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• W2023604322 abstract "Specific transport proteins mediate the packaging of neurotransmitters into secretory vesicles and consequently require targeting to the appropriate intracellular compartment. To identify residues in the neuron-specific vesicular monoamine transporter (VMAT2) responsible for endocytosis, we examined the effect of amino (NH2-) and carboxyl (COOH-)-terminal mutations on steady state distribution and internalization. Deletion of a critical COOH-terminal domain sequence (AKEEKMAIL) results in accumulation of VMAT2 at the plasma membrane and a 50% reduction in endocytosis. Site-directed mutagenesis shows that replacement of the isoleucine-leucine pair within this sequence by alanine-alanine alone reduces endocytosis by 50% relative to wild type VMAT2. Furthermore, the KEEKMAIL sequence functions as an internalization signal when transferred to the plasma membrane protein Tac, and the mutation of the isoleucine-leucine pair also abolishes internalization of this protein. The closely related vesicular acetylcholine transporter (VAChT) contains a similar di-leucine sequence within the cytoplasmic COOH-terminal domain that when mutated results in accumulation of VAChT at the plasma membrane. The VAChT di-leucine sequence also confers internalization when appended to two other proteins and in one of these chimeras, conversion of the di-leucine sequence to di-alanine reduces the internalization rate by 50%. Both VMAT2 and VAChT thus use leucine-based signals for efficient endocytosis and as such are the first synaptic vesicle proteins known to use this motif for trafficking. Specific transport proteins mediate the packaging of neurotransmitters into secretory vesicles and consequently require targeting to the appropriate intracellular compartment. To identify residues in the neuron-specific vesicular monoamine transporter (VMAT2) responsible for endocytosis, we examined the effect of amino (NH2-) and carboxyl (COOH-)-terminal mutations on steady state distribution and internalization. Deletion of a critical COOH-terminal domain sequence (AKEEKMAIL) results in accumulation of VMAT2 at the plasma membrane and a 50% reduction in endocytosis. Site-directed mutagenesis shows that replacement of the isoleucine-leucine pair within this sequence by alanine-alanine alone reduces endocytosis by 50% relative to wild type VMAT2. Furthermore, the KEEKMAIL sequence functions as an internalization signal when transferred to the plasma membrane protein Tac, and the mutation of the isoleucine-leucine pair also abolishes internalization of this protein. The closely related vesicular acetylcholine transporter (VAChT) contains a similar di-leucine sequence within the cytoplasmic COOH-terminal domain that when mutated results in accumulation of VAChT at the plasma membrane. The VAChT di-leucine sequence also confers internalization when appended to two other proteins and in one of these chimeras, conversion of the di-leucine sequence to di-alanine reduces the internalization rate by 50%. Both VMAT2 and VAChT thus use leucine-based signals for efficient endocytosis and as such are the first synaptic vesicle proteins known to use this motif for trafficking. Endocytosis promotes the rapid and efficient internalization of many plasma membrane proteins. In addition, endocytosis contributes to the trafficking of membrane proteins that do not normally reside at the cell surface. For example, endocytosis retrieves the trans-Golgi network (TGN) 1The abbreviations used are: TGN, trans-Golgi network; AP, adaptor protein complex; CMF-PBS, calcium/magnesium-free phosphate-buffered saline; FITC, fluorescein isothiocyanate; HA, hemaglutinin; LDCV, large dense core vesicle; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PNS, post-nuclear supernatant; Tac, interleukin-2 receptor α-subunit; VAChT, vesicular acetylcholine transporter; VMAT, vesicular monoamine transporter; ELISA, enzyme-linked immunosorbent assay; CHO, Chinese hamster ovary; BSA, bovine serum albumin. and endosomal proteins TGN38, furin, and the mannose 6-phosphate receptors from the plasma membrane, where they appear at low levels (1Kornfeld S. Annu. Rev. Biochem. 