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The Cytoplasmic Tail of the Vesicular Acetylcholine Transporter Contains a Synaptic Vesicle Targeting Signal

1998; Elsevier BV; Volume: 273; Issue: 15 Linguagem: Inglês

10.1074/jbc.273.15.9094

ISSN

1083-351X

Autores

H́el̀ene Varoqui, Jeffrey D. Erickson,

Tópico(s)

Photoreceptor and optogenetics research

Resumo

The human homologue of the vesicular acetylcholine transporter (hVAChT) and the neuronal isoform of the vesicular monoamine transporter (hVMAT2) are differentially targeted to two populations of regulated secretory organelles when expressed in PC12 cells. Western blot analysis of subcellular fractions from sucrose equilibrium density gradients and glycerol velocity gradients of homogenates from stably transfected cells revealed hVAChT immunoreactivity in fractions that contain synaptophysin, a marker of synaptic vesicles, while hVMAT2 immunoreactivity was confined to heavy fractions containing chromogranin B, a marker of large dense core vesicles. In cells treated with nerve growth factor, hVAChT immunoreactivity alone co-localized with synaptophysin and was abundantly expressed on synaptic vesicle clusters. Chimeras between hVMAT2 and hVAChT were utilized to identify the domain of hVAChT required for its expression on synaptic vesicles and which would shift the expression of hVMAT2 from large dense core vesicles to synaptic vesicles. Biochemical, immunocytochemical, and electron microscopic analyses revealed that a chimera in which the cytoplasmic tail of hVMAT2 was replaced with hVAChT sequences was now preferentially targeted to synaptic vesicles. In addition, hVAChT expression on synaptic vesicles was nearly abolished when the hVMAT2 cytoplasmic tail was present. Thus, structural information resides within the terminal cytoplasmic domain of VAChT, which specifically targets it to synaptic vesicles. The human homologue of the vesicular acetylcholine transporter (hVAChT) and the neuronal isoform of the vesicular monoamine transporter (hVMAT2) are differentially targeted to two populations of regulated secretory organelles when expressed in PC12 cells. Western blot analysis of subcellular fractions from sucrose equilibrium density gradients and glycerol velocity gradients of homogenates from stably transfected cells revealed hVAChT immunoreactivity in fractions that contain synaptophysin, a marker of synaptic vesicles, while hVMAT2 immunoreactivity was confined to heavy fractions containing chromogranin B, a marker of large dense core vesicles. In cells treated with nerve growth factor, hVAChT immunoreactivity alone co-localized with synaptophysin and was abundantly expressed on synaptic vesicle clusters. Chimeras between hVMAT2 and hVAChT were utilized to identify the domain of hVAChT required for its expression on synaptic vesicles and which would shift the expression of hVMAT2 from large dense core vesicles to synaptic vesicles. Biochemical, immunocytochemical, and electron microscopic analyses revealed that a chimera in which the cytoplasmic tail of hVMAT2 was replaced with hVAChT sequences was now preferentially targeted to synaptic vesicles. In addition, hVAChT expression on synaptic vesicles was nearly abolished when the hVMAT2 cytoplasmic tail was present. Thus, structural information resides within the terminal cytoplasmic domain of VAChT, which specifically targets it to synaptic vesicles. The organelles used for regulated biogenic amine and acetylcholine (ACh) 1The abbreviations used are: ACh, acetylcholine; CgB, chromogranin B (secretogranin I); DCV, dense core vesicle; LDCV, large dense core vesicle; SDCV, small dense core vesicle; NGF, nerve growth factor; NGS, normal goat serum; p38, synaptophysin; PC12, rat pheochromocytoma cell line; PBS, phosphate-buffered saline; PNS, postnuclear supernatant; TBS, Tris-buffered saline; TBZOH, dihydrotetrabenazine; TFR, transferrin receptor; VAChT, vesicular acetylcholine transporter; VMAT1, endocrine-specific isoform of the vesicular monoamine transporter; VMAT2, neuronal isoform of the vesicular monoamine transporter. secretion in neurons and endocrine cells are the large and small dense core vesicles (DCV) and small synaptic vesicles, respectively. The biogenesis and fate of these organelles and targeting of various proteins to them differ in several ways (1Navone F. Jahn R. DiGioia G. Stukenbrok H. Greengard P. De Camilli P. J. Cell Biol. 1986; 103: 2511-2527Crossref PubMed Scopus (557) Google Scholar, 2Patzak A. Winkler H. J. Cell Biol. 1986; 102: 510-515Crossref PubMed Scopus (123) Google Scholar, 3Südhof T.C. Jahn R. Neuron. 