Journal of Neurochemistry, 2001, 78, 1104±1113

Traf®cking of green ¯uorescent protein tagged-vesicular

acetylcholine transporter to varicosities in a cholinergic cell line

M. S. Santos,* J. Barbosa Jr,² G. S. Veloso,² F. Ribeiro,* C. Kushmerick,² M. V. Gomez,²S. S. G. Ferguson,³ V. F. Prado* and M. A. M. Prado²

*LaboratoÂrio de Neurobiologia Molecular, Departamento de BioquõÂmica-Imunologia, ²LaboratoÂrio de Neurofarmacologia,

Departamento de Farmacologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

³J.P. Robarts Research Institute, London, Ontario, Canada

Abstract

Synaptic vesicle proteins are suggested to travel from the

trans-Golgi network to active zones via tubulovesicular

organelles, but the participation of different populations of

endosomes in traf®cking remains a matter of debate. There-

fore, we generated a green ¯uorescent protein (GFP)-tagged

version of the vesicular acetylcholine transporter (VAChT) and

studied the localization of VAChT in organelles in the cell body

and varicosities of living cholinergic cells. GFP±VAChT is

distributed to both early and recycling endosomes in the cell

body and is also observed to accumulate in endocytic

organelles within varicosities of SN56 cells. GFP±VAChT

positive organelles in varicosities are localized close to

plasma membrane and are labeled with FM4-64 and GFP±

Rab5, markers of endocytic vesicles and early endosomes,

respectively. A GFP±VAChT mutant lacking a dileucine

endocytosis motif (leucine residues 485 and 486 changed to

alanine residues) accumulated at the plasma membrane in

SN56 cells. This endocytosis-defective GFP±VAChT mutant

is localized primarily at the somal plasma membrane and

exhibits reduced neuritic targeting. Furthermore, the VAChT

mutant did not accumulate in varicosities, as did VAChT. Our

data suggest that clathrin-mediated internalization of VAChT

to endosomes at the cell body might be involved in proper

sorting and traf®cking of VAChT to varicosities. We conclude

that genesis of competent cholinergic secretory vesicles

depends on multiple interactions of VAChT with endocytic

proteins.

Keywords: endocytosis, neurotransmitter release, neuro-

transmitter transporter, protein traf®c, synaptic vesicle, vesi-

cular acetylcholine transporter.

J. Neurochem. (2001) 78, 1104±1113.

Chemical transmission of signals at neuronal synapses

requires the regulated release of neurotransmitters stored

in synaptic vesicles. Synaptic vesicles are loaded with

neurotransmitter by means of speci®c vesicular transporters.

One family of vesicular transport proteins includes trans-

porters for monoamines (VMAT1 and VMAT2) and

acetylcholine (VAChT). The VAChT is a 12-transmembrane

domain protein that transports acetylcholine (ACh) synthe-

sized in cholinergic nerve terminals into synaptic vesicles in

exchange for H1 ions (for a review see Parsons 2000).

Alteration of transporter expression or traf®c to secretory

vesicles can alter neurotransmission (Pothos et al. 2000).

Targeting of synaptic vesicle proteins to synaptic sites is a

complicated process that appears to involve multiple

molecular determinants. For example, two distinct intra-

cellular traf®cking determinants are contained within the

cytoplasmic domain of synaptobrevin. One determinant

targets synaptobrevin to axons, whereas the other increases

the incorporation of the protein into hippocampal neuron

synaptic vesicles (Grote and Kelly 1996; West et al. 1997).

Furthermore, the incorporation of proteins into synaptic

1104 q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 1104±1113

Received April 17, 2001; revised manuscript received June 13, 2001;

accepted June 14, 2001.

Address correspondence and reprint requests to M. A. M. Prado,

LaboratoÂrio de Neurofarmacologia, Departamento de Farmacologia,

ICB, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627,

31270-901 Belo Horizonte, Minas Gerais, Brazil.

E-mail: mprado@icb.ufmg.br

Abbreviations used: ACh, acetylcholine; AP1, adaptor protein

complex 1; AP2, adaptor protein complex 2; DMEM, Dulbecco's

modi®ed Eagle's medium; GFP, green ¯uorescent protein; HB,

Hepes-buffered solution; HBSS, Hepes-buffered salt solution;

LDCVs, large dense core vesicles; SLMVs, synaptic-like microvesicles;

Tfn-TxR, Texas Red-labeled transferrin; TGN, trans-Golgi network;

VAChT, vesicular acetylcholine transporter; VMAT, vesicular mono-

amine transporter.

vesicles may represent a multistep process, as synaptophysin

passes from the trans-Golgi network (TGN) to the plasma

membrane and endosomes prior to being localized to

synaptic-like microvesicles (SLMVs) in non-differentiated

PC12 cells (ReÂgnier-Vigouroux et al. 1991; for a review see

Hannah et al. 1999).

The structural determinants that regulate the traf®cking of

VAChT and VMATs to SLMVs or large dense core vesicles

(LDCVs) appear to reside within their C-terminal tail

domains (Varoqui and Erickson 1998). In particular, both

VMAT2 and VAChT possess dileucine-like endocytosis

motifs in their C-terminal tails (Tan et al. 1998), whose

mutation reduces endocytosis and results in the accumula-

tion of both transporters in the plasma membrane, suggest-

ing a role for clathrin in their internalization (Tan et al.

1998). Amino acid residues proximal to the dileucine motif

appear to contribute to the differential sorting of VAChT

and VMATs in PC12 cells (Krantz et al. 2000). VAChT is

phosphorylated by protein kinase C (Barbosa et al. 1997;

Cho et al. 2000; Krantz et al. 2000), and phosphorylation

appears to regulate VAChT traf®c (Cho et al. 2000; Krantz

et al. 2000).

Although there is evidence that clathrin-mediated endo-

cytosis contributes to the regulation of VAChT distribution

in PC12 cells, the steps underlying sorting and traf®cking of

newly synthesized transporter protein from the TGN to

synaptic vesicles have yet to be determined. Biogenesis of

synaptic vesicles has been suggested to occur at nerve

endings (Nakata et al. 1998; Ahmari et al. 2000), with the

plasma membrane or endosomal compartments supposedly

as sources of synaptic vesicle proteins (FauÂndez et al. 1998;

Shi et al. 1998). However, it is not clear whether VAChT,

after exiting from the TGN, is targeted directly to nerve

terminals to be incorporated into endosomes or synaptic

vesicles or whether, as an alternative, the transporter uses

endocytic sorting machinery at the soma to be directed to its

®nal location. To examine these questions, we tagged

VAChT with the enhanced green ¯uorescent protein (GFP)

and studied its localization in living SN56 cells. Our results

provide evidence that cell body endocytic mechanisms and

organelles participate in targeting of VAChT to varicosities

in neuronal cells, and suggest that clathrin-mediated

endocytosis will have an important role in the genesis of

synaptic vesicles competent to release ACh.

