Journal of Neurochemistry, 2005, 94, 957–969
doi:10.1111/j.1471-4159.2005.03244.x
Structural requirements for steady-state localization of the vesicular acetylcholine transporter Lucimar T. Ferreira,* Magda S. Santos,,1 Natalia G. Kolmakova,§ Janaina Koenen, Jose Barbosa Jr.,* Marcus V. Gomez,* Cristina Guatimosim, Xiaodong Zhang,¶ Stanley M. Parsons,§ Vania F. Prado, and Marco A. M. Prado* *Program in Molecular Pharmacology, Departamento de Farmacologia, Departamento de Bioquı´mica-Imunologia and Departamento de Morfologia, ICB, UFMG, Belo Horizonte, Brazil §Department of Chemistry and Biochemistry and Neuroscience Research Institute, University of California, Santa Barbara, California, USA ¶Department of Cell Biology, Duke University Medical Center, Durham, North Carolina USA
Abstract The vesicular acetylcholine transporter (VAChT) regulates the amount of acetylcholine stored in synaptic vesicles. However, the mechanisms that control the targeting of VAChT and other synaptic vesicle proteins are still poorly comprehended. These processes are likely to depend, at least partially, on structural determinants present in the primary sequence of the protein. Here, we use site-directed mutagenesis to evaluate the contribution of the C-terminal tail of VAChT to the targeting of this transporter to synaptic-like microvesicles in cholinergic SN56 cells. We found that residues 481–490 contain the trafficking information necessary for VAChT localization and that within
this region L485 and L486 are strictly necessary. Deletion and alanine-scanning mutants lacking most of the carboxyl tail of VAChT, but containing residues 481–490, were still targeted to microvesicles. Moreover, we found that clathrin-mediated endocytosis of VAChT is required for targeting to microvesicles in SN56 and PC12 cells. The data provide novel information on the mechanisms and structural determinants necessary for VAChT localization to synaptic vesicles. Keywords: choline transporter 1, clathrin, PC12 cells, protein trafficking, SN56 cells, synaptic vesicle. J. Neurochem. (2005) 94, 957–969.
Synaptic vesicles are the most conspicuous type of secretory organelle in neurons, albeit other classes of secretory vesicles usually also are present in terminals, cell bodies and dendrites. Classical neurotransmitters, such as acetylcholine (ACh) and monoamines, are accumulated in secretory vesicles by specialized transporters. In many situations, vesicular transport appears to be rate limiting to formation of releasable neurotransmitter. Thus, vesamicol, which is a potent inhibitor of the vesicular acetylcholine transporter (VAChT), blocks storage of newly synthesized ACh by intact nerve terminals and transport of ACh by purified synaptic vesicles at similar concentrations (Parsons 2000). In addition, increased expression of VAChT in immature Xenopus neurons increases ACh release (Song et al. 1997). Rate limitation seems to be a conserved feature for this family of transporters, as careful analysis of whether the vesicular monoamine transporter (VMAT2) is rate limiting for dopamine release from cells suggests this to be the case
(Pothos et al. 2000). Therefore, understanding the determinants controlling localization of these transporters is of importance to determine how their expression levels in Received February 11, 2005; revised manuscript received March 31, 2005; accepted April 5, 2005. Address correspondence and reprint requests to Marco A. M. Prado, Departamento de Farmacologia or to Vania F. Prado, Departamento de Bioquı´mica-Imunologia, ICB, UFMG, Avenida Antonio Carlos 6627 Belo Horizonte, MG 31270-910, Brazil. E-mail:
[email protected] or
[email protected] 1 The present address of Magda S. Santos is Departments of Physiology and Neurology, University of California San Francisco, 600 16th Street, San Francisco, CA 94143–2140, USA. Lucimar T. Ferreira and Magda S. Santos contribute equally to this work. Abbreviations used: ACh, acetylcholine; AP, adaptor protein; CHT1, high affinity choline transporter; GFP, green fluorescent protein; HA, hemagglutinin; VAChT, vesicular acetylcholine transporter; VMAT2, vesicular monoamine transporter 2; VAMP2, vesicle-associated membrane protein 2.
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synaptic vesicles can be modulated to influence neurotransmitter release. VAChT is predominantly present in synaptic vesicles in cholinergic neurons and in synaptic-like microvesicles in model cell lines (Gilmor et al. 1996; Weihe et al. 1996; Liu and Edwards 1997; Prado et al. 2002). The C-terminal region of VAChT has been implicated in trafficking of the protein to microvesicles in PC12 cells (Varoqui and Erickson 1998; Krantz et al. 2000). VAChT is phosphorylated (Barbosa et al. 1997), and a phosphoserine residue in the C-terminal tail controls sorting of VAChT to the different types of secretory organelles present in PC12 cells (Cho et al. 2000; Krantz et al. 2000). At a finer level of resolution, a particular di-leucine motif (L485 and L486) in the C-terminal tail is required for VAChT endocytosis (Tan et al. 1998). Di-leucine motifs interact with clathrin adaptor proteins (Bonifacino and Traub 2003), and the C-terminal tail of VAChT interacts with adaptor proteins AP-1 and AP-2 (Barbosa et al. 2002; Kim and Hersh 2004). Whether clathrin-mediated endocytosis is involved in VAChT trafficking in vivo has not been explicitly tested, although it is known that dynamin is required for VAChT trafficking (Barbosa et al. 2002). Little is known whether other molecular determinants contribute to steady-state localization of VAChT. For example, a classical internalization motif containing tyrosine is found close to the endocytic motif L485 and L486. Another di-leucine (L474 and L475) also is present in the carboxyl terminal region of VAChT. In addition, recent experiments have suggested that a non-classical type of tyrosine-like motif in the C-terminal region of VAChT may participate in endocytosis by interacting with AP-2 (Kim and Hersh 2004). We have now used site-directed mutagenesis and interference with clathrin-mediated endocytosis to probe potential contributions of other regions in the C-terminal tail to VAChT trafficking and steady-state distribution in cholinergic SN56 cells.
