Molecular Biology of the Cell
Vol. 17, 2113–2124, May 2006
A Second SNARE Role for Exocytic SNAP25 in Endosome
Yoshikatsu Aikawa,* Kara L. Lynch, Kristin L. Boswell, and Thomas F.J. Martin*
Department of Biochemistry, University of Wisconsin, Madison, WI 53706
Submitted January 25, 2006; Revised February 7, 2006; Accepted February 8, 2006
Monitoring Editor: Sandra Schmid
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play key roles in membrane
fusion, but their sorting to specific membranes is poorly understood. Moreover, individual SNARE proteins can function
in multiple membrane fusion events dependent upon their trafficking itinerary. Synaptosome-associated protein of 25
kDa (SNAP25) is a plasma membrane Q (containing glutamate)-SNARE essential for Ca2?-dependent secretory vesicle–
plasma membrane fusion in neuroendocrine cells. However, a substantial intracellular pool of SNAP25 is maintained by
endocytosis. To assess the role of endosomal SNAP25, we expressed botulinum neurotoxin E (BoNT E) light chain in PC12
cells, which specifically cleaves SNAP25. BoNT E expression altered the intracellular distribution of SNAP25, shifting it
from a perinuclear recycling endosome to sorting endosomes, which indicates that SNAP25 is required for its own
endocytic trafficking. The trafficking of syntaxin 13 and endocytosed cargo was similarly disrupted by BoNT E expression
as was an endosomal SNARE complex comprised of SNAP25/syntaxin 13/vesicle-associated membrane protein 2. The
small-interfering RNA-mediated down-regulation of SNAP25 exerted effects similar to those of BoNT E expression. Our
results indicate that SNAP25 has a second function as an endosomal Q-SNARE in trafficking from the sorting endosome
to the recycling endosome and that BoNT E has effects linked to disruption of the endosome recycling pathway.
The distribution and restriction of proteins to appropriate
membrane compartments is essential for eukaryotic cell func-
tion. Compartments in the secretory pathway are dynamically
maintained by a balance of protein sorting and export via
vesicle formation and import of proteins by vesicle fusion. The
basic mechanisms of vesicle formation and consumption are
conserved with organelle-specific molecular components being
drawn from the families of soluble N-ethylmaleimide-sensitive
factor attachment protein receptor (SNARE), Rab and Sec 1
proteins (Bock et al., 2001; Zerial and McBride, 2001).
The ?35 members of the coiled-coil SNARE protein family
are classified as Q- (containing glutamate) or R (containing
arginine)-SNAREs based on the residue present in a central
hydrophilic layer of the coiled-coil domains (Fasshauer et al.,
1998; Weimbs et al., 1998). Cellular membrane compartments
contain a dynamic array of Q- or R-SNARE proteins character-
istic of the compartment (Chen and Scheller, 2001). The speci-
ficity of donor–acceptor membrane fusion resides in part with
the trans pairing of specific Q- and R-SNAREs into four-helix
bundles that generally consist of a 3Q–1R complex (McNew et
al., 2000). Assembly of the four-helix bundle draws the mem-
Because SNARE proteins recycle after fusion, they are present
in membrane intermediates in the recycling pathway, and in-
dividual SNARE proteins can participate in multiple fusion
events by engaging distinct SNARE protein partners (Nichols
and Pelham, 1998; Hay, 2001). It is important to map the
trafficking itinerary of a given SNARE protein to determine
potential steps at which it may function in fusion.
Synaptosome-associated protein of 25 kDa (SNAP25) is pre-
dominantly localized in the plasma membrane in neural and
neuroendocrine cells where it participates in Ca2?-dependent
fusion of secretory vesicles with the plasma membrane by
contributing two Q motifs to a complex with the plasma mem-
brane Q-SNARE syntaxin 1 and the vesicular R-SNARE vesi-
cle-associated membrane protein (VAMP) 2 (Sutton et al., 1998;
Jahn and Sudhof, 1999; Chen and Scheller, 2001). However,
SNAP25 is also found in intracellular membranes (Duc and
Catsicas, 1995; Marxen et al., 1997; Morgans and Brandstatter,
2000; Tao-Cheng et al., 2000; Aikawa et al., 2006). We found that
intracellular SNAP25 in PC12 cells localized to the recycling
endosome (RE) and trans-Golgi network (TGN) compartments,
which was dynamically maintained through endocytosis by a
dynamin-independent, ADP-ribosylation factor 6 (ARF6)-reg-
ulated pathway (Aikawa et al., 2006). Although syntaxin 1A,
the plasma membrane Q-SNARE partner for SNAP25, was not
cytic vesicles merged into a sorting endosome (SE) compart-
ment with the endosomal Q-SNARE syntaxin 13. SNAP25 was
subsequently trafficked to the RE-TGN compartment from
which it recycled back to the plasma membrane (Aikawa et al.,
This itinerary of SNAP25 through the endosome recycling
pathway raised the question as to whether this SNARE
protein has a second functional role in endosome fusion. The
endocytic trafficking pathway consists of a series of hetero-
This article was published online ahead of print in MBC in Press
on February 15, 2006.