Materials and methods

Cell culture

SN56 cells were a generous gift of Professor Bruce Wainer

(Department of Pathology, Emory University School of Medicine,

Atlanta, GA, USA). Cells were maintained in Dulbecco's modi®ed

Eagle's medium (DMEM; Sigma Chemical Co., St Louis, MO,

USA), 10% fetal bovine serum (Cultilab, Campinas, SP, Brazil),

2 mm l-glutamine, 1% penicillin/streptomycin in 50 mL culture

bottles at 378C in a 5% CO2 atmosphere. For differentiation we

incubated cells in the same medium as above but lacking fetal

bovine serum and supplemented with 1 mm dibutyril-cyclic-AMP

(Sigma) for at least 2 days. The medium was changed every 2 days.

For confocal analysis, cells were grown directly on coverslips and

were exposed to the same differentiation protocol as described

above. Cell passages up to 8 were used in the experiments.

Plasmid constructs

GFP±syntaxin 1A was a generous gift from Dr Ingo Leibiger

(Department of Molecular Medicine, Karolinska Institute, Stock-

holm, Sweden; Yang et al. 1999). GFP±Rab5 and Rab5:Q79L were

a gift of Professor Marc G. Caron (Department of Cell Biology,

Duke University and Howard Hughes Medical Institute).

The complete coding region of the mouse VAChT gene was

ampli®ed from genomic DNA by PCR using Pfu polymerase

(Barbosa et al. 1999). EcoRI and BamHI restriction sites were

added to 5 0 and 3 0 primers, respectively, in order to facilitate

cloning into the pEGFP±C2 vector (Living Colorse; Clontech

Laboratories, Palo Alto, CA, USA). The resulting clone (GFP±

VAChT) was used as a template to construct a mutant cDNA in

which the dileucine motifs previously shown to be important for

endocytosis (amino acid residues 485 and 486) were changed to

alanine residues (Fig. 1). Base changes were produced by

sequential PCR steps according to Ausubel et al. (1991). The

mutated cDNA was cloned in the EcoRI and BamHI restriction sites

of pEGFP±C2 vector and was denominated GFP±VAChT LLAA.

Sequences for all the primers are available upon request.

Plasmids were puri®ed using the Wizarde Plus Maxipreps DNA

Puri®cation System (Promega, Madison, WI, USA). To ensure that

no mutation had been introduced by the polymerase, all clones had

their sequence determined by automatic sequencing using the

dideoxynucleotide chain-termination method (Sanger et al. 1977).

Cell transfection

Approximately 5 � 104 SN56 cells were plated on coverslips 24 h

before transfection. Cells were transfected using 1 mg of DNA

employing liposome-mediated methods (either Tfxe-50 from

Promega or LipofectAMINE; Life Technologies, Gaithesburg,

Fig. 1 Schematic illustration of wild-type and mutant GFP±VAChT

fusion proteins. Hatched box corresponds to the N-terminal domain,

dotted box indicates the 12 transmembrane domains and open box

represents the C-terminal domain. Part of the C-terminal sequence

is indicated in order to show the dileucine to di-alanine mutation;

LLAA (L485A/L486A). Numbers indicate the position of residues that

border the C-terminal domain.

VAChT traf®c in living cells 1105

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 1104±1113

MD, USA) according to manufacturer instructions. SN56 cells were

maintained in serum-free medium and differentiation started 4 h

after transfection. GFP expression was examined 48±60 h after

transfection as suggested by Clontech.

Confocal imaging

Experiments were performed at room temperature (20±258C). Cells

on coverslips were incubated in HEPES-buffered salt solution

(HBSS in mm: 124 NaCl, 4 KCl, 2 CaCl2, 1.2 MgCl2, 10 glucose,

25 HEPES, pH 7.4 adjusted with NaOH). The coverslips were

washed in HBSS, then transferred to a custom holder in which the

coverslip formed the bottom of a 400-mL 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). The appropriate set of ®lters and laser lines was used to

avoid bleed-through between the different ¯uorophors used in this

work. Usually a Z-series of GFP-expressing cells was obtained

with an iris of , 3 mm. However, some optical sections of ®xed

cells were obtained with larger pinholes (6 mm). Image analysis

and processing were performed with the software Lasersharp

(Bio-Rad), Confocal Assistant, Adobe Photoshop and

Metamorph (Universal Imaging).

Labeling of organelles

In order to label endocytic organelles we used the styril dye

FM4-64. Cells were incubated with 6 mm of FM4-64 (Molecular

Probes, Eugene, OR, USA) for 30±90 min at 378C in 5% CO2 and

then visualized by confocal microscopy as described previously

(Barbosa et al. 1999). Labeling of endosomes was also performed

incubating cells with 30 mg/mL of Texas Red-labeled transferrin

(Tfn-TxR; Molecular Probes) at 378C in 5% CO2 for 5 min

(labeling of early endosomes) or 20 min (labeling of recycling

endosomes). After incubation, cells were washed three times with

ice-cold phosphate-buffered saline (PBS) and then ®xed with 3%

paraformaldehyde in PBS for 20 min

Western blotting

For preparation of post-nuclear supernatants, cells were collected in

homogenization buffer (HB) consisting of PBS (pH 7.4), 5 mm

EGTA and 200 mL of a protease inhibitor mixture (complete

protease inhibitor tablets; Boehringer Mannheim, Indianapolis, IN,

USA), and centrifuged for 10 min at 800 g. Cell pellets were

resuspended in HB, homogenized and nuclei and unbroken cells were

pelleted by centrifugation (800 g for 10 min at 48C). Aliquots of cell

extracts were frozen at 2808C until use. Proteins were resolved using

8% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and

transferred overnight to a nitrocellulose membrane as described

(Towbin et al. 1979; Barbosa et al. 1997). Nitrocellulose membranes

were blocked with PBs containing 0.1% Tween 20 and 5% non-fat

milk, incubated with commercial anti-serum against VAChT

(1 : 400; Phoenix Pharmaceuticals, Mountain View, CA, USA),

washed, and then incubated with a secondary goat anti-rabbit

antibody conjugated with peroxidase (1 : 50 000). Staining was

revealed by enhanced chemiluminescence (ECL from Amersham

Pharmacia Biotech, Piscataway, NJ, USA).