1992; 61: 307-330Crossref PubMed Scopus (934) Google Scholar, 2Reaves B. Horn M. Banting G. Mol. Biol. Cell. 1993; 4: 93-105Crossref PubMed Scopus (108) Google Scholar, 3Vorhees P. Deignan E. van Donselaar E. Humphrey J. Marks M.S. Peters P.J. Bonifacino J.S. EMBO J. 1995; 14: 4961-4975Crossref PubMed Scopus (187) Google Scholar). Retrieval from the plasma membrane thus contributes to the steady-state accumulation of these proteins in the TGN and endosomes. Another class of proteins appears at the cell surface only after stimulation and requires endocytosis to reform the specialized secretory vesicles in which they usually reside. Neurons contain two types of secretory vesicle that undergo regulated exocytosis. Synaptic vesicles, or synaptic-like microvesicles in endocrine cells, store classical neurotransmitters (4Sollner T. Rothman J.E. Trends Neurosci. 1994; 17: 344-348Abstract Full Text PDF PubMed Scopus (156) Google Scholar, 5Scheller R.H. Neuron. 1995; 14: 893-897Abstract Full Text PDF PubMed Scopus (196) Google Scholar, 6Sudhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1768) Google Scholar). Large dense core vesicles (LDCVs), or secretory granules in endocrine cells, store neuromodulators such as neural peptides, hormones, and the monoamine neurotransmitters (7Martin T.F.J. Curr. Opin. Neurobiol. 1994; 4: 626-630Crossref PubMed Scopus (85) Google Scholar). Despite a common function in regulated exocytosis, synaptic vesicles and LDCVs differ in their biogenesis. Synaptic vesicles form through recycling of their integral membrane proteins at the nerve terminal (8De Camilli P. Jahn R. Annu. Rev. Physiol. 1990; 52: 625-645Crossref PubMed Scopus (367) Google Scholar). Indeed, newly synthesized synaptic vesicle proteins traffick via the constitutive secretory pathway to the plasma membrane before they appear in synaptic vesicles (9Regnier-Vigouroux A. Tooze S.A. Huttner W.B. EMBO J. 1991; 10: 3589-3601Crossref PubMed Scopus (135) Google Scholar, 10Matteoli M. Takei K. Perin M.S. Sudhof T.C. De Camilli P. J. Cell. Biol. 1992; 117: 849-861Crossref PubMed Scopus (283) Google Scholar). In contrast, LDCVs derive directly from the TGN as part of the regulated secretory pathway (11Tooze S.A. Chanat E. Tooze J. Huttner W.B. Loh P. Mechanisms of Intracellular Trafficking and Processing of Proproteins. CRC Press, Boca Raton, FL1993: 157-177Google Scholar). In the TGN, LDCV proteins sort to the regulated secretory pathway and away from the constitutive secretory pathway (12Cutler D.F. Cramer L.P. J. Cell Biol. 1990; 110: 721-730Crossref PubMed Scopus (64) Google Scholar). Thus, endocytosis does not appear to have a direct role in LDCV formation. However, endocytosis presumably functions to retrieve LDCV membrane proteins after exocytosis. Indeed, the LDCV proteins glycoprotein III and ICA512 reappear in secretory granules after exposure at the cell surface (13Patzak A. Winkler H. J. Cell Biol. 1986; 102: 510-515Crossref PubMed Scopus (123) Google Scholar, 14Solimena M. Dirkyx Jr., R. Hermel J.M. Pleasic-Williams S. Shapiro J.A. Caron L. Rubin D.U. EMBO J. 1996; 15: 2102-2114Crossref PubMed Scopus (229) Google Scholar). Membrane proteins subject to efficient endocytosis contain specific, cytoplasmically disposed amino acid sequences for internalization (15Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (704) Google Scholar). Mutagenesis studies have shown that endocytic targeting often involves either a tyrosine- or leucine-based motif. For example, mutagenesis of tyrosine 807 in the low density lipoprotein receptor disrupts localization to clathrin-coated pits and so prevents the uptake of low density lipoprotein (16Davis C.G. Lehrman M.A. Russell D.W. Anderson R.G.W. Brown M.S. Goldstein J.L. Cell. 1986; 45: 15-24Abstract Full Text PDF PubMed Scopus (241) Google Scholar, 17Davis C.G. van Driel I.R. Russell D.W. Brown M.S. Goldstein J.L. J. Biol. Chem. 1987; 262: 4075-4082Abstract Full Text PDF PubMed Google Scholar). In addition, replacement of leucines 131 and 132 in CD3γ with alanine blocks endocytosis (18Letourneur F. Klausner R.D. Cell. 1992; 69: 1143-1157Abstract Full Text PDF PubMed Scopus (461) Google Scholar, 19Dietrich J. Kastrup J. Nielsen B.L. Odum N. Geisler C. J. Cell Biol. 1997; 138: 271-281Crossref PubMed Scopus (157) Google Scholar). Tyrosine and leucine-based motifs apparently bind to the clathrin adaptor protein AP-2 adaptor which directs the membrane proteins into clathrin-coated pits (19Dietrich J. Kastrup J. Nielsen B.L. Odum N. Geisler C. J. Cell Biol. 1997; 138: 271-281Crossref PubMed Scopus (157) Google Scholar, 20Glickman J.N. Conibear E. Pearse B.M.F. EMBO J. 1989; 8: 1041-1047Crossref PubMed Scopus (207) Google Scholar, 21Ohno H. Stewart J. Fournier M.-C. Bosshart H. Rhee I. Miyatake S. Saito T. Galluser A. Kirchhausen T. Bonifacino J. Science. 1995; 269: 1872-1875Crossref PubMed Scopus (826) Google Scholar, 22Heilker R. Manning-Krieg U. Zuber J.F. Speiss M. EMBO J. 1996; 15: 2893-2899Crossref PubMed Scopus (156) Google Scholar). Mutation of the di-leucine motif in CD3γ disrupts the interaction with AP-2, supporting a role for this interaction in endocytosis (19Dietrich J. Kastrup J. Nielsen B.L. Odum N. Geisler C. J. Cell Biol. 1997; 138: 271-281Crossref PubMed Scopus (157) Google Scholar). The sequences required for internalization of synaptic vesicle and LDCV membrane proteins have not previously been identified. We have now examined the endocytosis of two vesicular proteins that function to package classical neurotransmitters into secretory vesicles prior to regulated exocytosis (23Schuldiner S. Shirvan A. Linial M. Physiol. Rev. 1995; 75: 369-392Crossref PubMed Scopus (266) Google Scholar, 24Liu Y. Edwards R.H. Annu. Rev. Neurosci. 1997; 20: 125-156Crossref PubMed Scopus (237) Google Scholar). These proteins use a proton electrochemical gradient generated by the vacuolar H+-ATPase to drive the active transport of neurotransmitter into vesicles. Molecular cloning has identified two vesicular monoamine transporters (VMAT1 and 2) and a closely related vesicular acetylcholine transporter (VAChT) (25Liu Y. Peter D. Roghani A. Schuldiner S. Prive G.G. Eisenberg D. Brecha N. Edwards R.H. Cell. 1992; 70: 539-551Abstract Full Text PDF PubMed Scopus (524) Google Scholar, 26Erickson J.D. Eiden L.E. Hoffman B.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10993-10997Crossref PubMed Scopus (426) Google Scholar, 27Erickson J.D. Varoqui H. Schafer M.K. Modi W. Diebler M.F. Weihe E. Rand J. Eiden L.E. Bonner T.I. Usdin T.B. J. Biol. Chem. 1994; 269: 21929-21932Abstract Full Text PDF PubMed Google Scholar, 28Roghani A. Feldman J. Kohan S.A. Shirzadi A. Gunderson C.B. Brecha N. Edwards R.