Experimental procedures Cell culture SN56 cells were a generous gift of Professor Bruce Wainer (Department of Pathology, Emory University School of Medicine, Atlanta, GA, USA) and were maintained as previously described (Santos et al. 2001). Medium was changed every 2 days. Cells were differentiated in serum-free medium supplemented with 1 mM dibutyryl-cyclic AMP (Sigma Chemical Co., St Louis, MO, USA) for 2 days after transfection, as described (Barbosa et al. 1999). PC12 cells stably expressing VAChT with a luminal hemagglutinin (HA) tag (HA-VAChT) were a gift from Dr Robert H. Edwards (Departments of Neurology and Physiology, UCSF School of Medicine, USA) and were grown on Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum and 10% horse fetal serum. Stable PC12 cell clones expressing GFP-HA-VAChT
D491-530 and GFP-HA-VAChT were grown as described above in media containing 400 lg of geneticin. PC12A123.7 cells were obtained as a gift from Dr Louis Hersh, University of Kentucky. [3H]Vesamicol (20 Ci/mmol) and [3H]ACh (76 mCi/mmol) were obtained from New England Nuclear Division of Perkin Elmer Corp (Boston, MA, USA). Plasmid constructs The cDNA of mouse VAChT gene was amplified from genomic DNA by PCR using Pfu DNA polymerase (Barbosa et al. 1999; Santos et al. 2001). Detailed description of GFP-VAChT and GFP-VAChT L485A and L486A construction and characterization are given elsewhere (Santos et al. 2001). GFP-VAChT was used as a template to construct mutant cDNAs in the pEGFP-C2 vector using the EcoRI and BamHI restriction sites. The sequence of all primers is available upon request. The schematic illustration of wild type and mutant GFP-VAChT fusion proteins is shown in Fig. 1. Plasmids were purified using the Qiagen Plasmid Maxikit (Qiagen Inc., Valencia, CA, USA). All clones had their sequence confirmed by automatic sequencing using the dideoxynucleotide chain-termination method (Sanger et al. 1977). Alanine mutants were expressed in HEK 293 cells and SN56 cells and presented similar electrophoretic mobility as GFP-VAChT and the expected change in molecular mass when compared with HA-VAChT (increased by the mass of GFP, not shown). Myc-VAMP2 (synaptobrevin II) was a gift from Dr W. Volknandt (Biozentrum der J.W. Goethe-Universitat, Germany) and has also been previously characterized and shown to traffic similarly to endogenous VAMP2 (Volknandt et al. 2002). A rat VAChT construct with a luminal HA-tag was a gift from Dr Robert H. Edwards. This recombinant protein transports ACh and has been shown to traffic to synaptic-like microvesicles in PC12 cells (Tan et al. 1998; Krantz et al. 2000). This cDNA was used to generate a GFP-HA-VAChT and GFP-HA-VAChT D491-530 by PCR. An expression vector plasmid containing the C-terminal region of the adaptor protein AP180 (AP180-C) was a gift from Dr Benjamin J. Nichols (MRC Laboratory of Molecular Biology, UK). All PCR generated constructs had their sequence confirmed. Cell transfection For microscopy purposes, approximately 5 · 104 SN56 cells were plated on cover-slips 1 day before transfection and transfected using LipofectAMINE 2000 (Life Technologies, Gaithesburg, MD, USA) according to the manufacturer’s instructions. After 4 h of transfection, SN56 cells were differentiated as described above. In cotransfection experiments, we used 3–4 lg of DNA, following a plasmid ratio of 3:1 for GFP-VAChT constructs and myc-VAMP2, 1:4 for GFP-VAChT and AP180-C, and 1:1 for GFP-VAChT constructs and HA-VAChT wild type. For transfection of PC12 cells stably expressing HA-VAChT, approximately 1 · 105 cells were plated on a 35-mm tissue culture dish. We used 2.2 lg of DNA, following a plasmid ratio of 1:10 for GFP and AP180-C. GFP was used as a reporter for transfection. For characterization of [3H]vesamicol binding and [3H]ACh transport, GFP-VAChT from mouse and wild-type VAChT from rat were expressed in PC12A123.7 cells as described (Ojeda et al. 2003). The cells contain negligible levels of endogenous VAChT and therefore are suitable for such analysis (Shimojo et al. 1998). In
2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 957–969
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Fig. 1 Schematic illustration of wild type and mutant GFP-VAChT constructs. The black box represents GFP cDNA and the white box represents VAChT cDNA, as indicated. N corresponds to the N-terminal domain, 12TMD indicates the 12 transmembrane domains and C represents the C-terminal domain. Serial deletions of the VAChT cDNA were created in the C-terminal domain. Part of the C-terminal sequence is indicated in order to show the residues that were changed to alanine. The HA epitope is shown in two of the constructs and tags a luminal region in the loop between TMDs I and II. This is identical to a construct used previously by Edwards and collaborators (Tan et al. 1998; Krantz et al. 2000). GFP, green fluorescent protein; HA, hemagglutinin; VAChT, vesicular acetylcholine transporter.