Quanti®cation of the ¯uorescence

Image analysis was carried out to quantify the ¯uorescence

intensity of GFP±VAChT LLAA (n � 30) and GFP±syntaxin 1A

(n � 19) present in the plasma membrane at the cell body and

neurites. We obtained Z-series of the cells and chose optical slices

presenting the strongest ¯uorescent signal at the plasma membrane

for analysis. These regions were marked and the mean ¯uorescence

intensity was estimated using Metamorph software. Neurites were

examined only in a distance of at least 15 mm from the cell body.

In order to determine whether GFP±VAChT was accumulated in

varicosities, we analyzed ¯uorescence intensity in the brightest

early endosomes in the cell body and compared it with the

¯uorescence intensity of punctated labeling (presumably synaptic

vesicles or early endosomes) in the varicosity that presented the

strongest ¯uorescence signal for a given cell (n � 90). We chose to

analyze early endosomes (i.e. well de®ned ¯uorescent puncta

, 6 mm from the plasma membrane) because GFP±VAChT

present in the perinuclear region might represent newly synthesized

protein in addition to protein present in recycling endosomes.

Immuno¯uorescence

Cells were washed twice in 0.1 m phosphate buffer (pH 7.4) and

then ®xed with 3% paraformaldehyde in 0.1 m phosphate buffer for

20 min. Immunolabeling was performed as described elsewhere

(Barbosa et al. 1999). Primary antibody used was an anti-Syntaxin

(1 : 30) antibody (Alomone Labs, Jerusalem, Israel). The second-

ary antibody coupled to Alexa568e (Molecular Probes) was used

for immunostaining. Controls to test for speci®c labeling consisted

on omission of the primary antibody and adsorption of antibody

with a syntaxin peptide supplied by the manufacturer (not shown).

Results

Immunoblot analysis of SN56 cells transfected with GFP±

VAChT construct using an antibody against VAChT

indicated the presence of a major protein of 107 kDa. This

protein was not found in untransfected cells (Fig. 2a). In

addition, endogenous VAChT was identi®ed in both

transfected and non-transfected SN56 cells using the

VAChT antibody (Fig. 2a, arrow).

Confocal microscopy analysis of SN56 cells transfected

with GFP±VAChT (Fig. 2b) indicates that the protein is

expressed at the cell body in a perinuclear region and is

also accumulated in varicosities of neurites. Labeling of

GFP±VAChT was punctated and frequently found close to

the plasma membrane, specially in varicosities, where the

punctated labeling was most prominent in proximity to the

plasma membrane. Thus, GFP±VAChT labeling was con-

sistent with localization of the transporter in synaptic vesicle

or early endosomes. This pattern of labeling was similar to

that observed for native VAChT in SN56 cells (Barbosa

et al. 1999) and in PC12 cells (Liu and Edwards 1997) and it

was completely distinct from the pattern observed in SN56

cells expressing the GFP protein alone (Fig. 2d).

In order to investigate the nature of the compartments

visualized by GFP±VAChT we performed a series of

double-labeling experiments. Incubation of cells for

30 min with FM4-64, an endocytic dye used to visualize

synaptic vesicles and other endocytic organelles, showed

1106 M. S. Santos et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 1104±1113

labeling of the same punctated structures labeled by VAChT

in varicosities (Figs 3a and b, arrow). Higher magni®cation

view of a varicosity showed that the pattern of FM4-64 and

GFP±VAChT labeling was very similar (Figs 3c and d) and

illustrates the localization of GFP±VAChT labeled orga-

nelles in the proximity of the plasma membrane. Few

organelles labeled with GFP±VAChT did not incorporate

FM4-64 suggesting that some GFP±VAChT labeled organ-

elles in varicosities did not undergo spontaneous cycling

during incubation with the styril dye. At the cell body,

constitutive staining with FM4-64 was predominant at a

perinuclear region, and it also showed signi®cant colocaliz-

ation with GFP±VAChT (Figs 3a and b, arrowhead).

The above experiments suggest the presence of

GFP±VAChT in endocytic organelles. However, endosomes

are functionally heterogeneous organelles with distinct

morphological and biochemical features. We thus used

transferrin-Texas Red (Tfn-TxR) to investigate the nature of

endocytic compartments labeled by GFP±VAChT. Expo-

sure of SN56 cells to Tfn-TxR for 5 min at 378C labeled

Fig. 3 GFP2VAChT is present in endocytic organelles in varico-

sities and in a perinuclear region. (a) GFP±VAChT fusion protein

was transiently expressed in differentiated SN56 cells. Cells were

optically sectioned and the maximum Z projection is shown. Bar,

20 mm. (b) ± FM4-64 ¯uorescence of the same ®eld in A. Arrows

indicate colocalization of GFP±VAChT and FM4-64 in a growth cone

and arrowhead indicates colocalization of GFP±VAChT and FM4-64

in the soma of a transfected cell. The result is representative of 17

cells from 4 different experiments. (c) ± Magni®ed view of a varicos-

ity from another cell expressing GFP±VAChT. The ¯uorescence is

distributed close to the plasma membrane. (d) ± Staining of the

same varicosity with FM4-64. Bar, 5 mm.Fig. 2 Expression of VAChT and GFP2VAChT fusion protein in

SN56 cells. (a) Post-nuclear supernatant proteins of SN56 cells

transfected (line 1) or not transfected (line 2) with GFP2VAChT

were separated by SDS2PAGE and immunostained with the VAChT

antibody as described in the text. Major bands of < 80 kDa were

detected in both extracts corresponding to endogenous VAChT

(arrow). In transfected cells, a major band of < 107 kDa correspond-

ing to the predicted mobility of GFP2VAChT was detected. (b) Distri-

bution of GFP2VAChT transiently expressed in SN56 cells. Living

cells were examined using laser scanning confocal microscopy by

optical sectioning and the maximum Z-projection is shown. These

cells are representative of over 90 cells examined. (c) Differential

interference contrast (DIC) image of the cell in (b). (d) Distribution of

GFP protein transiently expressed in SN56 cells. (e) DIC image of

the cell in (d). Bars, 20 mm.