* Present address: Department of Pharmaceutical Technology,
Tokushima-bunri University, Saniki-city, Kagawa 769-2193, Japan.
Address correspondence to: Thomas F.J. Martin (firstname.lastname@example.org).
Abbreviations used: ARF6, ADP-ribosylation factor 6; BoNT, botu-
linum neurotoxin; CTxB, cholera toxin B; GFP, green fluorescent
protein; LC, light chain; RE, recycling endosome; SE, sorting endo-
some; SNAP25, synaptosomal associated protein of 25 kDa; SNARE,
soluble N-ethylmaleimide-sensitive factor attachment protein recep-
tor; Tf, transferrin; VAMP, vesicle-associated membrane protein.
© 2006 by The American Society for Cell Biology2113
geneous and dynamic organelles that have been character-
ized through studies of recycling membrane proteins such
as the transferrin (Tf) receptor (Gruenberg, 2001; Maxfield
and McGraw, 2004). Tf is internalized by clathrin-dependent
endocytosis into primary endocytic vesicles that merge into
a peripheral cellular compartment of SEs. Tf receptor recy-
cling back to the plasma membrane occurs either directly
from the SE compartment or from an RE compartment lo-
cated in the perinuclear centriolar region of the cell (Daro et
al., 1996; Sheff et al., 1999; Gruenberg, 2001; Maxfield and
McGraw, 2004). The mechanisms that govern trafficking be-
tween and fusion of endosomal compartments are incom-
pletely understood. A SNARE complex consisting of syn-
taxin 7, syntaxin 8, vti1b, and VAMP 8 was characterized as
mediating the homotypic fusion of late endosomes, and
syntaxin 13 has been implicated in the homotypic fusion of
early endosomes (McBride et al., 1999; Antonin et al., 2002).
However, the roles of numerous other SNARE proteins dis-
tributed on endosomes (Chen and Scheller, 2001; Hay, 2001),
and the specific fusion events they mediate in endosomal
trafficking, remain to be characterized.
To assess the role of SNAP25 in endosome trafficking in
PC12 cells, we expressed the light chain (LC) of botulinum
neurotoxin E (BoNT E), a highly selective protease for SNAP25
family proteins (Blasi et al., 1993; Schiavo et al., 1993). We found
that cleavage of SNAP25 arrested endosomal SNAP25 and
syntaxin 13 trafficking from the SE to the RE compartment.
This was accompanied by the inhibition of cargo (e.g., Tf)
transit to the RE and by the destabilization of SNAP25/syn-
trafficking was observed in PC12 cells lacking SNAP25. Our
results identify a second role for SNAP25 as a Q-SNARE at a
trafficking step in the endosomal recycling pathway, and they
imply that the effects of BoNT E on neural and neuroendocrine
cells are not restricted to regulated vesicle exocytosis at the
MATERIALS AND METHODS
Cell Culture and Transfections
PC12 cells were cultured and transfected with DNA constructs as described
previously (Aikawa and Martin, 2003). Cells were plated onto polylysine-
coated coverslips. In some experiments where indicated, PC12 cells were
induced to differentiate with 50 ng/ml nerve growth factor (NGF) treatment
for the indicated times. Hippocampal rat neurons were prepared from em-
bryonic (E)18 rats and cultured in Neurobasal/B27 medium.
Expression vectors encoding GFP-Rab-11 were generously provided by T. Balla
(National Institutes of Health, Bethesda, MD), human Tf receptor by F. R. Max-
field (Weill Medical College of Cornell University, New York, NY), green fluo-
rescent protein (GFP)-Rab5, GFP-Rab5Q79L, and GFP-Rab5S34N by S. Ferguson
(Robarts Research Institute, London, Ontario, Canada), myc-syntaxin 13 by H.