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10620-10624Crossref PubMed Scopus (173) Google Scholar). VMAT1 occurs in non-neural cells such as chromaffin cells of the adrenal medulla whereas VMAT2 occurs in monoamine neurons. The sequences of the VMATs and VAChT show 41% amino acid identity and predict 12 transmembrane segments flanked by cytoplasmic amino (NH2)- and carboxyl (COOH)-terminal domains. In addition, a large lumenal loop occurs between transmembrane segments 1 and 2. Despite these similarities in structure and function, VMATs and VAChT localize to distinct secretory vesicles. In PC12 cells, VMATs occur predominantly in LDCVs (29Liu 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) whereas VAChT resides predominantly in synaptic-like microvesicle (30Liu Y. Edwards R.H. J. Cell Biol. 1997; 139: 907-916Crossref PubMed Scopus (82) Google Scholar). Localization of these proteins to distinct compartments presumably derives from different sorting signals. To assess the role of endocytosis in the distribution of VMAT2, we have produced mutations within the cytoplasmic NH2 and COOH termini and examined their effect on internalization. We find that an isoleucine-leucine pair within the COOH-terminal domain is required for the intracellular localization and efficient endocytosis of VMAT2. Similarly, a leucine-leucine pair within the COOH-terminal domain of VAChT also functions as an endocytic signal. These transporters thus use leucine-based sequences for efficient endocytosis. All cells were maintained in 5% CO2 at 37 °C in medium containing penicillin and streptomycin. PC12 cells were grown in Dulbecco's modified Eagle's-H21 medium supplemented with 5% Cosmic calf serum and 10% equine serum (Hyclone, Logan, UT) and were transfected by electroporation at 250 V and 500 microfarads as described previously (31Grote E. Hao J.C. Bennett M.K. Kelly R.B. Cell. 1995; 81: 581-589Abstract Full Text PDF PubMed Scopus (145) Google Scholar). COS1 cells were grown in Dulbecco's modified Eagle's-H21 medium with 10% Cosmic calf serum and were transfected with 10 μg of DNA per 15-cm plate by electroporation at 300 V and 950 microfarads in phosphate-buffered saline (PBS). CHO cells were maintained in Ham's F-12 media supplemented with 5% Cosmic calf serum and were transfected using LipofectAMINE (Life Technologies, Inc., Grand Island, NY). For CHO transfection, 0.5–1 μg of plasmid DNA was incubated with 3 μl of LipofectAMINE in 20 μl of Opti-MEM media (Life Technologies, Inc.) for 20 min at room temperature. 500 μl of Opti-MEM media was then added and the lipid-DNA complexes were transferred to cells grown on poly-l-lysine-coated glass coverslips. After incubation of the cells at 37 °C for 6 h, an equal volume of Ham's F-12 media supplemented with 10% Cosmic calf serum without antibiotics was added. Following overnight incubation, the media was removed and replaced by regular CHO cell media. All cell lines were assayed 1.5 to 3 days after transfection. Mutagenesis was performed either by standard polymerase chain reaction (PCR) techniques using Pfu polymerase (Stratagene, La Jolla, CA) or by the Kunkel method (32Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-381Crossref PubMed Scopus (4558) Google Scholar) using single-stranded DNA prepared according to Ref. 33Tan P.K. Howard J.P. Payne G.S. J. Cell Biol. 1996; 135: 1789-1800Crossref PubMed Scopus (96) Google Scholar. The sequences of mutagenic oligonucleotides are available upon request. The dideoxy sequencing method was used to verify all the desired mutations and to exclude unwanted mutations. cDNAs were cloned into pcDNA1/Amp (Invitrogen, Carlsbad, CA). To facilitate the subcloning of VMAT2 mutants, we used a cDNA with two silent mutations that create aBglII and a SalI site at nucleotides 440 and 1302, respectively (34Finn III, J.P. Edwards R.H. J. Biol. Chem. 1997; 272: 16301-16307Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). A sequence encoding the hemaglutinin epitope, YPYDVPDYA, was inserted after the codon for glycine 96 in the VMAT2 cDNA (35Krantz D.E. Peter D. Liu Y. Edwards R.H. J. Biol. Chem. 1997; 272: 6752-6759Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and after the codon for glycine 105 in the VAChT cDNA (28Roghani A. Feldman J. Kohan S.A. Shirzadi A. Gunderson C.B. Brecha N. Edwards R.