The Vesicular Acetylcholine Transporter Interacts with Clathrin-associated Adaptor Complexes AP-1 and AP-2
2004; Elsevier BV; Volume: 279; Issue: 13 Linguagem: Inglês
10.1074/jbc.m310681200
ISSN1083-351X
AutoresMyoung Hee Kim, Louis B. Hersh,
Tópico(s)Nicotinic Acetylcholine Receptors Study
ResumoIn neuronal cells the neurotransmitter acetylcholine is transferred from the cytoplasm into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). The cytoplasmic tail of VAChT has been shown to contain signals that direct its sorting and trafficking. The role of clathrin-associated protein complexes in VAChT sorting to synaptic vesicles has been examined. A fusion protein between the VAChT cytoplasmic tail and glutathione S-transferase was used to identify VAChT-clathrin-associated protein adaptor protein 1, adaptor protein 2 and adaptor protein 180 complexes from a rat brain extract. In vivo coimmunoprecipitation confirmed adaptin α and adaptin γ complexes, but adaptor protein 180 complexes were not detected by this technique. Deletion and site directed mutagenesis show that the VAChT cytoplasmic tail contains multiple trafficking signals. These include a non-classical tyrosine motif that serves as the signal for adaptin α and a dileucine motif that serves as the signal for adaptin γ. A classical tyrosine motif is also involved in VAChT trafficking, but does not interact with any known adaptor proteins. There appear to be two endocytosis motifs, one involving the adaptor protein 1 binding site and the other involving the adaptor protein 2 binding site. These results suggest a complex trafficking pathway for VAChT. In neuronal cells the neurotransmitter acetylcholine is transferred from the cytoplasm into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). The cytoplasmic tail of VAChT has been shown to contain signals that direct its sorting and trafficking. The role of clathrin-associated protein complexes in VAChT sorting to synaptic vesicles has been examined. A fusion protein between the VAChT cytoplasmic tail and glutathione S-transferase was used to identify VAChT-clathrin-associated protein adaptor protein 1, adaptor protein 2 and adaptor protein 180 complexes from a rat brain extract. In vivo coimmunoprecipitation confirmed adaptin α and adaptin γ complexes, but adaptor protein 180 complexes were not detected by this technique. Deletion and site directed mutagenesis show that the VAChT cytoplasmic tail contains multiple trafficking signals. These include a non-classical tyrosine motif that serves as the signal for adaptin α and a dileucine motif that serves as the signal for adaptin γ. A classical tyrosine motif is also involved in VAChT trafficking, but does not interact with any known adaptor proteins. There appear to be two endocytosis motifs, one involving the adaptor protein 1 binding site and the other involving the adaptor protein 2 binding site. These results suggest a complex trafficking pathway for VAChT. The neurotransmitter acetylcholine (ACh) 1The abbreviations used are: ACh, acetylcholine; VAChT, vesicular acetylcholine transporter; TGN, trans-Golgi network; AP, adapter protein; GST, glutathione S-transferase; SV, synaptic vesicles; SLMV, synaptic-like microvesicles; DTSSP, 3,3-dithio-bis-(sulfosuccinimidylpropionate); CHAPS, 3-[3-cholamidopropyl-dimethylammonio]-1-propane-sulfonate; PBS, phosphate-buffered saline; HA, hemagglutinin. 1The abbreviations used are: ACh, acetylcholine; VAChT, vesicular acetylcholine transporter; TGN, trans-Golgi network; AP, adapter protein; GST, glutathione S-transferase; SV, synaptic vesicles; SLMV, synaptic-like microvesicles; DTSSP, 3,3-dithio-bis-(sulfosuccinimidylpropionate); CHAPS, 3-[3-cholamidopropyl-dimethylammonio]-1-propane-sulfonate; PBS, phosphate-buffered saline; HA, hemagglutinin. is synthesized in the cytosol of cholinergic nerve terminals, transported into synaptic vesicles, and secreted upon calcium influx triggered by an extracellular signal. The vesicular acetylcholine transporter (VAChT) transports this ACh from the cytoplasm into synaptic vesicles in exchange for two protons (1Varoqui H. Erickson J.D. Mol. Neurobiol. 