1991; 6: 665-677Abstract Full Text PDF PubMed Scopus (432) Google Scholar, 4Trimble W.S. Linial M. Scheller R.H. Annu. Rev. Neurosci. 1991; 14: 93-122Crossref PubMed Scopus (131) Google Scholar, 5Kelly R.B. Cell. 1993; 72: 43-53Abstract Full Text PDF PubMed Scopus (213) Google Scholar, 6Bauerfeind R. Jelinek R. Hellwig A. Huttner W.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7342-7346Crossref PubMed Scopus (47) Google Scholar). LDCVs (approximately 200 nm) are the product of the trans-Golgi network as they contain soluble glycoproteins, such as the chromogranins, and various neuropeptides. Following maturation and subsequent exocytosis at the plasma membrane, components of the LDCV membrane must recycle to the Golgi complex to replenish their soluble neurohormone content and re-enter the regulated secretory pathway. In contrast, synaptic vesicle proteins, such as synaptophysin (p38), appear to travel to the plasma membrane via the constitutive secretory pathway and are then endocytosed and sorted in endosomes, from which synaptic vesicles (40 nm) bud. Exocytosis/endocytosis and recycling of cholinergic synaptic vesicles takes place at the presynaptic nerve terminal, while neuropeptide- and monoamine-containing LDCVs, located throughout the cell, may release nonsynaptically (7Thureson-Klein A.K. Klein R.L. Zhu P.-C. Kong J.-Y. Zimmermann H. Cellular and Molecular Basis of Synaptic Transmission. Springer-Verlag, Berlin1988: 137-151Crossref Google Scholar, 8Matteoli M. Haimann C. Torri-Tarelli F. Polak J.M. Ceccarelli B. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7366-7370Crossref PubMed Scopus (192) Google Scholar, 9De Camilli P. Jahn R. Annu. Rev. Physiol. 1990; 52: 625-645Crossref PubMed Scopus (365) Google Scholar, 10Lundberg J.M. Franco-Cereceda A. Lou Y.P. Modin A. Pernow J. Stjärne L. Greengard P. Grillner S. Hökfelt T. Ottoson D. Molecular and Cellular Mechanisms of Neurotransmitter Release. Raven Press, New York1994: 223-234Google Scholar). Cloning and characterization of the family of vesicular transporters for biogenic amines and ACh has provided new tools for the study of the biogenesis of these regulated secretory organelles (11Varoqui H. Erickson J.D. Mol. Neurobiol. 1997; 15: 165-191Crossref PubMed Google Scholar, 12Liu Y. Edwards R.H. Annu. Rev. Neurosci. 1997; 20: 125-156Crossref PubMed Scopus (237) Google Scholar). The examination of the subcellular localization of these proteins indicates that they are associated with distinct vesicle populations in neurons and neuroendocrine cells. The endocrine-specific VMAT1 isoform is expressed on LDCVs of the rat adrenal medulla (13Liu Y. Schweitzer E.S. Nirenberg M.J. Pickel V.M. Evans C.J. Edwards R.H. J. Cell Biol. 1994; 127: 1419-1433Crossref PubMed Scopus (125) Google Scholar). Tetrabenazine-sensitive transport of dopamine in the central nervous system occurs in vesicle structures that resemble synaptic vesicles (14Philipu A. Beyer J. Naunyn-Schmiedebergs Arch. Pharmacol. 1973; 278: 387-402Crossref PubMed Scopus (57) Google Scholar, 15Erickson J.D. Masserano J.M. Barnes E.M. Ruth J.A. Weiner N. Brain Res. 1990; 516: 155-160Crossref PubMed Scopus (34) Google Scholar, 16Floor E. Leventhal P.S. Wang Y. Meng L. Chen W. J. Neurochem. 1995; 64: 689-699Crossref PubMed Scopus (75) Google Scholar), yet, in rat central monoaminergic neurons, higher levels of the neuronal isoform of VMAT (VMAT2) have been observed by immunoelectron microscopy on DCVs and tubulovesicular structures than on synaptic vesicles (17Nirenberg M.J. Liu Y. Peter D. Edwards R.H. Pickel V.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8773-8777Crossref PubMed Scopus (124) Google Scholar, 18Nirenberg M.J. Chan J. Liu Y. Edwards R.H. Pickel V.M. J. Neurosci. 1996; 16: 4135-4145Crossref PubMed Google Scholar). VAChT, on the other hand, is abundantly expressed on synaptic vesicles in cholinergic nerve terminals inTorpedo electric organ and in the rat central nervous system (19Varoqui H. Meunier F.-M. Meunier F.A. Molgo J. Berrard S. Cervini R. Mallet J. Israël M. Diebler M.-F. Prog. Brain Res. 1996; 109: 83-95Crossref PubMed Google Scholar, 20Gilmore M.L. Nash N.R. Roghani A. Edwards R.H. Yi H. Hersch S.M. Levey A.I. J. Neurosci. 1996; 16: 2179-2190Crossref PubMed Google Scholar). The trafficking of VMATs and VAChT and biogenesis of LDCVs and synaptic vesicles can be studied using the pheochromocytoma (PC12) cell line. PC12s are mixed monoaminergic/cholinergic cells that synthesize, store, and secrete ACh as well as dopamine (21Green L.A. Rein G. Nature. 