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10620-10624Crossref PubMed Scopus (173) Google Scholar). The MAc chimeric transporter was produced by first using PCR to introduce a ClaI site at nucleotide 1450 of the VMAT2 cDNA. A PCR fragment corresponding to nucleotides 1493–1860 of VAChT cDNA was then subcloned into the junction usingClaI and XbaI, replacing nucleotides 1456–1637 of VMAT2. This construct encodes amino acids 1–461 of VMAT2 (the NH2-terminal and 12 transmembrane domains) followed by the COOH-terminal residues 460–530 of VAChT. The Tac chimeras were produced by first using PCR to introduce anXbaI site at nucleotide 982 within the Tac (interleukin-2 receptor α-subunit) cDNA (36Leonard W.J. Depper J.M. Crabtree G.R. Rudikoff S. Pumphrey J. Robb R.J. Kronke M. Svetlik P.B. Peffer N.J. Waldmann T.A. Greene W.C. Nature. 1984; 311: 626-631Crossref PubMed Scopus (606) Google Scholar) (a gracious gift of Maria Warmerdam and Warner Greene, University of California, San Francisco). PCR fragments from either VMAT2 or VAChT were then subcloned into this site using XbaI. All chimeras encode the full-length unmodified Tac protein fused either to the VMAT2 COOH terminus beginning with Lys-477, the VAChT COOH terminus beginning with Arg-479, or the 8 amino acid peptides corresponding to the VMAT2 leucine-based endocytosis motif. The transport activity of VMAT2 mutants was measured in membranes from COS1 cells. One day before membrane preparation, the medium of transfected cultures was replaced by fresh medium. To prepare membranes, cells from a 10-cm plate at 80% confluency were washed in calcium/magnesium-free phosphate-buffered saline (CMF-PBS), detached from the plate with trypsin in CMF-PBS, collected by centrifugation, and resuspended in 200 μl of cold 10 mm HEPES-KOH, pH 7.4, 0.32 m sucrose containing 2 μg/ml leupeptin, and 0.2 mm diisopropyl fluorophosphate. The cell suspension was then disrupted in a chilled water bath sonicator (Branson, Danbury, CT) at medium intensity for 30 s and the cell debris removed by sedimentation at 1000 ×g for 5 min at 4 °C. The postnuclear supernatant (PNS) was then transferred to a fresh tube. To measure transport activity, the uptake of [1,2-3H]serotonin (NEN Life Science Products, Boston, MA) was assayed as described previously (34Finn III, J.P. Edwards R.H. J. Biol. Chem. 1997; 272: 16301-16307Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) using 10 μl of PNS. For immunoblotting, 50 μl of PNS from the transport assay was sedimented at 100,000 × g for 1 h at 4 °C. The membrane pellet was resuspended in 3 × SDS sample buffer (New England Biolabs, Beverly, MA), incubated at room temperature for 5 min, and 100 μg separated by electrophoresis through 7% SDS-polyacrylamide gel electrophoresis gels. After electrophoresis, the proteins were transferred to nitrocellulose and HA-tagged VMAT2 visualized by enhanced chemiluminescence (Pierce, Rockford, IL) using monoclonal anti-HA.11 antibodies (Babco, Berkeley, CA) at a 1:2000 dilution and secondary horseradish peroxidase-conjugated anti-mouse antibodies diluted 1:2000 (Amersham, Arlington Heights, IL). Immunofluorescence was performed using transfected PC12 or CHO cells grown to 20–50% confluence on poly-l-lysine-coated glass coverslips. For steady-state localization, cells were fixed with 4% paraformaldehyde in 0.1 m sodium phosphate, pH 7.2, at 4 °C for 20 min and permeabilized at room temperature for 40 min in CMF-PBS containing 0.02% saponin, 2% bovine serum albumin, and 1% fish skin gelatin (IF buffer). Cells were then incubated for 1 h with monoclonal anti-HA.11 antibodies diluted 1:250, polyclonal anti-VMAT2 antibodies diluted 1:100 (39Peter D. Liu Y. Sternini C. de Giorgio R. Brecha N. Edwards R.H. J. Neurosci. 