brief, 50 lg of recombinant expression vector was transiently transfected into PC12A123.7 cells by electroporation. Cells were harvested after 72 h and gently homogenized to 95% breakage in 0.32 M sucrose, 10 mM HEPES-KOH at pH 7.4 supplemented with protease inhibitors. Postnuclear supernatant (about 35 mg protein/ mL) containing synaptic-like microvesicles was collected, after centrifugal pelleting of cellular debris at 800 g for 10 min at 23C, and stored frozen at )80C until use. PC12 cells were used to generate stable transfectants with GFPHA-VAChT and GFP-HA-VAChT D491-530. Cells were electroporated with the appropriate cDNAs and the pEF6/his vector (Invitrogen to confer resistance to blasticidin) and then selected with geneticin and blasticidin (Invitrogen, Carlsbad, CA, USA). Individual clones were picked and expanded. GFP fluorescence was used to select clones with lower expression levels, and immunoblots were used to confirm expression of recombinant HA-tagged proteins with the correct molecular mass (detected with HA, GFP and VAChT antibodies, not shown). After clone isolation, cells were maintained in medium with geneticin only. Immunofluorescence Cells were washed three times in phosphate-buffered saline (pH 7.4) and then fixed with 3% paraformaldehyde in phosphate-buffered saline for 20 min. Fixed cells were washed three times and then blocked and permeabilized in phosphate-buffered saline containing 2.5% normal goat serum, 0.05% nonidet P-40 and 1% bovine albumin (blocking solution) for 15 min at room temperature. Cells were incubated with primary antibody (diluted into the blocking solution) for 1 h at room temperature. An anti-HA (1:600) monoclonal antibody (Roche Diagnostics, Indianapolis, IN, USA) and an antic-myc (1:500) monoclonal antibody (Molecular Probes Inc., Eugene, OR, USA) were used as primary antibodies. Afterward
cells were washed three times. For immunostaining, a secondary antibody coupled to Alexa568 (Molecular Probes) was used. Control experiments in which the primary antibody was omitted and cells were not transfected also were done and indicated that labeling was specific. Organelle labeling In order to label endocytic organelles, the styryl dye FM4-64 (Molecular Probes, Eugene, OR, USA) was used. Cells were incubated with 6 lM of FM4-64 for 30 min at 37C in 5% CO2 and then visualized by confocal microscopy as described (Barbosa et al. 1999). Labeling of endosomes was performed using 30 lg/mL of Alexa568-labeled transferrin (Molecular Probes) at 37C in 5% CO2 for 20 min. After incubation, cells were washed three times with icecold phosphate-buffered saline, fixed with 3% paraformaldehyde in phosphate-buffered saline for 20 min and imaged. Confocal and fluorescence imaging Experiments were performed at room temperature (20–25C). Cover-slips were transferred to a custom holder in which they formed the bottom of a 400-lL bath. Imaging was performed with a Bio-Rad MRC 1024 laser scanning confocal system running the software LASERSHARP 3.0 coupled to a Zeiss microscope (Axiovert 100) with a water immersion objective (40 ·, 1.2 NA) or a Zeiss LSM 510 with a oil immersion objective (63 ·, 1.4 NA). Alternatively, an Axioskop Zeiss microscope coupled to a Micromax CCD camera was used to obtain epifluorescence images. The appropriate set of filters and laser lines was used to avoid bleedthrough between the different fluorophors used. Image analysis and processing were performed with the software LASERSHARP (Bio-Rad), CONFOCAL ASSISTANT, ADOBE PHOTOSHOP and METAMORPH (Universal Imaging, West Chester, PA, USA).
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Co-localization between GFP-VAChT and myc-VAMP2 was quantified using METAMORPH. Images were threshold, and the numbers of fluorescently labeled organelles containing GFP-VAChT constructs (green fluorescence) or myc-VAMP2 (red fluorescence) were detected automatically and independently by the software. The percentage of pixels that detected both colors in each image was calculated by creating an overlay mask. At least three experiments were analyzed in each condition.
Table 1 Transport activity and vesamicol binding of vesicular acetylcholine transporter (VAChT) and GFP-VAChTa Form
KMb
Vmaxc
KVd
Bmaxe
Vmax/Bmaxf
Wild type GFP-VAChT
0.40 ± 0.20 1.2 ± 0.5
50 ± 6 70 ± 10
20 ± 1 25 ± 3
5.2 ± 0.1 2.6 ± 0.1
9.6 27
a
Expressed in PC12A123Æ7 cells. mM ACh. c pmol ACh/mg postnuclear supernatant/min. d nM vesamicol. e pmol VAChT/mg postnuclear supernatant. f min)1. ACh, acetylcholine; GFP, green fluorescent protein; VAChT, vesicular acetylcholine transporter. b
Vesamicol binding and acetylcholine transport assays These assays were carried out as described (Ojeda et al. 2003). In brief, postnuclear supernatant was incubated at 37C with different concentrations of either [3H]vesamicol or [3H]ACh plus 5 mM MgATP and 2 mM MgCl2 in 110 mM potassium tartrate and 20 mM HEPES-KOH at pH 7.4 supplemented with 1 mM ascorbic acid. After 10 min, two 90-lL portions were rapidly filtered through glass-fiber filters coated with polyethylenimine. Unbound radioactivity was removed immediately with four washes of each filter. Bound radioactivity was determined by liquid scintillation spectrometry. Nonspecific binding or transport was determined in the presence of 4 lM non-radioactive vesamicol. Data for duplicates were averaged, and appropriate equations were fitted to averaged data to determine Bmax and Kv for specific binding of vesamicol and Vmax and Km for specific transport of ACh.
Results
GFP-VAChT has been shown to localize properly in cultured cells (Santos et al. 2001; Barbosa et al. 2002). Because most of our experiments used VAChT constructs tagged with GFP, we tested whether the GFP-tag interferes with VAChT function. Wild-type VAChT and GFP-VAChT were expressed in a mutant strain of PC12 cells that lacks endogenous VAChT. Western blot analysis of postnuclear supernatants using an antibody directed against the N-terminus of VAChT detected diffuse species of apparent molecular weights about 68 and 95 kDa, respectively (not shown). As the molecular weights of the VAChT and GFP-VAChT polypeptides are expected to be 59 and 86 kDa, respectively, the detected VAChT species were full length and likely glycosylated (Varoqui et al. 1996). An antiGFP antibody also did not reveal significant amounts of proteolysis products (not shown). Vesamicol binding and ACh transport by postnuclear supernatant was characterized (Table 1). GFP-VAChT binds vesamicol with similar affinity as wild-type VAChT does, although the level of expression (Bmax) was lower. GFPVAChT transports ACh well. The Km value was higher, but the Vmax value normalized for the expression level (Vmax/ Bmax) was higher than for wild-type VAChT. Because essentially all GFP-VAChT in postnuclear supernatant was intact, vesamicol binding and ACh transport were due to GFP-VAChT per se and not to untagged VAChT released by proteolysis. Because GFP-VAChT also traffics to endosomes and synaptic-like microvesicles (Santos et al. 2001; Ribeiro et al. 2003), we conclude that the GFP tag does not interfere