1997; 15: 165-191Crossref PubMed Google Scholar, 2Schuldiner S. Shirvan A. Stern-Bach Y. Steiner-Mordoch S. Yelin S. Laskar O. Ann. N. Y. Acad. Sci. 1994; 15: 174-184Crossref Scopus (5) Google Scholar). VAChT is localized to the membrane of the small synaptic vesicle in central cholinergic neurons (3Gilmor 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) and synaptic-like microvesicles (SLMV) and endosomes in PC12 cells (4Liu Y. Edwards R.H. J. Cell Biol. 1997; 139: 907-916Crossref PubMed Scopus (82) Google Scholar, 5Weihe E. Tao-Cheng J.H. Schafer M.K. Erickson J.D. Eiden L.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 16: 3547-3552Crossref Scopus (272) Google Scholar, 6Eiden L.E. J. Neurochem. 1998; 70: 2227-2240Crossref PubMed Scopus (172) Google Scholar). The trafficking of VAChT has been most thoroughly studied in PC12 cells where it is believed to involve a multistep process. It has been proposed, although not proven, that VAChT, along with synaptophysin (7Régnier-Vigouroux A. Tooze S.A. Huttner W.B. EMBO J. 1991; 10: 3589-3601Crossref PubMed Scopus (134) Google Scholar), is trafficked from the trans-Golgi network (TGN) to the plasma membrane via the constitutive secretory pathway, then from the plasma membrane to an endosomal compartment, and finally from the endosomal compartment to SLMVs (6Eiden L.E. J. Neurochem. 1998; 70: 2227-2240Crossref PubMed Scopus (172) Google Scholar). Membrane trafficking between organelles of the endocytic and secretory pathways is mediated by transport vesicles that shuttle between different compartments. Both the generation of transport vesicles and the selection of protein cargo for inclusion in these vesicles are dependent on the function of coat proteins attached to the cytosolic face of the appropriate membrane (8Rothman J.E. Wieland F.T. Science. 1996; 272: 227-234Crossref PubMed Scopus (1023) Google Scholar, 9Schekman R. Orci L. Science. 1996; 271: 1526-1533Crossref PubMed Scopus (818) Google Scholar, 10Kirchhausen T. Bonifacino J.S. Riezman H. Curr. Opin. Cell Biol. 1997; 9: 488-495Crossref PubMed Scopus (352) Google Scholar). Clathrin-coated vesicles represent the best characterized system of membrane protein trafficking in eukaryotic cells. The major components of clathrin-coated vesicles are clathrin and adaptor protein (AP) complexes. Clathrin provides the structural component while the adaptor protein complexes select the vesicle cargo and promote clathrin-lattice formation onto the respective membrane (11Wilde A. Brodsky F.M. J. Cell Biol. 1996; 135: 635-645Crossref PubMed Scopus (132) Google Scholar) and recruit accessory proteins to the site of vesicle formation. There are five known adaptor protein complexes associated with clathrin; adaptor proteins 1 through 4 (AP-1, AP-2, AP-3, and AP-4) and adaptor protein 180 (AP-180). The AP-1 complexes are associated with the TGN and are involved in the transport of proteins to the endosomal/lysosomal system and to the cell surface. AP-2 complexes are found at the plasma membrane and participate in the internalization of cell surface proteins (12Keen J.H. Annu. Rev. Biochem. 1990; 59: 415-43813Crossref PubMed Scopus (170) Google Scholar, 13Kirchhausen T. Cell. 2002; 109: 413-416Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 14Robinson M.S. Curr. Opin. Cell Biol. 1994; 123: 1093-1105Google Scholar), while AP-180 is found in synaptic vesicles of neuronal cells (15Zhang B. Koh Y.H. Beckstead R.B. Budnik V. Ganetzky B. Bellen H.J. 