1995; 15: 6179-6188Crossref PubMed Scopus (260) Google Scholar), or polyclonal anti-VAChT antibodies diluted 1:500–1,000 (30Liu Y. Edwards R.H. J. Cell Biol. 1997; 139: 907-916Crossref PubMed Scopus (82) Google Scholar) in IF buffer. After three 10-min washes in IF buffer, cells were incubated with appropriate secondary antibodies conjugated to fluorescein isothiocyanate (FITC) or rhodamine (ICN/Cappell, Costa Mesa, CA) at 1:250 in IF buffer. Cells were then washed twice in IF buffer for 10 min each, rapidly rinsed twice in PBS, and the coverslips mounted in Slowfade (Molecular Probes, Eugene, OR). To assess endocytosis, intact cells were incubated at 4 °C for 1 h with monoclonal anti-HA or monoclonal anti-interleukin-2 (Tac) antibodies (Babco) diluted 1:250 in standard medium, and then washed three times in ice-cold PBS. The cells were then either fixed as described above or incubated in medium at 37 °C for 1 h before fixation. After fixation and permeabilization, cells were incubated with polyclonal antibodies to VMAT2 or VAChT followed by simultaneous incubation with FITC-conjugated antibodies to mouse Ig and rhodamine-conjugated antibodies to rabbit Ig. The mounted coverslips were examined by epifluorescence at × 400 magnification. The internalization assay was performed as described previously (37Smythe E. Redelmeier T.E. Schmid S.L. Methods Enzymol. 1992; 219: 223-234Crossref PubMed Scopus (40) Google Scholar, 38Lamaze C. Fujimoto L.M. Yin H.L. Schmid S.L. J. Biol. Chem. 1997; 272: 20332-20335Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar), with minor modifications. Affinity-purified monoclonal HA.11 antibody (Berkeley Antibody Co.) was biotinylated using the Molecular Probes FluoReporter mini-biotin XX protein labeling kit according to the manufacturer's instructions. The wells of 96-well ELISA plates (Nunc, Thousand Oaks, CA) were coated for 3 h at 37 °C with 200 μl of goat anti-mouse IgG (Calbiochem, La Jolla, CA) diluted 1:1000 in 50 mm sodium bicarbonate, pH 9.6. After rinsing twice in PBS, the wells were blocked for 1 h at 37 °C with 200 μl of 10 mm Tris, pH 7.4, 50 mm NaCl, 1% Triton X-100, 0.1% SDS, and 0.2% BSA (blocking buffer) and stored at 4 °C. To measure endocytosis, each 15-cm plate of transiently transfected COS1 cells at 80–90% confluence was rinsed in CMF-PBS and incubated for 5 min in CMF-PBS containing 5 mm EDTA. After addition of an equal volume of Dulbecco's modified Eagle's-H21 medium containing 20 mm HEPES-KOH, pH 7.2, and 0.2% BSA (SFM), the cells were harvested mechanically, sedimented at 1000 rpm for 3 min in a table top centrifuge (Beckman, Palo Alto, CA), resuspended in 1 ml of SFM prewarmed to 37 °C and transferred to 1.5 ml in Eppendorf tubes. 100 μl of biotinylated HA antibody (25 μg/ml) in SFM was then added and the cells were incubated at 37 °C on a rotator (Barnstead/Thermolyne, Dubuque, IA). At various times after addition of the antibody, a 200-μl cell suspension was transferred to pre-chilled Eppendorf tubes containing 5 μl of 1 m sodium azide and 5 μl of 1 m sodium fluoride to arrest endocytosis. The cells were then pelleted, the unbound antibody removed by washing in ice-cold PBS containing 0.2% BSA, and divided in two equal aliquots. One aliquot was incubated at 4 °C for 1 h in 0.1 ml of PBS, 0.2% BSA while the other aliquot was incubated at 4 °C for 1 h on a rotator with 0.1 ml of PBS, 0.2% BSA containing 50 μg/ml avidin to sequester cell surface-associated biotin. After this treatment, 10 μl of 0.5 mg/ml biocytin in PBS, 0.2% BSA was added and the cells incubated on the rotator