with the function of VAChT and can be used to follow the trafficking of mutants without major interference. We used cholinergic SN56 cells to study the motifs in the C-terminal tail of VAChT that contribute to trafficking. Upon differentiation, these cells express neurites containing synaptic-like microvesicles (Hammond et al. 1990). The differentiated cells also exhibit cholinergic features, such as increased expression of cholinergic proteins and neuronal voltage-gated calcium channels (Blusztajn et al. 1992; Barbosa et al. 1999; Kushmerick et al. 2001). We expressed a number of GFP-tagged VAChT mutants (Fig. 1) and compared their localizations with that of HA-tagged VAChT or myc-tagged VAMP2, which also traffic to synaptic-like microvesicles in cultured cells (Volknandt et al. 2002; Ribeiro et al. 2003). Figure 2(a) (GFP-VAChT) and Fig. 2(b) (HA-VAChT) indicate that, as expected, the two VAChT constructs co-localize in the cell body (94 ± 4% of red and green pixels overlap) and also in processes where synaptic-like microvesicles are labeled (arrows indicate clusters of microvesicles). In general, HA-VAChT labeling is less defined than GFP-VAChT labeling, likely due to background binding by the primary and/or secondary antibody. An excellent degree of co-localization also is observed between GFP-VAChT (Fig. 2c) and myc-VAMP2 (in Fig. 2d, arrows point to clusters showing co-localization, Table 2). In varicosities, the localization of the proteins was virtually identical, suggesting that these proteins are located in similar clusters of synaptic-like microvesicles (Figs 2e and f show labeling of a large varicosity for GFP-VAChT and myc-VAMP2, respectively, in higher magnification). In order to test whether the C-terminal tail of VAChT contributes to the distribution of the protein, we examined the localization of several C-terminal deletion mutants in SN56 cells. Deletion of the entire C-terminal tail abolished the punctate distribution (GFP-VAChT D471-530, Figs 3a and b), and the fluorescence was distributed throughout the cell, but not in any particular identifiable compartment. Importantly, the accumulation of green fluorescence in
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(a)
(c)
(e)
(b)
(d)
(f)
Fig. 2 GFP-VAChT co-localizes with HA-VAChT and myc-VAMP2 in synaptic-like vesicles. SN56 cells were transiently co-transfected with distinct plasmids fixed and processed for immunofluorescence as described in the text. (a) A representative cell visualized with green fluorescence from GFP-VAChT. (b) The red immunofluorescence detection of HA-VAChT for the same cell. (c) Another representative cell visualized with green fluorescence from GFP-VAChT. (d) Red immunofluorescence of myc-VAMP2 for the same cell. A large varicosity was imaged at higher magnification in (e) for GFP-VAChT and in
(f) for myc-VAMP2. Cells were examined by laser scanning confocal microscopy by optical sectioning, and the maximum Z-projection is shown in all panels. Arrows point to some of the clusters of vesicles presenting co-localization between recombinant proteins. Green and red fluorescence are shown in gray scale. The panels are representative of over 40 cells examined in at least three independent experiments. (b) and (d), scale bar ¼ 20 lm; (f) scale bar ¼ 10 lm. GFP, green fluorescent protein; HA, hemagglutinin; VAChT, vesicular acetylcholine transporter; VAMP2, vesicle-associated membrane protein 2.
Table 2 Percentage of co-localization constructs and myc-VAMP2a
Similar results also were obtained in PC12 cells with or without expression of wild-type VAChT (not shown, but see Fig. 8). In varicosities of SN56 cells, GFP-VAChT D491-530 localized to groups of synaptic-like microvesicles that also could be labeled with the vital fluorescent dye FM4-64 (Fig. 3e GFP-VAChT D491-530 and Fig. 3f FM4-64), similarly to what is observed for GFP-VAChT (Santos et al. 2001). Moreover, GFP-VAChT D491-530 is present in early (arrowheads) and recycling (arrow) endosomes in the soma (Fig. 3g GFP-VAChT D491-530 and Fig. 3h transferrin), as is also observed for GFP-VAChT (Santos et al. 2001). Importantly, the percentages of co-localization are similar for GFP-VAChT D491-530 and transferrin compared to GFPVAChT and transferrin (69 ± 5 and 78 ± 3%, respectively, of red and green pixels overlap, p > 0.05). To further test whether trafficking of GFP-VAChT D491530 indeed requires the first 20 amino acids of the C-terminal tail, we generated another construct in which these residues were deleted, but the last 40 amino acids of the VAChT tail were present (GFP-VAChT D471-490). GFP-VAChT D471490 did not co-localize with myc-VAMP2 (Figs 3i and j, respectively). Thus, residues 471–490 of the VAChT C-terminal tail contain major trafficking motifs. In order to investigate the determinants within residues 471–490, we mutated different groups of amino acids to alanine residues (Fig. 1). Mutation of the first five amino acids (GFP-VAChT 471-475A in Fig. 4a) did not impair normal trafficking in SN56 cells (myc-VAMP2 in Fig. 4b).
Construct GFP-VAChT GFP-VAChT GFP-VAChT GFP-VAChT GFP-VAChT
wt 481-485A 486-490A 481-484A D487A-E488A
between
GFP-VAChT
Mean percentage ± SEM
Number of cells
80 31 55 73 77
28 21 17 20 20
± ± ± ± ±
3 3 5 3 4
a
Co-transfected into SN56 cells. GFP, green fluorescent protein; VAChT, vesicular acetylcholine transporter; VAMP2, vesicle-associated membrane protein 2.
neurites and varicosities was abolished. This confirms previous results showing that the C-terminal tail of VAChT is required for normal trafficking and localization (Varoqui and Erickson 1998). We next examined a series of C-terminal deletion mutations of VAChT for altered distribution in SN56 cells. Surprisingly, deletion of the last 20 residues of the C-terminal tail (511–530, not shown) or the last 40 amino acids (GFPVAChT D491-530, Figs 3c, e and g) did not affect localization. Co-localization experiments showed that retention of the first 20 amino acids of the C-terminal region of VAChT was sufficient to endow the same pattern as observed for HA-VAChT (Fig. 3c GFP-VAChT D491-530 and Fig. 3d HA-VAChT, 92 ± 2% of red and green pixels overlap).
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(a)
(b)
(g)
(h)
(i) (c)
(d)
(j) (e)
(f)
Fig. 3 Contribution of the C-terminal tail to the localization of VAChT. SN56 cells were transiently transfected and examined by laser scanning confocal microscopy. (a) Green fluorescence from a representative cell (out of 30 examined) expressing GFP-VAChT D471-530. (b) The same field in transmitted light. (c) A representative cell (out of 29 cells) visualized with green fluorescence from the mutant GFP-VAChT D491-530. (d) The same cell as in (c) visualized by red HA immunofluorescence to detect co-transfected HA-VAChT. Arrows point to some of the clusters of vesicles presenting co-localization between GFP-VAChT D491-530 and HA-VAChT. (e) Green fluorescence from a representative living varicosity of a cell expressing GFP-VAChT D491-530 and incubated with FM4-64, and (f) red fluorescence from the same varicosity. Arrows point to some of the co-localization spots.
