/ phosphate transporter family consisting of eight genes that are 0800141105/DC1. classified into three distinct subfamilies: (i) SLC17A1–4 (NPT1, © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800141105 PNAS ͉ April 15, 2008 ͉ vol. 105 ͉ no. 15 ͉ 5683–5686 Downloaded by guest on September 28, 2021 . b A B C D A9 A 7 Ab. el 1 mSLC17A9 preabs. Control nti SLC sc mSLC17A9 abs. u C m Pre l late C A na dio Kidney s Heart KDa ti e niarB r le r g s r y Live n M Human e d h k u T A T S L SLC17A9 115 G3PDH 72 Mouse 57 M SLC17A9 G3PDH 39 Mit 34 Fig. 2. Expression of SLC17A9 and its association with chromaffin granules. (A) Northern blotting showing expression of SLC17A9 in human and mouse brain and adrenal gland. Expression of G3PDH is also shown as a control. (B) Immunohistochemical localization of SLC17A9 protein in mouse adrenal gland. (Inset) Shown is control staining with preabsorbed antibodies. C, cortex; M, medulla. (C) Immunoelectronmicroscopy showing that SLC17A9 proteins (gold particles) are associated with chromaffin granules. (Inset) Shown is background labeling with control serum. Mit, mitochondrion. (D) Western blot indicates the presence of a SLC17A9 counterpart in the membrane of bovine adrenal chromaffin granule with a relative mobility of 61 kDa. The preabsorbed antibodies did not bind to the protein. The positions of marker proteins are indicated on the left. (Scale bars: B,10␮m; C, 100 nm.) (Fig. 2A). In mouse adrenal gland, the mSLC17A9 protein was pothesis, the SLC17A9 protein was expressed in High Five cells, specifically expressed in the medulla and was associated with solubilized from the membranes, and purified by Ni-NTA col- chromaffin granules (Fig. 2 B and C). The presence of a umn chromatography (Fig. 3A). The purified fraction showed SLC17A9 counterpart in bovine adrenal chromaffin granule two major protein bands (68 and 63 kDa) on SDS polyacrylamide membrane has been confirmed (Fig.
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