for an additional 15 min. The cells were then solubilized by the addition of 100 μl of blocking buffer and 90 μl was transferred in duplicate to the ELISA plates for an overnight incubation at 4 °C. The next day, the wells were washed twice in PBS, once in blocking buffer for 5 min, twice more in PBS, and then incubated for 1 h in blocking buffer containing streptavidin-horseradish peroxidase conjugate diluted 1:5000 (Boehringer Mannheim, Indianapolis, IN). After washing in PBS, 0.2% BSA, blocking buffer and PBS, 0.2% BSA, the wells were incubated for 2 min in 200 μl of 2 mm o-phenyldiamine-HCl and 0.01% hydrogen peroxide in 50 mm dibasic sodium phosphate, pH 5.0, 27 mm sodium citrate. 50 μl of sulfuric acid was added to stop the reaction and the absorbance at 490 nm determined using a kinetic microplate reader (Molecular Devices Corp., Sunnyvale, CA). Absorbance readings for each sample were quantified from standard curves of biotinylated HA antibody on each ELISA plate using the SOFTmax PRO software program (Molecular Devices). Standard curves showed linearity up to 40 ng, and the amount of antibody associated with transfected cells not treated with avidin ranged from 5 to 20 ng. Control cells transfected with vector alone bound 0.2–1 ng of antibody. The percentage of internalized biotinylated antibody was calculated using the equation: [(Ca −Va)/(Ct −Vt)] × 100, where Ca andCt are amounts of antibody associated with transfected cells incubated with and without avidin, respectively, andVa and Vt the amounts of antibody associated with control cells incubated with and without avidin. To identify the sequences in VMAT2 responsible for internalization, we produced deletions and point mutations in the cytoplasmic NH2 and COOH termini (Fig. 1 A). Since COOH-terminal truncations eliminate the epitope recognized by available VMAT2 antibodies (39Peter D. Liu Y. Sternini C. de Giorgio R. Brecha N. Edwards R.H. J. Neurosci. 1995; 15: 6179-6188Crossref PubMed Scopus (260) Google Scholar), we inserted a hemaglutinin (HA) epitope tag into the large lumenal loop between transmembrane segments 1 and 2 (35Krantz D.E. Peter D. Liu Y. Edwards R.H. J. Biol. Chem. 1997; 272: 6752-6759Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and used this tagged gene to produce all of the VMAT2 constructs (Fig. 1 A). Importantly, the HA tag neither perturbs the activity of VMAT2 nor affects its subcellular localization in PC12 cells. 2P. K. Tan, C. Waites, Y. Liu, D. E. Krantz, and R. H. Edwards, unpublished observations. The lumenal orientation of this epitope also enables us to monitor plasma membrane localization and endocytosis of the wild type and mutant proteins. Fig. 1 A lists the informative mutations and summarizes the findings. To assess the effect of the mutations on VMAT2 processing, we analyzed the expression and activity of VMAT2 mutants from extracts of transiently transfected COS1 cells. Immunoblotting with anti-HA antibody (Fig. 2 A) reveals low and high molecular weight forms of VMAT2 (lanes 2–7) that are absent from untransfected cells (lane 1). The smaller 55-kDa form of wild type VMAT2 (lane 2, upper arrow) is sensitive to digestion with endoglycosidase H (endo H)2 and so presumably resides in the endoplasmic reticulum. In contrast, the larger 75-kDa species (arrowhead) is resistant to endo H digestion, indicating residence in post-endoplasmic reticulum compartments. Point mutations do not alter the mobility or amount of both VMAT2 species (L484A, lane 7). As anticipated, small deletions (lanes 3 and 6) increase slightly the mobility of the larger as well as smaller species (middle arrow) and larger deletions (lanes 4 and 5) increase the mobility further (lower arrow). However, the larger truncations also appear to reduce