(g) Green fluorescence for a representative cell transfected with GFPVAChT D491-530 and incubated in Alexa568-labeled transferrin. (h) Red fluorescence from early (arrowheads) and recycling (arrow) endosomes in the same cell. (i) The pattern of distribution of GFPVAChT D471-490 in a representative cell (out of 40 examined), and (j) the localization of synaptic-like microvesicles in the same cell visualized with red immunofluorescence from co-transfected myc-VAMP2. Some images were obtained from living cells (a, e and f). The maximum Z-projection is shown in all figures. Green and red fluorescence are shown in gray scale. At least three independent experiments were done in each condition. Bars ¼ 20 lm. GFP, green fluorescent protein; HA, hemagglutinin; VAChT, vesicular acetylcholine transporter; VAMP2, vesicle-associated membrane protein 2.
Arrows in Figs 4a and b indicate some of the co-localized organelles. Interestingly, GFP-VAChT 471-475A lacks one (L474 and L475) of the two di-leucine motifs present in the C-terminal region, suggesting that these leucines do not participate in VAChT trafficking. Similar results were obtained when HA-VAChT was co-expressed instead of myc-VAMP2 (not shown). Replacement of amino acids 476–480 by alanine (GFPVAChT 476-480A, Fig. 4c) also did not interfere with normal trafficking in SN56 cells, as the protein shows the same pattern of localization as HA-VAChT does (Fig. 4d, arrows indicate some of the co-localization spots). In contrast, replacement of amino acids 481–485 with alanine (the first L of the L485L486 di-leucine motif is mutated) changed trafficking (Fig. 4e). GFP-VAChT 481-485A did not accumulate in varicosity sites (arrow) and showed no localization to organelles labeled with myc-VAMP2 (Fig. 4f) in the soma. In fewer cells, fluorescence was more restricted to the plasma membrane (not shown). In other cells,
fluorescence was at the plasma membrane, but it was also abundant in non-identified structures in the cytoplasm, albeit it never seemed to label well-defined organelles (Fig. 4e). The mutant might be retained in the endoplasmic reticulum or other intracellular compartments. A similar difference in distribution of VAChT constructs was obtained when GFPVAChT 481-485A was expressed with HA-VAChT (not shown). Figure 4(g) shows the pattern of expression for another mutant, GFP-VAChT 486-490A, and Fig. 4(h) shows the corresponding localization of myc-VAMP2 in SN56 cells. Even though the second leucine in the L485-L486 di-leucine motif was mutated, some GFP-VAChT 486-490A was found in organelles that contained myc-VAMP2 (arrows). Some GFP-VAChT 486-490A also was distributed in non-identified organelles in the cytoplasm, and in this respect resembled GFP-VAChT 481-485A. A similar difference in apparent distribution of constructs was obtained when HA-VAChT was expressed instead of myc-VAMP2 (not
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shown). Thus, it is likely that this construct is at least partially mislocalized. Quantitative analysis of the extent of co-localization between GFP-VAChT 481-485A and GFP-VAChT 486490A on one hand and myc-VAMP2 on the other hand confirmed the above conclusions (Table 2). When the same analysis was repeated in cells expressing HA-VAChT instead of myc-VAMP2, virtually identical results were obtained (not shown). These results suggest that critical information required for GFP-VAChT trafficking in SN56 cells is present
Fig. 4 Residues 481–490 in the C-terminal tail of VAChT are necessary for correct localization. SN56 cells were transiently co-transfected with distinct GFP-VAChT constructs, HA-VAChT and myc-VAMP2, and images were obtained by laser scanning confocal microscopy. (a) The localization of green GFP-VAChT 471-475A in a representative cell, and (b) the localization of red myc-VAMP2 in the same cell. (c) The localization of green GFP-VAChT 476-480A in a representative cell, and (d) the localization of red HA-VAChT. (e) The localization of green GFP-VAChT 481-485A in a representative cell, and (f) the localization of red myc-VAMP2 in the same cell. (g) The localization of green GFP-VAChT 486-490A in a representative cell, and (h) labeling of red myc-VAMP2 for the same cell. Arrows present some co-localization regions. The localization of each GFP-VAChT mutant was analyzed in over 40 cells either co-expressing HA-VAChT or mycVAMP2 in at least six independent experiments. Green and red fluorescence are shown in gray scale. Scale bar ¼ 20 lm. GFP, green fluorescent protein; HA, hemagglutinin; VAChT, vesicular acetylcholine transporter; VAMP2, vesicle-associated membrane protein 2.