1977; 268: 349-351Crossref PubMed Scopus (186) Google Scholar, 22Greene L.A. Rein G. Brain Res. 1977; 129: 247-263Crossref PubMed Scopus (334) Google Scholar, 23Schubert D. Klier F.G. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5184-5188Crossref PubMed Scopus (102) Google Scholar, 24Robois R.V. Reynolds E.E. Toll L. Howard B.D. Biochemistry. 1980; 19: 1240-1248Crossref PubMed Scopus (92) Google Scholar). Subcellular fractionation of PC12 cells has revealed that the LDCVs possess a reserpine-sensitive transport system for monoamines (25Bauerfeind R. Régnier-Vigouroux A. Flatmark R. Huttner W.B. Neuron. 1993; 11: 105-121Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 26Clift-O'Grady L. Linstedt A.D. Lowe A.W. Grote E. Kelly R.B. J. Cell Biol. 1990; 110: 1693-1703Crossref PubMed Scopus (146) Google Scholar). Accordingly, VMAT1 is expressed almost exclusively on LDCVs in untreated and NGF-differentiated PC12 cells (13Liu Y. Schweitzer E.S. Nirenberg M.J. Pickel V.M. Evans C.J. Edwards R.H. J. Cell Biol. 1994; 127: 1419-1433Crossref PubMed Scopus (125) Google Scholar, 27Erickson J.D. Weihe E. Schäfer M.K.-H. Neale E. Williamson L. Bonner T.I. Tao-Cheng J.-H. Eiden L.E. Prog. Brain Res. 1996; 109: 69-82Crossref PubMed Google Scholar, 28Weihe E. Tao-Cheng J.-H. Schäfer M.K.-H. Erickson J.D. Eiden L.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3547-3552Crossref PubMed Scopus (273) Google Scholar). PC12 cells also contain synaptic vesicle-like structures that resemble rat brain synaptic vesicles in size, density, protein composition, and endocytotic origin (26Clift-O'Grady L. Linstedt A.D. Lowe A.W. Grote E. Kelly R.B. J. Cell Biol. 1990; 110: 1693-1703Crossref PubMed Scopus (146) Google Scholar, 29Wiedenmann B. Rehm H. Knierim M. Becker C.-M. FEBS Lett. 1988; 240: 71-77Crossref PubMed Scopus (54) Google Scholar, 30Cutler D.F. Cramer L.P. J. Cell Biol. 1990; 110: 721-730Crossref PubMed Scopus (64) Google Scholar, 31Cameron P.L. Südhof T.C. Jahn R. De Camilli P. J. Cell Biol. 1991; 115: 151-164Crossref PubMed Scopus (240) Google Scholar). These PC12 cell synaptic vesicles are cholinergic as they are able to specifically transport ACh and bind vesamicol (25Bauerfeind R. Régnier-Vigouroux A. Flatmark R. Huttner W.B. Neuron. 1993; 11: 105-121Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 32Blumberg D. Schweitzer E.S. J. Neurochem. 1992; 58: 801-810Crossref PubMed Scopus (18) Google Scholar). Previously, we have identified VAChT as a functional vesicular transporter for ACh (33Erickson J.D. Varoqui H. Schäfer M.K.-H. Diebler M.-F. Weihe E. Modi W. Rand J.B. Eiden L.E. Bonner T.I. Usdin T. J. Biol. Chem. 1994; 269: 21929-21932Abstract Full Text PDF PubMed Google Scholar, 34Varoqui H. Erickson J.D. J. Biol. Chem. 1996; 271: 27229-27232Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) and demonstrated that it is preferentially associated with synaptic vesicles in PC12 cells (27Erickson J.D. Weihe E. Schäfer M.K.-H. Neale E. Williamson L. Bonner T.I. Tao-Cheng J.-H. Eiden L.E. Prog. Brain Res. 1996; 109: 69-82Crossref PubMed Google Scholar, 28Weihe E. Tao-Cheng J.-H. Schäfer M.K.-H. Erickson J.D. Eiden L.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3547-3552Crossref PubMed Scopus (273) Google Scholar). In the present study, we examined whether VAChT sorting to synaptic vesicles relies on a unique targeting domain. We first compared the expression of the human homologues of VAChT and VMAT2 against the rat neuroendocrine PC12 cell background and find that these proteins are targeted to synaptic vesicles and LDCVs, respectively. The subcellular distribution of chimeric hVMAT2/hVAChT molecules was then examined, and the results demonstrate that the cytoplasmic tail of VAChT is necessary and sufficient for synaptic vesicle targeting in PC12 cells. Rat PC12 cells were maintained at 37 °C in an atmosphere of 95% air, 5% CO2 in Dulbecco's modified Eagle's medium containing 7% fetal bovine serum, 7% heat-inactivated horse serum, penicillin (100 units/ml), streptomycin (100 mg/ml), and glutamine (4 mm). The stable PC12 cell line expressing hVAChT used was described previously (34Varoqui H. Erickson J.D. J. Biol. Chem. 1996; 271: 27229-27232Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The hVMAT2 (35Erickson J.D. Schäfer M.K.