the amount of the larger species relative to the smaller (lanes 4 and 5, arrowhead), suggesting impaired transit through the endoplasmic reticulum. Functional analysis shows that all the mutants retain serotonin transport activity (Fig. 2 B), indicating that at least a fraction of each folds normally, exits the endoplasmic reticulum and sorts to an acidic compartment such as endosomes that can support function (25Liu Y. Peter D. Roghani A. Schuldiner S. Prive G.G. Eisenberg D. Brecha N. Edwards R.H. Cell. 1992; 70: 539-551Abstract Full Text PDF PubMed Scopus (524) Google Scholar). Consistent with the impaired processing of the larger truncations 476* and 2-18Δ/476*, these mutants exhibit reduced activity (Fig. 2 B). However, the reduced activity observed for many of the mutants may also result from impaired internalization. Since mutants defective in endocytosis should accumulate at the cell surface, we first examined the distribution of VMAT2 mutants in transfected PC12 cells. Taking advantage of the lumenal orientation of the epitope tag, we used a monoclonal HA antibody to detect cell surface VMAT2 in intact cells (Fig. 3, panels B, D, F,and G). After incubation with the HA antibody for 1 h at 4 °C, the cells were fixed, permeabilized, and incubated with polyclonal VMAT2 antibodies to identify transfectants (Fig. 3,panels A, C, and E), followed by the appropriate secondary antibodies. Cells expressing wild type VMAT2 (Fig. 3 A) show faint or absent cell surface staining (Fig. 3 B), consistent with previous results indicating that VMAT2 has a predominantly intracellular localization (25Liu Y. Peter D. Roghani A. Schuldiner S. Prive G.G. Eisenberg D. Brecha N. Edwards R.H. Cell. 1992; 70: 539-551Abstract Full Text PDF PubMed Scopus (524) Google Scholar,29Liu 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). 3Y. Liu and R. H. Edwards, manuscript in preparation. Deletion of the NH2 terminus (2–18Δ) does not affect this localization (data not shown), suggesting that this domain lacks signals for endocytosis. In contrast, deletion of the COOH-terminal 39 amino acids of VMAT2 (476*) results in high levels of expression at the cell surface (Fig. 3 G). To locate the endocytic signal within the COOH terminus, we examined mutants with smaller deletions. A mutant lacking the last 31 residues of the COOH terminus (484*) is not detectable at the plasma membrane (data not shown), suggesting that the region present in this mutant but absent from 476* contain an endocytosis signal. Indeed, the internal deletion 476–484Δ (Fig. 3 C) appears at high levels on the cell surface (Fig. 3 D). Since the 9 residues deleted in this mutant (AKEEKMAIL, Fig. 1 B) contain an isoleucine-leucine sequence that resembles leucine-based endocytosis motif, we replaced both residues with alanine. This double point mutant (I483A/L484A, Fig. 3 E) also occurs at high levels on the plasma membrane (Fig. 3 F). Since VMAT2 resides in endocytic compartments in non-neuronal cells (25Liu Y. Peter D. Roghani A. Schuldiner S. Prive G.G. Eisenberg D. Brecha N. Edwards R.H. Cell. 1992; 70: 539-551Abstract Full Text PDF PubMed Scopus (524) Google Scholar, 29Liu Y. Schweitzer E.S. Nirenberg M.J. Pickel V.M. Evans C.J. Edwa" @default.
• W2023604322 created "2016-06-24" @default.
• W2023604322 creator A5006681144 @default.
• W2023604322 creator A5027616900 @default.
• W2023604322 creator A5033640601 @default.
• W2023604322 creator A5054228916 @default.
• W2023604322 creator A5089917527 @default.
• W2023604322 date "1998-07-01" @default.
• W2023604322 modified "2023-09-27" @default.
• W2023604322 title "A Leucine-based Motif Mediates the Endocytosis of Vesicular Monoamine and Acetylcholine Transporters" @default.
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