within residues 481–490 and that the di-leucine motif (L485 and L486) is part of the critical information. We thus generated GFP-VAChT mutants intended to define the importance of the amino acids surrounding the di-leucine motif. In some proteins, residues on the N-terminal side of a di-leucine motif are recognized as part of the motif (Bonifacino and Traub 2003). Indeed, the targeting of VAChT to synaptic-like microvesicles in PC12 cells is decreased by mutation of serine 480 to glutamic acid (S480E), a change that mimics phosphorylation of S480 by protein kinase C (Krantz et al. 2000). However, confirming previous observations (Krantz et al. 2000), mutation of this serine residue to alanine (S480A) does not affect VAChT trafficking in PC12 or SN56 cells (Barbosa Jr et al., unpublished results). GFP-VAChT 481-484A was constructed to examine the effects of mutating residues immediately before the di-leucine motif. The mutant had similar steadystate distribution (Fig. 5a) as myc-VAMP2 did in SN56 cells (Fig. 5b and Table 2). Thus, mutation of the four residues preceding the di-leucine motif has no effect on internalization of VAChT to microvesicles and endosomes when both leucines are present. Likewise, mutation of amino acids after the di-leucine motif, namely in GFP-VAChT D487A-E488A, did not affect trafficking of the transporter (Fig. 5d) when compared to myc-VAMP2 (Fig. 5e and Table 2). Therefore, these results suggest that the di-leucine motif alone is one of the major determinants for trafficking of VAChT to microvesicles and endosomes in SN56 cells. Within the di-leucine motif, the first leucine (L485) seems to be more important, as GFPVAChT 481-485A failed to localize to endosomes and microvesicles, whereas GFP-VAChT 486-490A failed only partially. To further test the importance of L485, a point mutation (L485A) in GFP-VAChT was generated. The mutant was not
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Fig. 5 Amino acid residues other than the di-leucine motif (485 and 486) did not affect VAChT localization. SN56 cells were co-transfected with distinct GFP-VAChT mutants and myc-VAMP2 and were analyzed by laser scanning confocal microscopy. (a) A representative cell (out of 36 examined) expressing green fluorescence from GFPVAChT 481-484A, and (b) the distribution of red fluorescence from myc-VAMP2 for the same cell. (d) A representative cell (out of 31 examined) expressing green fluorescence from GFP-VAChT
D487A-E488A, and (e) the same cells stained with red immunofluorescence for myc-VAMP2. Arrows point to some of the clusters of vesicles presenting the two recombinant proteins. (c) and (f) Same field of cells in the left in transmitted light. Green and red fluorescence are shown in gray scale. Representative optical sections are shown in all figures for three distinct experiments. Scale bar ¼ 20 lm. GFP, green fluorescent protein; VAChT, vesicular acetylcholine transporter; VAMP2, vesicle-associated membrane protein 2.
targeted to microvesicles or endosomes, and it was present mainly at the plasma membrane (Fig. 6a, arrows) in the cell body and neurites. Nearly no co-localization between this mutant and myc-VAMP2 (Fig. 6b) could be detected. The pattern of expression of GFP-VAChT L485A resembles that for the di-leucine mutant (GFP-VAChT L485A L486A, Fig. 6d), which also was present mainly at the plasma membrane and showed no co-localization with myc-VAMP2 (Fig. 6e). The C-terminal region of the clathrin adaptor protein AP180 (AP180-C) inhibits internalization of transferrin and EGF receptor (Ford et al. 2001), probably by sequestering clathrin (Zhao et al. 2001). In contrast, it spares non-clathrin dependent endocytosis (Nichols et al. 2001; Nichols 2002). AP180-C and soluble GFP were transiently co-expressed in undifferentiated PC12 cells stably expressing HA-VAChT, and the effect on endocytosis of HA-VAChT was determined to test whether interference with clathrin-mediated endocytosis has similar effects as mutation of the critical di-leucine motif. Because the HA-tag in this construct is luminal (Tan et al. 1998), block of endocytosis is expected to allow the HA antibody to recognize VAChT at the plasma
membrane of non-permeabilized cells. Figure 7a shows that transfected PC12 cells (identified by the presence of GFP, Fig. 7b) accumulated HA-VAChT at the plasma membrane. Permeabilization and staining of non-transfected cells (i.e. without AP180-C) shows typical punctate labeling for VAChT (Fig. 7c). No labeling by HA antibody at the plasma membrane is seen in non-transfected cells (Fig. 7d), even when the exposure time for the CCD camera is increased fivefold (note the increase in background). Thus, AP180-C inhibits endocytosis of HA-VAChT. In another set of experiments under similar conditions, transfection with AP180-C blocked 70% of the uptake of fluorescently labeled transferrin and thus clathrin-mediated endocytosis (not shown). To examine how AP180-C affects GFP-VAChT traffic in differentiated cholinergic cells, we overexpressed both constructs in SN56 cells (Figs 7e and f). Cells transfected with AP180-C did not differentiate as well as non-transfected cells did, and many cells expressing AP180-C (which has a myc epitope revealed by staining with a myc antibody, Fig. 7f) showed shorter neurites, similarly to what has been observed by overexpressing the dynamin I mutant K44A
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Fig. 6 The L485A and L486A mutations alter localization of VAChT in SN56 cells. Cells were transiently co-transfected with GFP-VAChT L485A or GFP-VAChT L485A-L486A and myc-VAMP2 and analyzed by laser scanning confocal microscopy. (a) The distribution of green fluorescence in a representative cell (out of 35 examined) from GFPVAChT L485A. (b) The red immunofluorescence from myc-VAMP2 in the same cell. Arrows show localization of GFP-VAChT L485A at the plasma membrane. (d) The green fluorescence in a representative cell
from GFP-VAChT L485A-L486A (out of 18 examined), and (e) the red immunofluorescence from myc-VAMP2 in the same cell. (c) and (f) Same field of cells in the left in transmitted light. Representative optical sections are shown for three independent experiments. Scale bar ¼ 20 lm. GFP, green fluorescent protein; HA, hemagglutinin; VAChT, vesicular acetylcholine transporter; VAMP2, vesicle-associated membrane protein 2.