-H. Bonner T.I. Eiden L.E. Weihe E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5166-5171Crossref PubMed Scopus (361) Google Scholar) and chimeric cDNAs were subcloned into Rc/CMV (Invitrogen) at the HindIII and NotI sites. PC12 cells were transfected with the various constructs using Lipofectin (10 mg/ml; Life Technologies, Inc.), and stable transformants were selected with 0.5 mg/ml Geneticin (G418; Life Technologies, Inc.). G418-resistant colonies were picked and grown overnight on collagen-coated four-chamber permanox slides. Cells were fixed for 2 h in 10% formalin/phosphate-buffered saline (PBS) and incubated with species-specific hVAChT or isoform-specific hVMAT2 antipeptide polyclonal antisera at a final dilution of 1:2000 in PBS, 0.1% Triton, 3% normal goat serum. Primary antibodies were visualized using VectaStain kit (Vector Laboratories Inc.). Polyclonal antipeptide antibodies against the C-terminal amino acids of rVMAT1, hVMAT2, and hVAChT (35Erickson J.D. Schäfer M.K.-H. Bonner T.I. Eiden L.E. Weihe E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5166-5171Crossref PubMed Scopus (361) Google Scholar, 36Weihe E. Schäfer M.K.-H. Erickson J.D. Eiden L.E. J. Mol. Neurosci. 1994; 5: 149-164Crossref PubMed Scopus (192) Google Scholar, 37Schäfer M.K.-H. Weihe E. Erickson J.D. Eiden L.E. J. Mol. Neurosci. 1995; 6: 225-235Crossref PubMed Scopus (78) Google Scholar) were affinity-purified using the immunogenic peptide and SulfoLink kit (Pierce). Polyclonal antibodies against chromogranin B (CgB) were a generous gift of Reiner Fischer-Colbrie (Innsbrück). Ian S. Trowbridge (The Salk Institute, La Jolla, CA) kindly provided the monoclonal antibody (H68.4) against human transferrin receptor (TFR). Monoclonal antibodies directed against synaptophysin (p38) were purchased from Sigma. A unique BstBI restriction site was introduced into the hVAChT cDNA at the junction between the putative 12th transmembrane domain and the cytoplasmic tail (amino acids RN; see Fig. 6) using the method of Deng and Nickoloff (38Deng W.P. Nickoloff J.A. Anal. Biochem. 1992; 200: 81-85Crossref PubMed Scopus (1079) Google Scholar). An equivalent site is found in the wild-type hVMAT2 cDNA. The Escherichia coli BMH 71–18mutS bacterial strain and buffers were provided with the transformer site-directed mutagenesis kit (CLONTECH). Mutagenesis was performed on theSalI/XbaI fragment of hVAChT in pUC18. The mutagenic primer included two nucleotide changes in hVAChT (C1849 to T; C1852 to A) such that a silentBstBI restriction site was added. Transformants were screened by restriction analysis and verified by cDNA double-stranded sequencing using the Sequenase kit (U. S. Biochemical Corp.). The mutated SalI/XbaI fragment was then reintroduced into the hVAChT DNA. The BstBI-NotI fragments containing the cytoplasmic tails of hVAChT and hVMAT2 were then exchanged, and the chimeric cDNAs were subcloned into pRc/CMV for transfection into PC12 cells. Control and PC12 cell lines expressing hVAChT, hVMAT2, and the chimeras were grown to confluence, rinsed with PBS, collected in PBS containing 10 mm EDTA (pH 7.4), and centrifuged at 800 ×g for 10 min. The cell pellets were resuspended and homogenized (Dounce, type B pestle) in ice-cold buffer containing 110 mm potassium tartrate, 20 mm HEPES, 1 mm EGTA, 0.5 mm MgCl2, adjusted to pH 7.0 with KOH, and supplemented with a protease inhibitors mixture (0.2 mm phenylmethylsulfonyl fluoride, 2 mg/ml leupeptin, 2 mg/ml aprotinin, 2 mg/ml pepstatin). The nuclei and unbroken cells were spun out at 800 × g for 10 min, resuspended, homogenized, and spun as before. The two low speed supernatants were combined, and the protein content was measured using the Bradford assay (39Bradford M.M. Anal. Biochem. 1976; 73: 248-254Crossref Scopus (216357) Google Scholar). The levels of expression of the human proteins were assessed on Western blot and with [3H]vesamicol (l-[piperidinyl-3,4-3H]vesamicol) and [3H]TBZOH (α-[O-methyl-3H]dihydrotetrabenazine) binding measurements. For [3H]vesamicol or [3H]TBZOH binding assays, aliquots of PNS (30 μg of protein in 50 μl) or aliquots of the fractions collected from glycerol gradients (50 ml) were mixed with buffer containing 20 mm potassium tartrate, 20 mm HEPES at pH 7.4 (50–100 μl), and various concentrations of [3H]vesamicol (31 Ci/mmol, NEN Life Science Products) or [3H]TBZOH (152 Ci/mmol, Amersham Pharmacia Biotech). After 1-h incubation at 20 °C, the reactions were stopped by vacuum filtration through GF/C glass fiber filters (Whatman) presoaked in 0.5% polyethyleneimine, followed by a 5-ml wash with ice-cold buffer containing 110 mm potassium tartrate and 20 mmHEPES (pH 7.4). Radioactivity bound to the filters was solubilized in 1 ml of 1% SDS and measured by scintillation counting in 10 ml of EcoScint (National Diagnostics). Nonspecific