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Cell. 1995; 82: 773-783Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 22Simpson F. Peden A.A. Christopoulou L. Robinson M.S. J. Cell Biol. 1997; 137: 835-845Crossref PubMed Scopus (306) Google Scholar). Yet another recently described adaptor protein, AP-4, has no known function (24Dell'Angelica E.C. Mullins C. Bonifacino J.S. J. Biol. Chem. 1999; 274: 7278-7285Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 25Hirst J. Bright N.A. Rous B. Robinson M.S. Mol. Biol. Cell. 1999; 10: 2787-2802Crossref PubMed Scopus (217) Google Scholar). Selection of cargo membrane proteins by adaptor protein complexes is dependent on a “sorting signal,” usually located in a cytoplasmic domain of the cargo membrane protein. There are two known sorting signals for interaction with clathrin-coated vesicles, a tyrosine-based sorting signal, YXXØ or NPXY (where X is any amino acid and Ø is a large hydrophobic amino acid (leucine, isoleucine, phenylalanine, methionine, valine) and the dileucine-based sorting signal. Both AP-1 and AP-2 have been found to recognize tyrosine-based and dileucine-based sorting signals (26Mellman I. Curr. Opin. Cell Biol. 1996; 8: 497-498Crossref PubMed Scopus (50) Google Scholar, 27Marks M.S. Ohno H. Kirchhausen T. Bonifacino J.S. Trends Cell Biol. 1997; 7: 124-128Abstract Full Text PDF PubMed Scopus (277) Google Scholar). AP-3 can also recognize the dileucine-sorting motif (28Vowels J.J. Payne G.S. EMBO J. 1998; 17: 2482-2493Crossref PubMed Scopus (93) Google Scholar, 29Darsow T. Burd C.G. Emr S.D. J. Cell Biol. 1998; 142: 913-922Crossref PubMed Scopus (120) Google Scholar, 30Cowles C.R. Odorizzi G. Payne G.S. Emr S.D. Cell. 1997; 91: 109-118Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). It is believed that at least two different sorting machineries are necessary for the trafficking of newly synthesized VAChT from the TGN to its final destination on the synaptic vesicle. Sorting from the TGN to the plasma membrane is suggested to involve the constitutive secretory pathway, and would not require a special sorting system. However, endocytosis from the plasma membrane to an endosomal compartment and intracellular trafficking from the endosomal compartment to synaptic vesicles likely require sorting machineries. Although there is evidence that clathrin-mediated endocytosis contributes to the sorting of VAChT in PC12 cells, the steps and pathway underlying the sorting and trafficking of newly synthesized transporter has yet to be determined. We now report on the identification of clathrin-coated vesicles that are involved in VAChT trafficking. DNA Constructs and Mutagenesis—A cDNA corresponding to the 60 amino acid cytoplasmic carboxyl tail of VAChT (residues 471–530), VAChTCTD, was generated by PCR and subcloned into the EcoR1/NotI restriction sites of the bacterial expression vector pGEX5X-3 by adding appropriate restriction sites to the PCR primers. Wild-type rat VAChT, subcloned into the EcoRI and XbaI sites of pBluescript KS+ (Invitrogen), was used as a template for mutagenesis. Site-directed mutagenesis was performed using the QuikChange Site-directed Mutagenesis kit (Invitrogen). To prepare deletion mutants a stop codon was inserted at the appropriate site of the mutagenic primer. To verify that only the desired mutation was introduced, the mutated portion was sequenced by the ThermoSequenase Radiolabeled Terminator Cycle Sequencing Kit (Amersham Biosciences). Full-length mutant VAChTs were subcloned into the mammalian expression vector pcDNA3 or pcDNA3.1 (Invitrogen), and the 60 amino acid cytoplasmic carboxyl tail of mutant VAChT was subcloned into pGEX5X-3. Cell Culture and Preparation of Stable Transfectants—PC12 cells were cultured in 5% CO2 at 37 °C in RPMI 1640 media containing l-glutamine (Invitrogen), 10 mm HEPES, 1 mm sodium pyruvate, 0.45% glucose, 10% horse serum, 5% bovine calf serum, and 1% penicillin/streptomycin. Electroporation was used for transfection of wild type and mutant VAChT cDNAs into PC12 cells. For electroporation, cells were detached from plates with trypsin/EDTA, washed with ice-cold phosphate-buffered saline (PBS), and resuspended in 800 μl of cold PBS at a cell density of ∼6 × 107 cells/ml. The resuspended cells were mixed with 50 μg of plasmid DNA. After a 10-min incubation on ice, the cell-DNA mixture was transferred to a 0.4-cm gap cuvette (Bio-Rad), electroporated (0.2 kV, 975 microfarads) using a Bio-Rad Gene Pulser II, then replated in culture media and cultured for 48–72 h. Stable transformants were selected with 0.5 mg/ml of Geneticin (Invitrogen) or 0.5 mg/ml of Zeocin (Invitrogen). GST Fusion Protein Production and in Vitro Binding Assays—Wild type and mutant VAChT cytoplasmic tail constructs in pGEX5X-3 were transformed into E. coli BL21. 