(Barbosa et al. 2002). GFP-VAChT in AP180-C expressing cells was present at the plasma membrane, similarly to what was observed for the di-leucine mutants (L485, and L485 plus L486). However, there was also an accumulation in the perinuclear region that was less evident with the di-leucine mutants (Figs 7e and g compared with Fig. 6). In the few transfected cells that presented longer neurites and varicosities (Fig. 7h), GFP-VAChT was evenly distributed in the plasma membrane of processes, with no evidence of preferential accumulation in varicosities, as normally observed for GFP-VAChT in the absence of AP180-C (compare Fig. 7g with Figs 2 and 8). Previous work has suggested that a non-classical tyrosine motif in the extreme C-terminus of VAChT may contribute to AP-2 binding and endocytosis of VAChT by PC12 cells (Kim and Hersh 2004). Those results contrast with the apparently normal trafficking of GFP-VAChT D491-530 in SN56 cells seen in the current work. One possibility to explain the discrepancy is that GFP-VAChT D491-530 distributes similarly to VAChT but uses an alternative pathway, such as non-clathrin-mediated endocytosis, to reach endocytic organelles. To test this possibility, HA-tagged versions of GFP-VAChT and GFP-VAChT D491-530 were generated and used to investigate whether inhibition of clathrin-mediated endocytosis affects the distribution of GFP-VAChT D491-530. The steady-state distributions of the constructs were similar to each other (as determined by the GFP fluorescence, Figs 8a and e), as
expected from the experiments shown in Fig. 3. The luminal-HA epitope was not detected in non-permeabilized cells, suggesting that there would be only negligible amounts of the proteins at the cell surface (Figs 8b and f, same cells as in Figs 8a and e, respectively). In contrast, cells expressing GFP-HA-VAChT or GFP-HA-VAChT D491-530 that also were transfected with AP180-C presented GFP fluorescence (Figs 8c and g) and HA epitope (Figs 8d and h) predominantly at the plasma membrane. The results of these experiments suggest that both GFP-HA-VAChT D491-530 and GFP-HA-VAChT use clathrin-mediated endocytosis during their trafficking. Another possibility to explain the discrepancy in results is that VAChT D491-530 traffics differently in SN56 and PC12 cells. Therefore, we generated PC12 cells stably expressing GFP-HA-VAChT or GFP-HA-VAChT D491-530. The cells were differentiated with NGF for 7 days, and distribution of GFP fluorescence was examined for both constructs (Figs 8i and j for GFP-HA-VAChT and GFP-HA-VAChT D491-530, respectively). Fluorescence was accumulated predominantly in varicosities of PC12 cells in both cases, similarly to what is observed in SN56 cells (Figs 8i and j, arrows). In the cell bodies, both constructs presented punctate distribution of fluorescence, and there was no apparent accumulation of GFP-VAChT D491-530 at the plasma membrane. Therefore, the absence of tyrosines 524, 526 and 527 did not seem to affect endocytosis and localization of VAChT in either PC12 or SN56 cell lines.
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Fig. 7 Inhibition of clathrin-mediated endocytosis decreases VAChT internalization and alters localization. PC12 cells stably expressing VAChT tagged with a luminal HA epitope were or were not transiently co-transfected with AP180-C and free GFP. (a) Non-permeabilized cells accumulate red immunofluorescence from HA-VAChT at the plasma membrane in cells co-transfected with AP180-C and GFP (representative epifluorescence image for 73 cells imaged in 4 independent experiments). (b) The green fluorescence from free GFP for the same cells indicating co-transfection. (c) Permeabilized cells not co-transfected contain punctate red immunofluorescence from HAVAChT. In (d), PC12 cells like those in (c) were treated similarly except they were not permeabilized. The acquisition time of this image was fivefold longer than for the previous panels. (e) Green fluorescence in a representative SN56 cell (out of 85 examined) transiently co-transfected with GFP-VAChT and AP180-C, and (f) red myc-staining (AP180-C has a myc epitope) for the same cell. (g) The green fluorescence of another SN56 cell transiently co-transfected with GFPVAChT and AP180-C. (h) The image in transmitted light of the cell in (g). Green and red fluorescence are shown in gray scale. These images are representative of three independent experiments. Scale bar ¼ 20 lm. GFP, green fluorescent protein; HA, hemagglutinin; VAChT, vesicular acetylcholine transporter.
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Discussion
The carboxyl terminal tail of VAChT has been shown to mediate trafficking to synaptic-like microvesicles in PC12 cells (Varoqui and Erickson 1998; Krantz et al. 2000). Our findings here confirm those observations, as a mutant lacking the entire C-terminal tail of VAChT (GFP-VAChT D471-530) did not accumulate in any identifiable compartment and appeared to distribute evenly throughout the cell. Here we also have generated an extensive collection of VAChT mutants to determine which parts of the carboxyl terminal tail of this transporter contribute to its steady-state distribution. Two experiments narrowed the critical region. Firstly, deletion of the last 40 amino acids of the C-terminal tail (GFPVAChT D491-530) still allowed the truncated protein to localize to endosomes and synaptic-like microvesicles. Secondly, deletion of the first 20 residues of the C-terminal tail (GFP-VAChT D471-490) impaired trafficking to endosomes
and synaptic-like microvesicles and distributed fluorescence throughout the cell. Within amino acids 471–490, alanine scanning mutagenesis revealed that the important residues are L485 and L486. Mutation of the amino acids surrounding the di-leucine motif did not significantly affect trafficking of VAChT, as long as the di-leucine motif was intact. On the other hand, concomitant mutation of L485 and the preceding four amino acids (GFP-VAChT 481-485A) is more disruptive to trafficking than mutation of L485 alone or L485 and L486, which stranded VAChT on plasma membrane. GFP-VAChT 481-485A distributed fluorescence in nonidentified compartments (i.e distinct from early endosomes and microvesicles) instead of a defined organelle, although the protein also was observed at the plasma membrane. Similarly, concomitant mutation of L486 and the trailing amino acids (GFP-VAChT 486-490A) also distributed fluorescence in less defined cytoplasmic organelles, although some fluorescence did localize to endosomes. These data suggest that amino acids other than the di-leucine motif (L485 and L486) may have accessory roles during intracellular trafficking, but the di-leucine motif is the major determinant involved in the localization of VAChT. Moreover, it would seem that L485 is more critical than L486, although both are important. Previous work has suggested that Y524, Y526 and Y527 participate in VAChT trafficking by interacting with the AP-2 clathrin adaptor complex (Kim and Hersh 2004). These authors suggest that this non-canonical tyrosine-based motif (524YNYY527) could, in addition to the di-leucine motif (L484 and L485), participate in the internalization of the transporter. Our data suggest that, if this is the case, the tyrosine-based motif does not participate in endocytic processes that take place during VAChT trafficking to
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Fig. 8 GFP-VAChT D491-530 localization depends on clathrin-mediated endocytosis. In (a) to (h), SN56 cells were stained with red immunofluorescence for HA epitope under non-permeabilizing conditions. (a) The green fluorescence of a representative cell transiently transfected with GFP-HA-VAChT. (b) The same field imaged for HA epitope. In (c), a representative cell (out of 22 imaged in three experiments) co-transfected with GFP-HA-VAChT and AP180-C was imaged for green fluorescence. (d) The same cell imaged for the HA epitope. (e) A similar experiment as for (a) in cells transfected with GFP-HA-VAChT D491-530. (f) The same field imaged for HA epotope.