Fig. 4 GFP2VAChT is present in early and recycling endosomes.

SN56 cells were transfected with GFP2VAChT and examined by

confocal microscopy (a and c). (b) Labeling of cells in (a) with Tfn-

TxR for 5 min at 378C. Arrows indicate some of the structures

labeled by both GFP2VAChT and Tfn-TxR. Bar, 10 mm. (d) Label-

ing of cells in (c) with Tfn-TxR for 20 min at 378C. Arrows indicate

colocalization of GFP2VAChT and transferrin in recycling endo-

somes. The arrowhead shows colocalization in early endosomes.

Bar, 20 mm.

VAChT traf®c in living cells 1107

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 1104±1113

organelles close to the plasma membrane, i.e. early

endosomes (Fig. 4b). A 20-min incubation with Tfn-TxR

led to labeling of a perinuclear region in SN56 cells that

has been described in other cells as the recycling

endosomal compartment (Fig. 4d, Yamashiro and Max®eld

1984).

Analysis of GFP±VAChT transfected cells that were

exposed for 5 min to Tfn-TxR showed that GFP±VAChT

was colocalized with Tfn-TxR ¯uorescence in punctated

intracellular structures (Figs 4a and b). Moreover, a large

part of perinuclear GFP±VAChT was also present within the

recycling endosomal compartment visualized after 20 min

incubation with Tfn-TxR (Figs 4c and d). Although weak

labeling of varicosities with Tfn-TxR was observed in some

cells (not shown), Tfn-TxR labeling was predominantly

localized to the soma of SN56 cells. Thus, different from

FM4-64, Tfn-TxR could not be used to identify VAChT

positive organelles in varicosities. Lack of labeling of

neurites with Tfn-TxR is consistent with the absence of

transferrin receptors in neurites as described before

(Chilcote et al. 1995).

Early endosomes have been shown to participate in the

genesis of synaptic vesicles. Therefore, to gain further

insight into the nature of the intracellular organelles labeled

by GFP±VAChT and FM4-64 in neurites of SN56 cells, we

tested whether Rab5, a GTP-binding protein involved in

early endosomal function and traf®cking, was also present in

VAChT-positive intracellular organelles. In differentiated

SN56 cells, GFP±Rab5 was located in punctated structures

Fig. 5 GFP2VAChT is present in Rab5-positive endosomes. (a)

Cells were transfected with GFP2Rab5 construct, and living cells

were examined by serial Z-sectioning. The maximum Z projection is

presented. (b) Image of the same cell as in (a) stained with FM4-64

for 30 min. Arrows indicate colocalization of GFP2Rab5 and FM4-

64. (c) Coexpression of GFP2VAChT and the constitutively acti-

vated Rab5 mutant Q79L. Cells were examined by serial Z-section-

ing and the maximum Z projection is shown. Expression of

Rab5:Q79L results in larger endosomes visualized after incubation

with FM4-64 (c2). GFP2VAChT (c1) also appears in structures lar-

ger than that observed normally (e.g. Figs 1, 2, 3 and 4) which have

FM4-64 (c2). Bars, 20 mm.

Fig. 6 Subcellular distribution of GFP2VAChT LLAA in SN56 cells.

(Upper) Digital reconstruction (maximum Z projection) of cells

expressing GFP2VAChT LLAA. Note that ¯uorescence is present at

the plasma membrane. Cell A has ¯uorescence in neurites, whereas

cell B does not. Arrow indicates a varicosity labeled with the mutant

protein and arrowhead a neurite from cell B that is not labeled.

(Lower) DIC image of the same cells. Bar, 20 mm.

1108 M. S. Santos et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 1104±1113

at the cell body and in varicosities of neurites (Fig. 5a).

Organelles labeled with GFP±Rab5 in varicosities showed

colocalization with FM4-64 (Figs 5a and b, arrows). Thus,

the localization of GFP±Rab5 is consistent with previous

reports demonstrating that Rab5 is localized to the early

endosomal membrane compartment of neurons and may also

be distributed to synaptic vesicles (de Hoop et al. 1994;

Fischer von Mollard et al. 1994; SoÈnnichsen et al. 2000).

In the cell body, there was also some colocalization of

GFP±Rab5 and FM4-64, however, it was dif®cult to

determine if the organelles that had GFP±Rab5 were

the same as those that had GFP±VAChT. To test whether

in cell bodies GFP±VAChT organelles also recruit Rab5,

we cotransfected GFP±VAChT with a Rab5 mutant

(Rab5:Q79L) defective in GTP hydrolysis which has been

shown to facilitate endosome homotypic fusion (Stenmark

et al. 1994). This experiment shows that GFP±VAChT

positive organelles at the cell body are greatly enlarged in

this condition (Fig. 5c1), consistent with the action of the

Rab5:Q79L protein. In these enlarged endosomal vesicles,

GFP±VAChT ¯uorescence (Fig. 5c2) is easily discernible at

the endosomal membrane, whereas FM4-64 appears to be

restricted to the interior of the endosome (Fig. 5c2).

The presence of GFP±VAChT in endocytic organelles at

the cell body of differentiated SN56 cells suggests that,

similar to synaptophysin, GFP±VAChT may cycle through

the plasma membrane before reaching endocytic organelles.

That is, VAChT may be targeted to the somal plasma

membrane, internalized by endocytosis and only then be

sorted to varicosities. However, the presence of GFP±

VAChT in endosomal compartments in the soma does not

exclude the possibility that GFP±VAChT endocytosis

occurs primarily at varicosities. Therefore, to test whether

VAChT endocytosis is required for the localization of

VAChT to somal endosomes, we generated a GFP±VAChT

mutant in which the dileucine motif (amino acid residues

485 and 486) previously shown to be necessary for

endocytosis was replaced by di-alanine (Tan et al. 1998;

GFP±VAChT LLAA). Previous experiments have examined

the localization of VAChT presenting a similar mutation by

immuno¯uorescence in non-neuronal cells (Tan et al. 1998).

However, to our knowledge the distribution of this VAChT

mutant has not been investigated in living neuronal cells.

Consistent with the results of Tan et al. (1998), GFP±VAChT

LLAA was located mainly at the plasma membrane (Fig. 6).

Surprisingly, we observed two distinct labeling patterns; in

< 60% of the cells, varicosities were labeled at the plasma

membrane (Fig. 6, cell A), whereas in the remaining 40%

little or no GFP±VAChT LLAA labeling was detected in

varicosities or processes (Fig. 6, cell B, arrowheads).