5 ml of bacteria, grown overnight in LB media containing 100 μg/ml of ampicillin was transferred to 500 ml of 2× YT media (1.6% tryptone, 1% yeast extract, 0.5% NaCl) containing ampicillin and cultured at 37 °C an additional 3–5 h until the OD600 reached 0.5–0.7. Protein expression was then induced by the addition of 0.1 mm isopropyl β-d-thiogalactoside (IPTG) for 1–2 h at 27 °C. After induction bacteria were pelleted, washed, and resuspended in cold PBS containing a protease inhibitor mixture (Roche Applied Science) and 0.2 mm phenylmethylsulfonyl fluoride (Sigma). Bacteria were disrupted with a French Pressure Cell at 500–1000 p.s.i. Cell debris was removed by centrifugation at 14,000 × g for 30 min, and the resulting bacterial lysate was either used immediately or stored at -80 °C until use. GST fusion proteins were purified by incubation with glutathione-agarose beads (Sigma) for 2 h at 4 °C followed by washing with cold PBS. In most experiments the fusion proteins were not eluted from the beads. The amount of bound fusion protein was quantitated with an aliquot after elution from the beads with Coomassie Plus protein assay reagent (Pierce). Rat brain extracts were prepared by homogenization of frozen rat brain (Pel-Freez Biologicals) using a Potter-Elvejhem homogenizer by several up and down strokes in Cytosol Buffer (25 mm Hepes, pH 7, 125 mm potassium acetate, 2.5 mm magnesium acetate, 1 mm dithiothreitol, and 0.1% glucose) containing protease inhibitor mixture (Roche Applied Science), 0.2 mm phenylmethylsulfonyl fluoride (Sigma), 10 mm EDTA, and 0.1% of CHAPS. The homogenate was then centrifuged at 100,000 × g for 30 min and the resulting supernatant used immediately. In vitro interaction assays were performed by incubating 40 μl of glutathione agarose beads containing ∼10 μg of bound GST fusion protein with 1 ml (2 mg) of rat brain homogenate for 14 h at 4 °C. After binding, beads were washed five times in cytosol buffer containing 1% Triton X-100. Bound proteins were then eluted with SDS gel loading buffer (62 mm Tris-HCl, pH 6.8, 1 mm EDTA, 10% glycerol, 5% SDS, and 5% 2-mercaptoethanol) and analyzed by SDS-PAGE and Western blotting. Monoclonal antibodies against adaptin α, adaptin γ, and β-NAP (Transduction Laboratories) and AP-180 (Sigma) were used to detect AP-2, AP-1, AP-3, and AP-180 complexes, respectively. Preparation of Postnuclear Supernatants—Postnuclear supernatants were prepared as described in Kim et al. (31Kim M.H. Lu M. Lim E.J. Chai Y.G. Hersh L.B. J. Biol. Chem. 1999; 274: 673-680Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Briefly, transfected cells were trypsinized and rinsed with ice-cold PBS containing 10 mm EDTA. Cell pellets were resuspended in homogenization buffer containing 0.32 m sucrose, 10 mm HEPES-KOH (pH 7.4), 4% protease inhibitor mixture (Roche Applied Science), and 0.2 mm phenylmethylsulfonyl fluoride. The cell suspension was homogenized on ice with a Potter-Elvejhem homogenizer by several up and down strokes within a 5-min period. Postnuclear supernatants were then collected by centrifugation at 800 × g for 10 min. The protein concentration was measured with the Coomassie Plus protein assay reagent based on the Bradford method (Pierce). Immunoprecipitation—1 ml of postnuclear supernatant (∼5 mg of total protein) was incubated with the cross-linking reagent 3,3-dithiobis-(sulfosuccinimidyl)propionate (DTSSP) at 5 mm for2hat4 °C. After cross-linking, 100 μl of 10% Nonidet P-40 was added and insoluble material removed by centrifugation at 20,000 × g for 20 min. The