(g) The green fluorescence of a representative cell that was co-transfected with GFP-HA-VAChTD491-530 and AP180-C. (h) The same cell stained for the HA epitope. Confocal parameters were identical on (b), (d), (f), and (h). (i) Representative PC12 cells stably expressing GFP-VAChT that were differentiated with NGF for 7 days. (j) PC12 cells stably expressing GFP-HA-VAChTD491-530 that were differentiated with NGF for 7 days. Arrows show neurite tips and varicosities. Representative optical sections are shown in all figures. Scale bar ¼ 20 lm. GFP, green fluorescent protein; HA, hemagglutinin; VAChT, vesicular acetylcholine transporter.
synaptic vesicles, or alternatively, this tyrosine-based motif is not as important as L485 and L486 are for the steadystate localization of VAChT. One possibility, which remains to be tested, is that this motif might be relevant during synaptic vesicle endocytosis, but not during endosomal trafficking. Synaptic vesicle proteins, like other axonal proteins, could in principle be selectively delivered to axonal carriers directly from the trans-Golgi network (Nakata et al. 1998; Sampo et al. 2003), or could be delivered non-specifically to the plasma membrane and then targeted to synaptic vesicle precursors by selective retrieval (Sampo et al. 2003). VAMP2 is one protein that appears to use selective retrieval
from other compartments in the cell to be delivered to axons and synaptic vesicles. Indeed, mutation of an amino acid that is important for endocytosis of VAMP2 (M46A) impairs its polarized localization in neurons (Sampo et al. 2003). This agrees with previous experiments in Caenorhabditis elegans where in an unc-11/AP-180 mutant, VAMP trafficking is disrupted and the protein is found in dendrites and the cell body (Nonet et al. 1999). These observations suggest that retrieval by endocytosis is necessary for trafficking of VAMP2 to synaptic vesicles. However, an interaction with synaptophysin also has been shown to be of importance for retention of VAMP in vesicles (Pennuto et al. 2003). Previous experiments have suggested that synaptophysin
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also is internalized in endosomes in the cell body prior to its targeting to synaptic-like microvesicles (Regnier-Vigouroux et al. 1991). Overexpression of a dynamin I mutant (K44A) impairs the trafficking of VAChT and leads to accumulation of VAChT in endocytic intermediates close to the plasma membrane. Moreover, this effect is not observed for GFP-VAChT L485A L486A, suggesting that the sorting of VAChT at the cell body to the endocytic intermediates takes advantage of the di-leucine motif (Barbosa et al. 2002). Although it has been reported that dynamin I and L485 and L486 are important for VAChT trafficking, there has been no direct test of whether the endocytic process, necessary for VAChT trafficking, involves clathrin. This seems to be of importance since several nonclathrin-mediated processes are also dependent on dynamin I activity (Nichols and Lippincott-Schwartz 2001). Thus, we overexpressed a fragment of the adaptor protein AP-180 that has been shown to sequester clathrin (Zhao et al. 2001) and specifically disrupt clathrin-mediated endocytosis (Ford et al. 2001; Nichols et al. 2001) to investigate VAChT endocytosis and steady-state distribution. We found that overexpression of AP-180-C impairs VAChT endocytosis as well as trafficking. Importantly, these effects are not cell specific, as PC12 cells stably expressing different VAChT constructs present identical results as SN56 cells. Moreover, steady-state distribution of GFP-VAChT D491-530, which lacks the last 40 residues at the carboxyl terminal tail (including Y524, Y526 and Y527), was also disrupted by overexpression of AP-180-C. These results suggest that steady-state distribution of VAChT depends on clathrin-mediated endocytosis and that the major determinant required for endocytic retrieval is the di-leucine motif. One possibility is that certain proteins are not targeted directed from the trans-Golgi network to synaptic vesicles. Therefore, clathrin-mediated endocytosis could contribute to select proteins that enter ‘preformed packages’, used to carry synaptic proteins to nerve terminal (Ahmari et al. 2000). Clearly a second selection step would be necessary to sort synaptic vesicle proteins to axonal carriers after clathrinmediated endocytosis. Di-leucine motifs are present in a wide variety of proteins where they can interact with multiple adaptor protein complexes to mediate protein trafficking (Bonifacino and Traub 2003). It is noteworthy, however, that other proteins that use a di-leucine-like signal for trafficking in neurons, such as the sodium channel NAv 1.2, use selective endocytic retrieval to maintain their steady-state localization in neurons (Garrido et al. 2001). Importantly, the high-affinity choline transporter CHT1, which is predominantly present in cholinergic synaptic vesicles and co-localizes with VAChT in SN56 cells (Ribeiro et al. 2003), presents a di-leucine-like motif at its C-terminal tail that seems to be the major determinant for its endocytosis (Ribeiro et al. 2005). Endocytosis of CHT1 is, similarly to what was observed here for VAChT, dependent on dynamin I and clathrin (Ribeiro et al. 2005). Therefore, it seems that the
two transporters controlling the synthesis and storage of ACh may use, at least in part, similar trafficking motifs. Another possibility is that oligomerization has a role in VAChT trafficking. Preliminary experiments (Prado and Prado, unpublished results) have indicated that HA-tagged VAChT failed to immunoprecipitate GFP-VAChT. Moreover, overexpression of VAChT with mutants did not seem to alter the localization of either protein when compared to the pattern observed when the proteins were expressed individually (our unpublished observations). Thus, it is unlikely that oligomerization influences VAChT trafficking signifficantly. In summary, our data indicate that amino acids 481–490 contain the necessary information in the C-terminal tail of VAChT for proper targeting to microvesicles. Within this region, residues L485 and L486 appear to be absolutely necessary. We also provide novel evidence that supports the need for clathrin-mediated retrieval of VAChT as an obligatory step during the trafficking of this transporter to synapticlike microvesicles. Acknowledgements We thank Drs Robert Edwards, W. Volknandt, Louis Hersh and B. J. Nichols for their gifts of reagents. This work was supported by CNPq, CAPES, FAPEMIG, IBRO and a Center for Excellence grant (Pronex-MG). Part of this work was carried out during a sabbatical period taken by MAMP and VFP at the Department of Cell Biology, Duke University, and they wish to thank Marc Caron for his continuous support and encouragement during this period. MAMP is a fellow of the Guggenheim Foundation (2004–05).
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2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 957–969