A detailed analysis of ¯uorescence in a large number of

cells transfected with GFP±VAChT or the mutant (Table 1)

indicated that there were signi®cantly fewer cells with the

protein in varicosities (identi®ed morphologically) when the

GFP±VAChT LLAA construct was expressed compared with

GFP±VAChT. GFP±VAChT LLAA labeling of recycling

endosomes was also well diminished when compared with

GFP±VAChT.

Fig. 7 Expression of syntaxin 1A and GFP2syntaxin 1A in SN56

cells. (a) Immuno¯uorescence analysis of syntaxin 1A localization in

SN56 cells. Cells were cultured and processed for immuno¯uores-

cence as described in Materials and methods and imaged by confo-

cal microscopy. An optical section of representative cells stained

with an anti-syntaxin 1A antibody is shown. (b) Subcellular distri-

bution of GFP2syntaxin 1A fusion protein transiently expressed in

differentiated SN56 cells. Living cells were examined using laser

scanning confocal microscopy. GFP2syntaxin 1A is present in the

membrane of the cell body and neurites. Bars, 10 mm.

Table 1 Comparison of the subcellular distribution of GFP2VAChT and GFP2VAChT LLAA

GFP±VAChT GFP±VAChT LLAA

Total of cells 90 82

Fluorescence in varicosities 84 cells (93�.3%) 49 cells (59�.8%)

Fluorescence in recycling endosomes 75 cells (83�.3%) 14 cells (17%)

Images of cells were inspected and the number of cells presenting GFP±VAChT ¯uorescence in varicosities or perinuclear region was recorded for

the wild-type or mutated ¯uorescent protein.

VAChT traf®c in living cells 1109

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 1104±1113

We measured the ¯uorescence intensity of the brightest

region at the plasma membrane in the soma and neurites of

cells expressing GFP±VAChT LLAA to ascertain whether

the protein was evenly distributed or whether one of these

compartments presented preferential accumulation of the

protein. We excluded from the analysis cells presenting no

¯uorescence in neurites, i.e. 40% of the cells. The remaining

cells expressing the mutant were analyzed and the

¯uorescence intensity was compared individually. The data

indicate that ¯uorescence in the soma plasma membrane

was on average 36% higher than in neurites (Fig. 8a).

In order to test whether this analysis would discriminate

if the ¯uorescence intensity in plasma membrane of the

cell body and varicosities were homogeneous, we analyzed

the expression pattern of GFP2syntaxin 1A. We chose

syntaxin 1A because this plasma membrane protein is

present in axons and soma of cultured hippocampal neurons

without evidence of preferential accumulation (Garcia et al.

1995; reviewed by Galli et al. 1995).

Immuno¯uorescence analysis of SN56 cells shows that

endogenous syntaxin 1A is found at the plasma membrane

(Fig. 7a) in a pattern similar to that described for cultured

hippocampal neurons (Galli et al. 1995; Garcia et al. 1995).

Cells transfected with a GFP±syntaxin 1A construct showed

the same subcellular distribution of endogenous syntaxin

(Fig. 7b). Importantly, in contrast to the results obtained

with the GFP±VAChT LLAA, the intensity of ¯uorescence

at the plasma membrane for GFP±syntaxin was identical for

the brightest regions of the cell body and processes of SN56

cells (Fig. 8b).

We also analyzed whether GFP±VAChT ¯uorescence

was similar in early endosomes in the cell body and

organelles in varicosities (endocytic organelles or synaptic

vesicles). GFP±VAChT labeled organelles in varicosities

were on average 33% more ¯uorescent than early endo-

somes in the cell body. This suggests that contrary to the

experiments with the mutant GFP±VAChT LLAA, which is

more concentrated in the cell body, GFP±VAChT appears to

be more concentrated in varicosities.

Discussion

In neurons, synaptic vesicle genesis appears to occur at

nerve terminals (reviewed by Hannah et al. 1999). It has

been suggested that distinct synaptic proteins travel via

tubulovesicular or irregular organelles by axonal transport to

their ®nal destination (Nakata et al. 1998; Ahmari et al.

2000). In one of the models proposed, proteins travel

independently using tubulovesicular organelles from the

TGN to nerve endings (Nakata et al. 1998). However, recent

results suggest that synaptic proteins may travel together, as

a `pre-assembled' proto-terminal (Ahmari et al. 2000).

Whereas in the ®rst proposal, the participation of cell body

endocytic organelles is supposedly negligible (Nakata et al.

1998), the suggestion of formation of proto-terminals may

involve the participation of endosomes and sophisticated

sorting process in the soma. In agreement with the latter

model of synaptic vesicle protein traf®cking, some synaptic

Fig. 8 Quanti®cation of ¯uorescence intensity of GFP2VAChT

LLAA, GFP2syntaxin 1A and GFP2VAChT in the soma or neurite

of SN56 cells. (a) Average ¯uorescence intensity of the brightest

region in the cell body membrane for 30 randomly chosen cells

expressing GFP2VAChT LLAA (dark bars) and average of the ¯uor-

escence intensity of the brightest region in the membrane of neurites

(clear bars). The intensity of ¯uorescence of GFP2VAChT LLAA in

neurites is < 36% less than that at the cell body (see details in

methodology). This difference is statistically signi®cant ( p , 0.05,

Student's t-test). (b) Similar analysis was performed in 19 cells

expressing GFP2syntaxin 1A. The ¯uorescence intensity in the

membrane of the cell body or neurites is similar; error bars represent

SEM. (c) Analysis of ¯uorescence intensity of GFP2VAChT present

in the brightest early endosome in the cell body and in endocytic

organelles in varicosities (n � 90). Fluorescence in varicosities was

33% higher than in early endosomes ( p , 0.05, Student's t-test).

1110 M. S. Santos et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 1104±1113

vesicle proteins sort to endosomes in non-neuronal cells

(reviewed by Hannah et al. 1999).