binding was determined in the presence of 30 μml-vesamicol or 1 μm unlabeled tetrabenazine and was subtracted from the total binding. For equilibrium density fractionation, a PNS (2–3 mg of protein) was loaded on a continuous 11-ml 0.6–1.6m (20–46%) sucrose gradient buffered with 20 mm HEPES (pH 7) for sedimentation at 40,000 rpm at 4 °C for 3 h in a SW40 rotor. Twelve 1-ml fractions were collected from the bottom using a fraction recovery system (Beckman). Glycerol velocity fractionation was performed according to the method described by Kelly and co-workers (40Grote E. Hao J.C. Bennett M.K. Kelly R.B. Cell. 1995; 81: 581-589Abstract Full Text PDF PubMed Scopus (145) Google Scholar) with minor modification. Briefly, the PNS was centrifuged at 10,000 × g for 10 min, and the resulting supernatant (about 2 mg of protein) was loaded onto a 4.6-ml 5–25% glycerol gradient prepared in homogenization buffer for a 45-min-long centrifugation at 55,000 rpm in a SW55 rotor. A first fraction equivalent to the volume-loaded, then successive 350-ml fractions, were collected from the top. The linearity of the gradients was verified by refractometry. Equal volumes of each gradient fraction were processed for Western blot analysis (41Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207200) Google Scholar). High speed pellets were prepared from diluted gradient fractions; the sucrose gradient fractions were brought to 320 mm sucrose with 20 mm HEPES (pH 7) and centrifuged for 3 h at 40,000 rpm in a Ti70.1 rotor, while the glycerol gradient fractions were diluted 5-fold in the same buffer and spun for 1 h at 100,000 rpm in a TLA100.2 rotor. Pellets were solubilized in sample buffer containing 62 mm Tris-HCl (pH 6.8), 1 mm EDTA, 10% glycerol, 5% SDS, and 50 mm dithiothreitol. Membrane proteins were separated on 8% polyacrylamide mini-gels and electrotransferred onto nitrocellulose (Hybond-ECL, Amersham Pharmacia Biotech). (CgB and p38 could also be detected on aliquots directly taken from gradient fractions and dissolved in sample buffer.) Following a 1-h preincubation in TBS (20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 0.1% Tween 20) containing 5% non-fat dry milk, the blots were incubated for 2 h at room temperature with affinity-purified hVAChT or hVMAT2 antibodies (1 μg/ml), rVMAT1 antisera (1:500), anti-CgB (1/800), anti-p38 (1/5000), or anti-TFR (8 μg/ml) in TBS-1% bovine serum albumin. Bound primary antibodies were visualized using a monoclonal anti-rabbit antibody or a polyclonal anti-mouse antibody, each coupled to peroxidase (Sigma) and an ECL (Amersham Pharmacia Biotech). Cells were plated on polyornithine (100 μg/ml)-coated glass coverslips and treated with NGF (50 ng/ml; Collaborative Biomedical Products) for 4 days. After fixation in 10% formalin/PBS, cells were washed extensively and permeabilized with 0.1% Triton X-100 in 3% NGS/PBS. Double staining for the transporters and p38 was then performed using the affinity-purified anti-hVAChT or anti-hVMAT2 antisera (1 μg/ml) and anti-p38 (1/300) diluted in the blocking/permeabilization buffer. Primary antibodies were applied for 2 h at room temperature, then the cells were washed with PBS and incubated with fluorescein isothiocyanate-coupled goat anti-rabbit (1/80; Sigma) and tetramethylrhodamine B isothiocyante-coupled goat anti-mouse (1/128; Sigma) in 3% NGS/PBS, for 1 h at room. After final washes, the coverslips were mounted with Cytoseal 60 (Stephens Laboratories) and examined under epifluorescence. We used the method described by Tao-Cheng and co-workers (42Tanner V.A. Ploug T. Tao-Cheng J.-H. J. Histochem. Cytochem. 1996; 44: 1481-1488Crossref PubMed Scopus (60) Google Scholar). Briefly, cells were grown on Matrigel (Collaborative Biomedical Products)-coated eight-well chamber slides and treated with NGF (50 ng/ml) for 14 days. Following fixation in 4% formaldehyde, 0.01% glutaraldehyde (hVAChT labeling) or 2% formaldehyde, 0.1% glutaraldehyde (hVMAT2 labeling), the preparations were washed extensively and stained for hVAChT or hVMAT2 using 3 μg/ml affinity-purified antibodies in 0.1% saponin, 5% NGS/PBS. A secondary anti-rabbit antibody coupled to a 1.4 nm gold particle (Nanoprobe) was applied thereafter and the particles amplified by silver enhancement (HQ kit, Nanoprobes). The human vesicular transporters could be distinguished from the endogenously expressed rVAChT or rVMAT1 in stably transfected PC12 cell lines using species-specific or isoform-specific antisera. In addition, the human proteins were assayed biochemically as the level of specific [3H]vesamicol binding is very low in naive PC12 cells (34Varoqui H. Erickson J.D. J. Biol. Chem. 1996; 271: 27229-27232Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), and the VMAT2 isoform selectively binds TBZOH (35Erickson J.D. Schäfer M.K.