resulting cell lysate was subjected to immunoprecipitation. The cell lysate was first precleared with 30 μl of protein G-agarose for 1 h at 4 °C, then incubated with anti-VAChT antibody for 1 h, and then for an additional hour after adding protein G-agarose. Immunoprecipitates were collected by centrifugation and washed four times with homogenization buffer supplemented with 100 mm NaCl and 1% Nonidet P-40. The immunoprecipitated proteins were eluted with SDS sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting. Organelle Immunoisolation—Post-nuclear supernatants (500 μl containing ∼250 μg of total protein) derived from wild type or mutant VAChT transfected PC12 cells were precleared with 10 μl of mouse preimmune serum and 10 μl of Dynabeads® M-450 (Dynal ASA, Oslo, Norway) for 1 h at 4 °C. The precleared post nuclear supernatant was incubated with 2 μg of monoclonal antibody to synaptophysin (Chemicon) for 1 h at 4 °C. Dynabeads® M-450 (25 μl bed volume) conjugated to rabbit anti-mouse antibody (Dynal ASA) was added and incubated for 2 h at 4 °C followed by four washes in homogenization buffer. The immunoisolated vesicles were then extracted in SDS sample buffer and subjected to SDS-polyacrylamide gel electrophoresis and Western analysis. Cell Fractionation (Linear Sucrose Gradient Centrifugation)—Post-nuclear supernatant (500 μl; ∼5 mg of total protein) was loaded onto a 10-ml linear sucrose gradient from 0.6 to 1.6 m sucrose in 10 mm HEPES buffer (pH 7.4) and centrifuged at 30,000 rpm for 6 h in an SW40 Ti rotor in a Beckman LE-80 centrifuge at 4 °C. Fractions of 500 μl were collected from the bottom of the tube, 10 μl of each fraction was mixed with 10 μl of 2× SDS sample buffer and subjected to SDS-polyacrylamide gel electrophoresis and Western blotting. A mouse monoclonal antibody against synaptophysin (Chemicon) or a rabbit antiserum against secretogranin II was used to detect synaptic-like microvesicles and large dense core vesicles, respectively. The latter antiserum was a generous gift from Dr. Jonathan Scammell, University of Alabama. A goat polyclonal antibody against VAChT (Chemicon) was used. SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis—For the detection of mutant protein expression, 5 μl of postnuclear supernatant (50 μg of total protein) containing the mutant VAChT was diluted to 500 μl with homogenization buffer and pelleted by centrifugation at 50,000 rpm for 1 h in a TLA-100–3 rotor using a Beckman TL-100 ultracentrifuge. The pellet was resuspended in 20 μl of SDS sample buffer and separated by 10% SDS-PAGE. Separated proteins were electrophoretically transferred to an Immobilon P membrane and blocked with Tris-buffered saline containing 10% nonfat dry milk and 0.1% Tween 20 for 1 h at room temperature. The membrane was sequentially incubated with primary antibody (goat anti-rVAChT antibody), then with peroxidase-conjugated porcine anti-goat antibody for 1 h each at room temperature. Between each incubation, the blot was washed three times for 10 min with Tris-buffered saline and 0.1% Tween 20. Immune complexes were visualized by enhanced chemiluminescence (ECL) (Amersham Biosciences). Immunofluorescence Microscopy—For immunostaining, cells were plated onto glass coverslips coated with 5 μg/ml laminin (Sigma), fixed for 20 min with cold methanol and blocked in PBS containing 5% bovine calf serum and 5% horse serum. The cells were then incubated with primary antibody in the same buffer for 20 min at room temperature, washed three times, incubated an additional 20 min at room temperature with the appropriate secondary antibody in the same buffer, and washed again three times with blocking buffer and three times with PBS. The coverslip were mounted on glass slides with Vectorshield mounting solution. Cells were viewed on a Nikon Eclipse E600 Microscope. To detect the VAChT primary antibody, the secondary antibody was a fluorescein-conjugated sheep anti-goat antibody (Chemicon). For the monoclonal antibody to synaptophysin (Chemicon), Texas red-conjugated bovine anti-mouse antibody (Vector Laboratories) was used. The C-terminal Cytoplasmic Tail of VAChT Interacts with AP Complexes—The cytoplasmic tail of a number of membrane proteins has been shown to contain information for targeting to various intracellular destinations (32Sorkin A. McKinsey T. Shih W. Kirchhausen T. Carpenter G. J. Biol. Chem. 1995; 270: 619-625Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 33Shin J. Doyle C. Yang Z. Kappes D. Strominger J.L. EMBO J. 1990; 9: 425-434Crossref PubMed Scopus (87) Google Scholar, 34Denzer K. Weber B. Hille-Rehfeld A.V. Figura A. Pohlmann R. Biochem. J. 1997; 326: 497-505Crossref PubMed Scopus (32) Google Scholar, 35Varoqui H. Erickson J.D. J. Biol. Chem. 1998; 273: 9094-9098Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The VAChT cytoplasmic carboxyl tail fits into this group as it has been shown to be involved in the targeting of this transporter to synaptic vesicles (35Varoqui H. Erickson J.D. J. Biol. Chem. 1998; 273: 9094-9098Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). This segment of VAChT contains two putative sorting motifs; a dileucine motif preceded by a phosphorylation site and two possible tyrosine-based sorting motifs, Fig. 1A. To analyze for an interaction of the VAChT cytoplasmic tail with adaptor protein (AP) complexes, we initially utilized a fusion protein consisting of the carboxyl cytoplasmic domain of rVAChT fused to GST (GST-VAChTCTD) to pull-down complexes from a rat brain extract. Thus GST-VAChTCTD was mixed with a rat brain extract, precipitated with glutathione agarose, washed, and subjected to SDS-PAGE followed by Western blot analysis. The resolved proteins were probed with antisera to adaptin α (a subunit of the AP-2 complex), adaptin γ (a subunit of the AP-1 complex), β-NAP (a subunit of the AP-3 complex), and AP-180. As shown in Fig. 2, adaptin α, adaptin γ and AP-180, but not β-NAP were co-precipitated with GST-VAChTCTD. GST alone did not form any detectable complexes. To confirm the interaction of the VAChT cytoplasmic tail with specific AP complexes we sought to demonstrate the presence of these complexes in vivo. Although PC12 cells express endogenous VAChT mRNA as detected by PCR, VAChT protein expression is too low to be detected by immunoprecipitation and Western blot analysis. Therefore, as done in other studies (4Liu Y. Edwards R.H. J. Cell Biol. 1997; 139: 907-916Crossref PubMed Scopus (82) Google Scholar, 35Varoqui H. Erickson J.D. J. Biol. Chem. 1998; 273: 9094-9098Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) full-length VAChT was stably transfected into PC12 cells to produce PC12VAChT. In general cargo protein-adaptor protein complex interactions are transient and unstable. A post-nuclear supernatant prepared from PC12VAChT was therefore treated with the homobifunctional cross-linking reagent DTSSP for 2 h at 4 °C to fix complexes. After lysis of the vesicles, samples were immunoprecipitated with anti-VAChT antisera and subjected to SDS-PAGE followed by Western blot analysis. This procedure led to the identification of adaptin α and adaptin γ complexes, Fig. 3. No complexes or VAChT protein were detected with non-transfected PC12 cells, nor was an AP-180 complex detected by this procedure. Since PC12 cells contain far less AP-180 than rat brain (data not shown), this experiment cannot exclude VAChT interacting with AP-180 in neurons. Analysis of VAChT Deletion Mutants—Based on the experiments described above, it was possible to demonstrate that the VAChT C-terminal cytoplasmic tail forms specific AP complexes. We next set out to identify the sequence(s) within the C-terminal tail of VAChT that interacts with each AP complex. As noted above and shown in Fig. 1, VAChT contains both putative dileucine and tyrosine motifs that could be involved in adaptor complex formation. A series of deletion mutants were constructed and are shown in Fig. 1B. The mutant VAChTΔC11 has the most C-terminal