The VAChT provides an attractive model protein to both

characterize and understand the pathways involved with

synaptic vesicle biogenesis, as the transporter is predomi-

nantly distributed to synaptic vesicles (Gilmor et al. 1996;

Weihe et al. 1996; Liu and Edwards 1997; reviewed by

Erickson and Varoqui 2000). Moreover, the determinants

required for synaptic vesicle targeting of VAChT appear to

be localized at the C-terminal domain of the transport

protein (Varoqui and Erickson 1998; Krantz et al. 2000). In

this study, we used a GFP-tagged version of the transporter

and a cholinergic model system to explore the pathways

followed by the protein in living cells. GFP±VAChT was

found predominantly in endocytic organelles at the cell body

and in varicosities of neurites from SN56 cells. The protein

labeled punctated organelles that could be double stained by

short incubation periods with FM4-64, a general endocytic

and synaptic vesicle marker. Mutation of the dileucine motif

at positions 485±486 to alanine changed the traf®cking of

the recombinant protein, suggesting that VAChT amino

acid sequences are primarily responsible for targeting of

GFP±VAChT in SN56 cells.

Previous experiments have shown that brain cholinergic

neurons present somatic staining for VAChT at a perinuclear

region (SchaÈfer et al. 1998). Strong soma staining for

endogenous VAChT is also observed in SN56 cells (Barbosa

et al. 1999). In addition, in PC12 cells stably expressing low

amounts of VAChT, the protein is also found in intracellular

structures at a perinuclear location and has been found in

endosomes by subcellular fractionation (Liu and Edwards

1997). Hence, tagging VAChT with GFP does not appear to

impair or change the traf®c of the protein in SN56 cells. It is

thus unlikely that the presence of GFP±VAChT in endo-

somes is a result of transient overexpression of the protein.

Endosomal populations show differences in their mole-

cular composition and localization in cells (SoÈnnichsen et al.

2000). In polarized cells, endosomal sorting of proteins can

help to distribute them to their appropriate subcellular

location and to maintain cell polarity (reviewed by Winckler

and Mellman 1999). In our experiments, the incubation of

SN56 cells with Tfn-TxR for 5 and 20 min was used to

discriminate the identity of early and recycling endosomal

vesicular populations as described previously in other model

systems (SoÈnnichsen et al. 2000). We found that there was

clear colocalization of GFP±VAChT and Tfn-TxR in

populations of early and recycling endosomes at the cell

body. The localization of GFP±VAChT in early endosomes

was also con®rmed by cotransfection of GFP±VAChT with

a Rab5 mutant constitutively activated that enhances

homotypic endosome fusion. The perinuclear presence of

GFP±VAChT seems to indicate that at least part of the

protein is found in the recycling endosomal population

(labeled with Tfn-TxR for 20 min), but it does not discount

the possibility that part of the perinuclear labeling is

composed of newly synthesized protein passing through

the Golgi.

GFP±Rab5 stained FM4-64 positive organelles in both

varicosities and neurites of SN56 cells. It appears that

FM4-64 labeling in varicosities is primarily contained in

organelles that recruit Rab5. As GFP±VAChT in varicos-

ities is present in organelles that are labeled with FM4-64,

we conclude that GFP±VAChT is localized to Rab5-positive

organelles.

It is not clear at present whether differentiated SN56 cells

would recognize axonal targeting signals, but several lines

of evidence support the notion that these cells present a

neuronal phenotype. Differentiated SN56 cells project long

neurites containing varicosities, and this is dependent on

dynamin 1 activity (J. Barbosa Jr et al. unpublished

observations), as reported for extension of axons from

cultured neurons (Masur et al. 1990; Torre et al. 1994). The

majority of organelles present in varicosities of SN cells are

identical to true synaptic vesicles (Hammond et al. 1990).

Moreover, varicosities accumulate synaptotagmin, synapto-

physin and also VAChT as observed by confocal micro-

scopy (Barbosa et al. 1999), suggesting that the vesicles

contain the necessary proteins involved in transmitter

release. This study has shown that the organelles present

in varicosities (presumably synaptic vesicles seen by

Hammond et al. 1990) undergo cycles of spontaneous

exo-endocytosis and recruit Rab5, a marker or early

endosomes which is also found in synaptic vesicles.

Together these observations provide support for the use of

SN56 cells as a model to study traf®cking of cholinergic

proteins. However, it should be noted that SN56 cells are a

model of immature cholinergic neurons (Hammond et al.

1990), and thus may present small differences in exo-

endocytosis activity.

The fact that GFP±VAChT is present in early and

recycling endosomes in the cell body and in early

endosomes or synaptic vesicles in varicosities does not

reveal the site(s) of GFP±VAChT endocytosis in SN56

cells. However, two alternative mechanisms can be

proposed. VAChT could leave the TGN in tubulovesicular

organelles that are targeted to growth cones and varicosities,

and by constitutive exo-endocytosis the protein would reach

synaptic vesicles before being returned to endosomes in the

cell body by retrograde transport (Nakata et al. 1998). One

alternative view is that VAChT leaves the TGN in

constitutive vesicles, fuses to the plasma membrane at the

cell body and then internalizes to early and recycling

endosomes before sorting to organelles that are targeted to

growth cones and varicosities. By following the localization

of GFP±VAChT LLAA, we gained novel insights on the

journey of VAChT. The mutated protein appeared to

preferentially accumulate at the cell body, although the

presence of the protein in neurites was also detected. There

VAChT traf®c in living cells 1111

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 1104±1113

were some striking features observed with this mutant. First,

labeling in neurites was uniformly distributed at the plasma

membrane, which is in sharp contrast to the predominant

labeling of varicosities and punctated structures seen with

GFP±VAChT. Secondly, there were fewer cells with GFP

labeling in varicosities when the mutant was expressed

compared with the wild-type protein. Thirdly, just a small

percentage of cells expressing the mutant presented some

degree of perinuclear labeling, suggesting that GFP±

VAChT LLAA failed to reach recycling endosomes. Finally,

the maximum ¯uorescence intensity in neurites for the

mutant was 36% lower than that at the plasma membrane in

the soma. The lack of difference for the ¯uorescence

measurements with GFP±syntaxin 1A suggests that prefer-

ential accumulation of GFP±VAChT in the somal plasma

membrane is not an artifact. Moreover, this result is in sharp

contrast with the accumulation of GFP±VAChT in varicos-

ities when compared with early endosome ¯uorescence

(33% more GFP±VAChT in varicosities). Thus, interference

with GFP±VAChT endocytosis will lead to signi®cant

accumulation of the protein in the soma. We thus conclude

that part of GFP±VAChT primarily recycles at the cell body

before targeting to varicosities. However, we cannot discount

the existence of other mechanisms that will carry some of the

protein directly to neurites without passing by soma endocytic

intermediates as suggested by Nakata et al. (1998). Com-

plementary approaches such as metabolic labeling and

subcellular fractionation may have to be used in future

experiments to discriminate between these two possibilities.