-H. Bonner T.I. Eiden L.E. Weihe E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5166-5171Crossref PubMed Scopus (361) Google Scholar). Chimeras of hVAChT and hVMAT2 in which the cytoplasmic tails were exchanged retained the ability to bind these specific inhibitors. The affinity of the V/2 chimera (Kd = 15 ± 3.4 nm; n = 3) for [3H]vesamicol was slightly lower than that of hVAChT (Kd = 4.2 ± 0.7 nm; n = 6) while the affinity of the 2/v chimera (Kd = 5.9 ± 0.3;n = 3) for [3H]TBZOH was not significantly different from that observed for hVMAT2 (Kd = 7.3 ± 0.3 nm;n = 3). The level of expression of hVAChT (Bmax = 7.4 ± 0.7 pmol/mg) was similar to that of hVMAT2 (Bmax = 5.0 ± 1.2 pmol/mg) and approximately four to five times higher than levels of expression of the chimeras (2/v; Bmax = 1.6 ± 0.5 pmol/mg and V/2; Bmax = 1.3 ± 0.4 pmol/mg). This difference in levels of wild-type versuschimeric protein expression was considered advantageous considering the reported saturability of the protein machinery for synaptic vesicle biogenesis (43Grote E. Kelly R.B. J. Cell Biol. 1996; 132: 537-547Crossref PubMed Scopus (75) Google Scholar). To characterize the subcellular distribution of hVAChT and hVMAT2 in PC12 cells, we have fractionated the cells by equilibrium sedimentation through sucrose density gradients (Fig. 1). Immunoblots of the fractions stained with antibodies to chromogranin B (CgB), a soluble protein of LDCVs, and p38 indicated that these proteins predominantly reside as expected in the heavier LDCV and lighter synaptic vesicle fractions of the gradient. In addition, the antipeptide antibody to the endogenous rVMAT1 detected immunoreactive material migrating in the heavy fractions containing CgB. When these immunoblots were stained with antibodies directed against hVMAT2 and hVAChT, these proteins predominantly reside in LDCV- and synaptic vesicle-containing fractions of the gradient, respectively. To localize the hVAChT immunoreactivity that appeared in lighter density fractions on the sucrose gradient, we have used glycerol gradients that separate by sedimentation rate and not by equilibrium density (Fig. 2). Furthermore, velocity sedimentation through glycerol enables separation of endosomal membranes from synaptic vesicles (26Clift-O'Grady L. Linstedt A.D. Lowe A.W. Grote E. Kelly R.B. J. Cell Biol. 1990; 110: 1693-1703Crossref PubMed Scopus (146) Google Scholar). We found that when high speed supernatants of PC12 cells were centrifuged through glycerol, hVAChT sedimented in synaptic vesicle fractions containing p38. The transferrin receptor (TFR) also shuttles between early endosomes and the plasma membrane in PC12 cells. However, the endosomal membranes containing the TFR were excluded from fractions containing synaptic vesicles. A large fraction of the p38 and VAChT in PC12 cells was present in fractions that also contain the transferrin receptor and is consistent with the model that PC12 synaptic vesicles bud from endosomes (31Cameron P.L. Südhof T.C. Jahn R. De Camilli P. J. Cell Biol. 1991; 115: 151-164Crossref PubMed Scopus (240) Google Scholar, 44Linstedt A.D. Kelly R.B. Neuron. 1991; 7: 309-317Abstract Full Text PDF PubMed Scopus (100) Google Scholar, 46Regnier-Vigouroux A. Tooze S.A. Huttner W.B. EMBO J. 1991; 10: 3589-3601Crossref PubMed Scopus (135) Google Scholar). PC12 cells were treated with NGF to induce formation of neural processes. Morphologic examination of hVAChT and hVMAT2 expression of NGF-treated PC12 cells (4 days) by light microscopy and immunofluorescence revealed co-localization of hVAChT and p38 and a distinct pattern of distribution for hVMAT2 (Fig. 3). Double staining with antibodies to hVAChT and p38 showed intense perinuclear localization which was not observed for hVMAT2. Immunoelectron microscopic analysis was performed on cultures of cells treated with NGF for prolonged periods of time (14 days) to generate synaptic vesicle clusters (42Tanner V.A. Ploug T. Tao-Cheng J.-H. J. Histochem. Cytochem. 1996; 44: 1481-1488Crossref PubMed Scopus (60) Google Scholar). Under these conditions, an abundance of hVAChT-immunoreactive gold particles was observed on synaptic vesicles, while hVMAT2 was relatively excluded from these organelles (Fig. 4).Figure 4Localization of hVAChT and 2/v chimera to synaptic vesicle clusters. NGF-treated (14 days), stably transfected PC12-cells were fixed and processed for pre-embedding immunoelectron microscopy. The hVAChT, hVMAT2, and 2/v chimera were visualized using affinity-purified antisera (anti-hVAChT or anti-hVMAT2), 1.4-nm gold particle-coupled anti-rabbit antibody and silver enhancement.