Hirling (Laboratorie de Neurobiologie Cellulaire, Lausanne, Switzerland), and
cDNAs encoding rat and human SNAP23 by P. A. Roche (National Institutes of
Health). The N-terminal enhanced green fluorescent protein (EGFP) construct
encoding wild-type mouse SNAP25b, rat and human SNAP23, and rat syntaxin
13 were prepared by PCR amplification of open reading frames and cloning into
pEGFP-C1 (Clontech, Mountain View, CA). The following oligonucleotides were
used as primers: 5?-GATCTCGAGACATGTATCGG-3? and 5?-CGCGAAT-
TCATTTAGAAGCAA-3? to make the EGFP expression vector encoding rat
syntaxin 13. The C-terminal EGFP and enhanced cyan fluorescent protein con-
structs encoding rat VAMP 2 were prepared by PCR amplification of the open
reading frame and ligation into pEGFP-N1 (Clontech). Site-directed mutagenesis
Small hairpin RNA (shRNA) vectors for down-regulation of SNAP25 were
constructed in the Expression Arrest pSM2 vector (Open Biosystems, Huntsville,
AL) with 22mers that target nucleotides 335–356 (SNAP25 shRNA 1) and 473–
494 (SNAP25 shRNA 2) of SNAP25b using designs based on the method of
Paddison et al. (2004). All constructs were checked by sequencing.
Immunofluorescence and Endocytosis Assays
The uptake of 30 ?g/ml Alexa568-conjugated Tf (Molecular Probes, Carlsbad,
CA) was conducted in PC12 cells expressing human Tf receptor at 37°C in
calcium-free Locke’s solution for the indicated times. Cells were moved to an
ice bath, and unbound Tf was removed by extensive washing with phos-
phate-buffered saline (PBS) and fixed with 4% formaldehyde for 30 min at
room temperature. The uptake of Alexa 647-cholera toxin B (CTxB) (Molec-
ular Probes) and all immunofluorescence were conducted as described pre-
viously (Aikawa and Martin, 2003). The primary antibodies used were mono-
clonal anti-SNAP25 (Sternberger Monoclonals, Lutherville, MD), monoclonal
anti-hemagglutinin (HA) (BAbCO, Richmond, CA), monoclonal anti-myc
9E10 (Covance, Princeton, NJ), and monoclonal anti-TGN-38 (K. Howell,
University of Colorado, Denver, CO).
Immunoprecipitation and Western Blotting
Transfected cells were washed with PBS before harvesting in lysis buffer (50 mM
Tris, pH 7.5, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 1 mM phenylmeth-
ylsulfonyl fluoride, and Boehringer complete protease inhibitor cocktail). Lysates
were clarified by sedimentation, and supernatants were adsorbed with protein
G-Sepharose beads before conducting 3-h incubations with protein G-Sepharose
bead-immobilized SNAP25 monoclonal antibody. Beads were washed five times
with lysis buffer and eluted in sample buffer containing 0.2 M dithiothreitol for
analysis by Western blotting. Primary antibodies used for Western blotting were
Rab 3A monoclonal (BD Transduction Laboratories, Lexington, KY); GFP mono-
clonal JL-8 (BD Biosciences, San Jose, CA), SNAP25 monoclonal (Sternberger
Monoclonals), monoclonal anti-HA (BAbCO), and monoclonal anti-myc 9E10
Confocal Microscopy and Image Analysis
Cells were examined using an MRC-600 (Bio-Rad, Hercules, CA) or C1 (Nikon,
Tokyo, Japan) laser scanning confocal microscope with a 63? oil immersion
objective. For the determination of fluorescence intensities of SNAP25, syntaxin
13, and Tf in the perinuclear region, images were thresholded, and regions of
interest were quantitated using MetaMorph imaging software (Molecular De-
vices, Sunnyvale, CA). Fifty to 150 cells were examined per condition, and
significance of differences was assessed by Student’s t test.
Quantification of Neurite Outgrowth
Transfected PC12 cells were fixed after 2 d of NGF treatment. Transfected
(GFP-positive) cells were measured and grouped into different categories
based on neurite length. Classes were defined as cells with processes of 0–0.5,
0.5–1, and 1–3 times cell body diameter. Three independent series of trans-
fections were carried out and analyzed. Significance of differences was as-
sessed by Student’s t test.