putative tyrosine-sorting motif deleted, the mutant VAChTΔC37 has both C-terminal putative tyrosine-sorting motifs deleted, while the mutant VAChTΔC53 has the above deletions plus the dileucine motif and the phosphorylation site deleted. The interaction with adaptin α was essentially eliminated in all of the mutants, even VAChTΔC11, in which the C-terminal 11 amino acids that contain a putative tyrosine motif was deleted. This finding suggests that the C-terminal 11 amino acids contain the AP-2 interaction signal. The adaptin γ interaction was diminished in the VAChTΔC11 and VAChTΔC37 deletion constructs, and was absent in the VAChTΔC53 construct in which the phosphorylation site and the dileucine motif were deleted (Fig. 4). However, all of these mutants exhibited interaction with AP-180 and since together they represent deletion of all but the first 8 amino acids of the C-terminal tail we consider the interaction with AP-180 as being nonspecific. Supporting this suggestion is the finding that a construct in which the first 8 amino acids (residues 471–478) of VAChT were deleted still bound to AP-180. We examined the consequence of eliminating the interaction with adaptin α or adaptin γ by examining the trafficking of these deletion mutants in PC12 cells. Full-length VAChT containing each of the deletion mutants was constructed in the expression vector pcDNA3.1. Since the C-terminal truncations eliminate the epitope recognized by available VAChT antibodies, the hemagglutinin epitope (HA), YPYDVPDYA, was attached to the N-terminal of VAChT for detection purposes. Since the HA epitope contains tyrosine residues that might affect trafficking we examined trafficking of wild type and VAChTΔC11 with and without the HA epitope. The VAChTΔC11 mutant reacted with the anti-C-terminal VAChT antibody. We found no difference. This observation is in agreement with the data of Tan et al. (36Tan P.K. Waites C. Liu Y. Krantz D.E. Edwards R.H. J. Biol. Chem. 1998; 273: 17351-17360Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) who also showed that the HA epitope did not affect VAChT subcellular localization in PC12 cells. Immunofluorescence was used to compare the trafficking of the deletion mutants. As shown in Fig. 5, all of the mutants exhibit a different subcellular localization from wild type VAChT, which is found in a perinuclear region colocalized with synaptophysin, the marker for synaptic-like microvesicles. PC12 cells expressing the VAChTΔC11 mutant, in which the interaction with AP-2 was eliminated and the interaction with AP-1 was diminished, showed staining mostly on the plasma membrane, but a small fraction still colocalized with synaptophysin. This expression pattern appeared very similar to what we observed with a dileucine mutant, Fig. 5. However, PC12 cells expressing the VAChTΔC37 or the VAChTΔC53 mutant exhibited staining throughout the cell. The mis-sorting of the VAChTΔC11 and VAChTΔC37 mutants was further confirmed by sucrose density gradient fractionation of transiently transfected PC12 cells. As shown in Fig. 6, transiently transfected wild type VAChT colocalized with synaptophysin the marker for synaptic-like microvesicles. In agreement with immunofluorescent staining the VAChTΔC11 mutant was partially colocalized with synaptophysin, but was reproducibly shifted one to two fractions in the direction of the more dense part of the sucrose gradient. In a similar analysis the VAChTΔC37 mutant was clearly mislocalized appearing as two distinct peaks in different parts of the sucrose gradient. Neither peak co-migrated with either secretogranin II or synaptophysin.Fig. 6Sucrose linear gradient fractionation of wild type and deletion mutant VAChT. Post-nuclear supernatants from wild type or rVAChT mutant-transfected PC12 cells were fractionated on a 0.6–1.6 M continuous sucrose gradient. Fractions were probed with anti-secretogranin II antisera (SgII) to locate large dense core vesicles, anti-sy
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