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Since hVAChT and hVMAT2 contain information within their primary sequences that selectively target them different secretory organelles in PC12 cells, chimeric molecules can be used to identify the relevant sorting domains. Fractionation of cells expressing chimeras of hVAChT and hVMAT2 in which the entire cytoplasmic tail of these proteins was switched revealed a marked shift in their subcellular distribution. When hVAChT contained the cytoplasmic tail of hVMAT2 (V/2 chimera), its distribution on sucrose density gradients was more similar to that observed for CgB or hVMAT2 than that seen with hVAChT or p38, although some immunoreactivity in intermediate fractions was also seen (Fig. 1). The V/2 chimera was excluded from p38 containing synaptic vesicle fractions on glycerol gradients, indicating that the cytoplasmic tail of hVAChT was necessary for synaptic vesicle targeting (Fig. 2). On the other hand, when the hVMAT2 protein had the cytoplasmic tail of hVAChT (2/v chimera), it co-localized in equilibrium density and glycerol velocity gradient fractions that contain p38, as was observed for hVAChT (Figs.1 and 2). Likewise, the immunocytochemical and electron microscopic distribution of the 2/v chimera resembled hVAChT with intense perinuclear co-localization with p38 (Fig. 3) and presence on synaptic vesicle clusters in NGF-treated cultures (Fig. 4). A further evaluation of the shift in the expression of hVMAT2 from LDCVs to synaptic vesicles due to the presence of the cytoplasmic tail of hVAChT was performed by analyzing the binding of [3H]TBZOH and [3H]vesamicol on membrane fractions from NGF-treated cultures obtained following centrifugation through glycerol (Fig. 5). Synaptic vesicle fractions containing hVAChT could selectively bind high levels of [3H] vesamicol. However, the binding of [3H]TBZOH to synaptic vesicle fractions collected from hVMAT2-expressing cells was low. On the other hand, the binding of [3H]TBZOH to synaptic vesicle fractions containing the 2/v chimera was similar to the level of [3H]vesamicol binding to hVAChT. The binding recovered in the synaptic vesicle fractions represents approximately 13, 3, and 25% of the total binding for hVAChT, hVMAT2, and 2/v chimera, respectively. Thus, while the total level of hVMAT2 expression is more than three times greater than that of the 2/v chimera in the stably transfected cells, the amount of [3H]TBZOH binding to the 2/v chimera in the synaptic vesicle fractions is considerable higher (approximately 4-fold) than that recovered for hVMAT2. These data indicate that the presence of cytoplasmic tail of hVAChT was sufficient for synaptic vesicle targeting of hVMAT2. The trafficking of vesicular transporters into regulatedversus constitutive vesicles and mechanisms of sorting and recycling of regulated secretory organelles can be studied using PC12 cells that express both LDCV and synaptic vesicle proteins. To identify the domain of VAChT, which is required for its preferential targeting to the cholinergic vesicles in PC12 cells, we have (i) examined the steady-state distribution of stably expressed hVAChT and hVMAT2 and found that they differentially sort to synaptic vesicles and LDCVs and (ii) demonstrated that the cytoplasmic domain of hVAChT is necessary for its expression on synaptic vesicles and sufficient to redirect the expression of hVMAT2 from LDCVs to synaptic vesicles. We have shown previously that the endogenously expressed rVAChT is localized to synaptic vesicle clusters in NGF-treated PC12 cells (27Erickson J.D. Weihe E. Schäfer M.K.-H. Neale E. Williamson L. Bonner T.I. Tao-Cheng J.-H. Eiden L.E. Prog. Brain Res. 1996; 109: 69-82Crossref PubMed Google Scholar,28Weihe E. Tao-Cheng J.-H. Schäfer M.K.-H. Erickson J.D. Eiden L.