Dileucine motifs similar to that found in VAChT have

been shown to interact with adaptor protein complexes 1

and 2 (AP1 and AP2, Dietrich et al. 1997), in particular

with the b subunit (Rapoport et al. 1998). Interaction with

other AP complexes (AP3 for example) has also been

suggested (Blagoveshchenskaya et al. 1999; Bonifacino and

Dell'Angelica 1999). The lack of VAChT mutant internal-

ization perhaps results from impaired interaction of VAChT

with the AP2 complex, as VAChT remains in the plasma

membrane. However, it is possible that, as suggested before,

the dileucine motif in VAChT may have multiple functions

(Krantz et al. 2000).

The lack of intracellular labeling with the GFP±VAChT

LLAA also suggests that clathrin-mediated endocytosis will

have a fundamental role in the incorporation of VAChT into

synaptic vesicles. Internalization of plasma membrane

occurs in a very fast way in varicosities of SN56 cells, as

observed by FM4-64 labeling. However, GFP±VAChT

LLAA is excluded from internalization. Thus, it seems

that no alternative mechanism (such as non-clathrin

mediated endocytosis) is available for the internalization

of GFP±VAChT.

We have extended previous observations on the role of

cell body organelles for synaptic vesicle protein traf®cking

in living cells. Our data indicate that in cholinergic cells, at

least part of VAChT may traf®c to varicosities via

endosomal intermediates, after passage through the plasma

membrane in the neuronal cell body. Much discussion has

been presented in the last few years in favor (reviewed by

Cremona and De Camilli 1997) or against (reviewed by

Palfrey and Artalejo 1998) the role of clathrin mediated

process in synaptic vesicle recycling. Our results suggest,

albeit indirectly, that formation of synaptic vesicles

competent for ACh release will depend on the interaction

of VAChT with clathrin associated proteins.

Acknowledgements

This work received support from CNPq, FAPEMIG, PRONEX,

PADCT and a CAPES fellowship to M.S.S. We thank Ms A. A.

Pereira and E. E. P. Silva for technical help. We thank Drs

Bruce Wainer, Marc Caron and Ingo Leibiger for the gift of

cells and plasmids, respectively.

References

Ahmari S. E., Buchanan J. and Smith S. J. (2000) Assembly of

presynaptic active zones from cytoplasmic transport packets. Nat.

Neurosci. 3, 445±451.

Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G.,

Smith J. A. and Struhl K. (1991) Mutagenesis of cloned DNA, in

Current Protocols in Molecular Biology, Section 8.5, 5±9.Greene

Publishing Associates and Wiley-Interscience, New York.

Barbosa J. Jr, Clarizia A. D., Gomez M. V., Romano-Silva M. A., Prado

V. F. and Prado M. A. M. (1997) Effect of protein kinase C

activation on the release of [3H]acetylcholine in the presence of

vesamicol. J. Neurochem. 69, 2608±2611.

Barbosa J. Jr, Massensini A. R., Santos M. S., Meireles S. I., Gomez

R. S., Gomez M. V., Romano-Silva M. A., Prado V. F. and Prado

M. A. M. (1999) Expression of the vesicular acetylcholine

transporter, proteins involved in exocytosis, and functional

calcium signaling in varicosities and soma of a murine septal

cell line. J. Neurochem. 73, 1881±1893.

Blagoveshchenskaya A. D., Hewitt E. W. and Cutler D. F. (1999)

Di-leucine signals mediate targeting of tyrosinase and synapto-

tagmin to synaptic-like microvesicles within PC12 cells. Mol.

Biol. Cell 10, 3979±3990.

Bonifacino J. S. and Dell'Angelica E. C. (1999) Molecular bases for the

recognition of tyrosine-based sorting signals. J. Cell Biol. 145,

923±926.

Chilcote T. J., Galli T., Mundigl O., Edelmann L., McPherson P. S.,

Takei K. and De Camilli P. (1995) Cellubrevin and synapto-

brevins: similar subcellular localization and biochemical proper-

ties in PC12 cells. J. Cell Biol. 129, 219±231.

Cho G. W., Kim M. H., Chai Y. G., Gilmor M. L., Levey A. I. and

Hersh L. B. (2000) Phosphorylation of the rat vesicular acetylcho-

line transporter. J. Biol. Chem. 275, 19942±19948.

Cremona O. and De Camilli P. (1997) Synaptic vesicle endocytosis.

Curr. Opin. Neurobiol. 7, 323±330.

Dietrich J., Kastrup J., Nielsen B. L., Odum N. and Geisler C. (1997)

Regulation and function of the CD3gamma DxxxLL motif: a

binding site for adaptor protein-1 and adaptor protein-2 in vitro.

J. Cell Biol. 138, 271±281.

Erickson J. D. and Varoqui H. (2000) Molecular analysis of vesicular

amine transporter function and targeting to secretory organelles.

FASEB J. 14, 2450±2458.

1112 M. S. Santos et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 1104±1113

FauÂndez V., Horng J. T. and Kelly R. B. (1998) A function for the AP3

coat complex in synaptic vesicle formation from endosomes. Cell

93, 423±432.

Fischer von Mollard G., Stahl B., Walch-Solimena C., Takei K.,

Daniels L., Khoklatchev A., De Camilli P., SuÈdhof T. C. and Jahn

R. (1994) Localization of Rab5 to synaptic vesicles identi®es

endosomal intermediate in synaptic vesicle recycling pathway.

Eur. J. Cell Biol. 65, 319±326.

Galli T., Garcia E. P., Mundigl O., Chilcote T. J. and De Camilli P.

(1995) v- and t-SNAREs in neuronal exocytosis: a need for

additional components to de®ne sites of release. Neuropharma-

cology 34, 1351±1360.

Garcia E. P., McPherson P. S., Chilcote T. J., Takei K. and De Camilli P.

(1995) rbSec1A and B colocalize with syntaxin 1 and SNAP-25

throughout the axon, but are not in a stable complex with syntaxin.

J. Cell Biol. 129, 105±120.

Gilmor M. L., Nash N. R., Roghani A., Edwards R. H., Yi H., Hersch

S. M. and Levey A. I. (1996) Expression of the putative vesicular

acetylcholine transporter in rat brain and localization in

cholinergic synaptic vesicles. J. Neurosci. 16, 2179±2190.

Grote E. and Kelly R. B. (1996) Endocytosis of VAMP is facilitated by

a synaptic vesicle targeting signal. J. Cell Biol. 132, 537±547.

Hammond D. N., Lee H. J., Tonsgard J. H. and Wainer B. H. (1990)

Development and characterization of clonal cell lines derived

from septal cholinergic neurons. Brain Res. 512, 190±200.

Hannah M. J., Schimd A. A. and Huttner W. B. (1999) Synaptic vesicle

biogenesis. Annu. Rev. Cell. Biol. 15, 733±798.

de Hoop M. J., Huber L. A., Stenmark H., Williamson E., Zerial M.,

Parton R. G. and Dotti C. G. (1994) The involvement of the small

GTP-binding protein Rab5a in neuronal endocytosis. Neuron 13,

11±22.

Krantz D. E., Waites C., Oorschot V., Liu Y., Wilson R. I., Tan P. K.,

Klumperman J. and Edwards R. H. (2000) A phosphorylation site

regulates sorting of the vesicular acetylcholine transporter to

dense core vesicles. J. Cell Biol. 149, 379±396.

Liu Y. and Edwards R. H. (1997) Differential localization of vesicular

acetylcholine and monoamine transporters in PC12 cells but not

CHO cells. J. Cell Biol. 139, 907±916.

Masur S. K., Kim Y. T. and Wu C. F. (1990) Reversible inhibition of

endocytosis in cultured neurons from the Drosophila temperature-

sensitive mutant shibirets1. J. Neurogenet. 6, 191±206.

Nakata T., Terada S. and Hirokawa N. (1998) Visualization of the

dynamics of synaptic vesicle and plasma membrane proteins in

living axons. J. Cell Biol. 140, 659±674.

Palfrey H. C. and Artalejo C. R. (1998) Vesicle recycling revisited: rapid

endocytosis may be the ®rst step. Neuroscience 83, 969±989.

Parsons S. M. (2000) Transport mechanisms in acetylcholine and

monoamine storage. FASEB J. 14, 2423±2434.

Pothos E. N., Larsen K. E., Krantz D. E., Liu Yj Haycock J. W., Setlik

W., Gershon M. D., Edwards R. H. and Sulzer D. (2000) Synaptic

vesicle transporter expression regulates vesicle phenotype and

quantal size. J. Neurosci. 20, 7297±7306.

Rapoport I., Chen Y. C., Cupers P., Shoelson S. E. and Kirchhausen T.

(1998) Dileucine-based sorting signals bind to the beta chain of

AP-1 at a site distinct and regulated differently from the tyrosine-

based motif-binding site. EMBO J. 17, 2148±2155.

ReÂgnier-Vigouroux A., Tooze S. A. and Huttner W. B. (1991) Newly

synthesized synaptophysin is transported to synaptic-like micro-

vesicles via constitutive secretory vesicles and the plasma

membrane. EMBO J. 10, 3589±3601.

Sanger F., Nicklen S. and Coulson A. R. (1977) DNA sequencing with

chain-terminating inhibitors. Proc. Natl Acad. Sci. USA 74,

5463±5467.

SchaÈfer M. K., Eiden L. E. and Weihe E. (1998) Cholinergic neurons

and terminal ®elds revealed by immunohistochemistry for the

vesicular acetylcholine transporter. I. Central nervous system.

Neuroscience 84, 331±359.

Shi G., FauÂndez V., Roos J., Dell'Angelica E. C. and Kelly R. B. (1998)

Neuroendocrine synaptic vesicles are formed in vitro by both

clathrin-dependent and clathrin-independent pathways. J. Cell

Biol. 143, 947±955.

SoÈnnichsen B., De Renzis S., Nielsen E., Rietdorf J. and Zerial M.

(2000) Distinct membrane domains on endosomes in the recycling

pathway visualized by multicolor imaging of Rab4, Rab5, and

Rab11. J. Cell Biol. 149, 901±914.

Stenmark H., Parton R. G., Steele-Mortimer O., Lutcke A., Gruenberg J.

and Zerial M. (1994) Inhibition of rab5 GTPase activity stimulates

membrane fusion in endocytosis. EMBO J. 13, 1287±1296.

Tan P. K., Waites C., Liu Y., Krantz D. E. and Edwards R. H. (1998) A

leucine-based motif mediates the endocytosis of vesicular

monoamine and acetylcholine transporters. J. Biol. Chem. 273,

17351±17360.

Torre E., McNiven M. A. and Urrutia R. (1994) Dynamin 1 antisense

oligonucleotide treatment prevents neurite formation in cultured

hippocampal neurons. J. Biol. Chem. 269, 32411±32417.

Towbin H., Staehelin T. and Gordon J. (1979) Electrophoretic transfer

of proteins from polyacrylamide gels to nitrocellulose sheets,

procedure and some applications. Proc. Natl Acad. Sci. USA 76,

4350±4354.

Varoqui H. and Erickson J. D. (1998) The cytoplasmic tail of the

vesicular acetylcholine transporter contains a synaptic vesicle

targeting signal. J. Biol. Chem. 273, 9094±9098.

Weihe E., Tao-Cheng J. H., Schafer M. K., Erickson J. D. and Eiden

L. E. (1996) Visualization of the vesicular acetylcholine

transporter in cholinergic nerve terminals and its targeting to a

speci®c population of small synaptic vesicles. Proc. Natl Acad.

Sci. USA 93, 3547±3552.

West A. E., Neve R. L. and Buckley K. M. (1997) Targeting of the

synaptic vesicle protein synaptobrevin in the axon of cultured

hippocampal neurons: evidence for two distinct sorting steps.

J. Cell Biol. 139, 917±927.

Winckler B. and Mellman I. (1999) Neuronal polarity: controlling the

sorting and diffusion of membrane components. Neuron 23,

637±640.

Yamashiro D. J. and Max®eld F. R. (1984) Acidi®cation of endocytic

compartments and the intracellular pathways of ligands and

receptors. J. Cell Biochem. 26, 231±246.

Yang S. N., Larsson O., Branstrom R., Bertorello A. M., Leibiger B.,

Leibiger I. B., Moede T., Kohler M., Meister B. and Berggren

P. O. (1999) Syntaxin 1 interacts with the L(D) subtype of

voltage-gated Ca(21) channels in pancreatic beta cells. Proc. Natl

Acad. Sci. USA 96, 10164±10169.

VAChT traf®c in living cells 1113

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 1104±1113