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3547-3552Crossref PubMed Scopus (273) Google Scholar). The localization of hVAChT also to synaptic vesicles in PC12 cells indicates that VAChT contains within its primary amino acid sequence information important for its selective targeting. On the other hand, hVMAT2 is preferentially targeted to LDCVs when expressed in PC12 cells. The localization of VMAT2 to LDCVs in PC12 cells suggests that the sorting mechanisms for VMAT2 are similar to those of VMAT1 and that information required for targeting to DCVs may be contained within regions of the VMAT molecule that are common to both VMAT1 and VMAT2. The soluble proteins and membrane-associated proteins that are absent from the constitutive pathway and synaptic vesicles are thought to sort into the regulated pathway through aggregation at thetrans-Golgi (47Burgess T.L. Kelly R.B. Annu. Rev. Cell Biol. 1987; 3: 243-293Crossref PubMed Scopus (750) Google Scholar). LDCV targeting information may therefore be in the luminal portion of the VMAT molecules as is the case for peptidyl-amidating monooxygenase, a type 1 membrane protein that is found in several forms, including a soluble variant (48Milgram S.L. Johnson R.C. Mains R.E. J. Cell Biol. 1992; 117: 717-728Crossref PubMed Scopus (103) Google Scholar). The lumenal glycosylated loop of hVMAT2 does not interfere with the targeting to synaptic vesicles though when it is present in a hVAChT chimera. 2H. Varoqui and J. D. Erickson, unpublished observation. Information affecting internalization from the plasma membrane may be found in cytoplasmic domains as has been observed for peptidyl-amidating monooxygenase and other exogenous granular membrane proteins such as P-selectin and furin when they are expressed in PC12 cells (49Disdier M. Morrissey J.H. Fugate R.D. Bainton D.F. McEver R.P. Mol. Biol. Cell. 1992; 3: 309-321Crossref PubMed Scopus (127) Google Scholar, 50Milgram S.L. Mains R.E. 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The absence of VMAT2 from synaptic vesicles in transfected PC12 cells suggests that PC12 cells lack some component that is required for VMAT2 targeting to synaptic vesicles. Indeed, SDCVs, the small synaptic vesicles of monoaminergic neurons, may be a third type of regulated secretory vesicle which is absent in PC12 cells (6Bauerfeind R. Jelinek R. Hellwig A. Huttner W.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7342-7346Crossref PubMed Scopus (47) Google Scholar). While studies of synaptic vesicle biogenesis in PC12 cells have led to the conclusion that p38 trafficks from the trans-Golgi to the cell surface via a constitutive vesicle, it is not known whether VAChT trafficks via this route or by some other organelle, such as the LDCV. Some VAChT-asssociated gold particles are observed on LDCVs along the fibers of control and hVAChT-expressing PC12 cells (11Varoqui H. Erickson J.D. Mol. Neurobiol. 1997; 15: 165-191Crossref PubMed Google Scholar, 54Tao-Cheng J.-H. Eiden L.E. Adv. 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Synaptic vesicle membrane proteins may have at least two distinct targeting signals that act sequentially: a plasma membrane internalization signal and a signal for sorting from the endosome to the synaptic vesicle. The information required for endocytosis of p38 lies in its cytoplasmic domain (56Linstedt A.D. Kelly R.B. J. Physiol. (Paris). 1991; 85: 90-96PubMed Google Scholar), but a synaptic vesicle sorting signal has not been identified. Synaptic vesicle biogenesis at the endosome has been suggested to be a result of selective association of synaptic vesicle proteins with one another to form an aggregate that excludes other membrane proteins (26Clift-O'Grady L. Linstedt A.D. Lowe A.W. Grote E. Kelly R.B. J. Cell Biol. 1990; 110: 1693-1703Crossref PubMed Scopus (146) Google Scholar). Proteins in such aggregates might then bud off from the endosomal membranes into vesicles of the correct dimensions. Kelly and co-workers (40Grote E. Hao J.C. Bennett M.K. Kelly R.B. Cell. 1995; 81: 581-589Abstract Full Text PDF PubMed Scopus (145) Google Scholar) have suggested that VAMP-2 (synaptobrevin) is targeted to synaptic vesicles in PC12 cells by associating, via a hydrophobic amphipathic helix in the cytoplasmic domain, with other synaptic vesicle membrane proteins. In the case of VAMP-2, this synaptic vesicle targeting signal is also used for endocytosis (43Grote E. Kelly R.B. J. Cell Biol. 1996; 132: 537-547Crossref PubMed Scopus (75) Google Scholar). The targeting of VAChT to synaptic vesicles in PC12 cells may occur by interaction of the cytoplasmic tail with specific escort proteins of a novel protein-sorting machinery. Several conserved amino acid residues within the cytoplasmic tail of hVAChT (dileucine and tyrosine-based motifs and putative serine phosphorylation site) are potential candidates (Fig. 6) for endocytosis and/or synaptic vesicle targeting signals (57Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. 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