Botulinum Neurotoxin E Inhibits the Endosomal
Trafficking of SNAP25
SNAP25 dynamically recycles between the plasma mem-
brane and a RE-TGN compartment in PC12 cells (Aikawa et
al., 2006). Although SNAP25 has a well characterized role as
a SNARE protein in regulated secretory vesicle exocytosis at
the plasma membrane (Banerjee et al., 1996; Chen et al., 1999;
Jahn and Sudhof, 1999), a function for intracellular SNAP25
has not been identified. In vitro studies suggested that
SNAP25 might function in homotypic early endosome fu-
sion (Sun et al., 2003), but this has not been assessed in vivo.
To investigate an intracellular role for SNAP25, we ex-
pressed the light chain of BoNT E (BoNT E LC) in PC12 cells.
This neurotoxin protease cleaves a 26 amino acid C-terminal
fragment from SNAP25 and inactivates the protein for
exocytic SNARE complex formation (Hayashi et al., 1994;
Banerjee et al., 1996; Chen et al., 1999). Under our expression
conditions, BoNT E LC efficiently cleaved SNAP25 as indi-
cated by the detection of a SNAP25 (1-180) fragment in the
portion of cells that were transfected (Figure 1A, bottom
row, asterisk) and by the complete conversion of GFP-
SNAP25 coexpressed with BoNT E LC to the GFP-SNAP25
(1-180) fragment (Figure 1A, top row, asterisk).
BoNT E LC expression markedly altered the cellular dis-
tribution of SNAP25. In the absence of BoNT E LC expres-
sion (Figure 1C, top row), GFP-SNAP25 localized to the
Y. Aikawa et al.
Molecular Biology of the Cell 2114
plasma membrane (?75%) as well as to a perinuclear region
(?25%) that corresponds to the RE and TGN (Aikawa et al.,
2006). Expression of BoNT E LC shifted the intracellular
distribution of GFP-SNAP25 to peripherally distributed en-
dosomes and strongly reduced GFP-SNAP25 in the perinu-
clear RE-TGN compartment (Figure 1C, bottom row, arrow-
heads) by 62% (Figure 1D).
To assess whether the intracellular redistribution of
SNAP25 was specifically dependent on its cleavage by BoNT
E, we determined the cellular distribution of the BoNT E-re-
sistant D179K mutant of GFP-SNAP25. Confirming our pre-
vious studies (Zhang et al., 2002), cleavage of GFP-SNAP25
D179K by BoNT E LC was not detected (Figure 1A). Impor-
tantly, the perinuclear RE distribution of GFP-SNAP25
D179K was unaltered by coexpression of BoNT E LC (Figure
1C, middle, and D). Additional studies showed that endog-
enous SNAP25 detected by immunocytochemistry exhibited
a similar redistribution as GFP-SNAP25 in BoNT E-express-
ing cells (our unpublished data). Overall, the results indicate
that the cleavage of SNAP25 by BoNT E LC per se is respon-
sible for the altered endosomal trafficking of SNAP25.
We next addressed whether SNAP23, a closely related
homologue of SNAP25, was involved in the BoNT E-
induced redistribution of SNAP25. SNAP23 exhibits ?58%
sequence identity with SNAP25 (Radvichandran et al., 1996;
Wang et al., 1997), and rat SNAP23 contains a conserved
BoNT E scissile bond (Macaulay et al., 1997; Vaidyanathan et
al., 1999). Because of this similarity, SNAP23 might function
redundantly with SNAP25 in rat PC12 cells, and its cleavage
by BoNT E could be involved in the altered trafficking of
SNAP25. Because SNAP23 is found at very low levels in
PC12 cells (Grant et al., 1999), we expressed GFP-SNAP23.
However, we found that neither rat nor human SNAP23 was
cleaved in BoNT E LC-expressing PC12 cells (Figure 1B).
Lack of cleavage of rat SNAP23 is consistent with the re-
ported need to use high BoNT E concentrations for cleavage
in vitro, whereas human SNAP23 is known not to be a
substrate for BoNT E (Macaulay et al., 1997; Vaidyanathan et
al., 1999). GFP-SNAP23 localized to the plasma membrane
and to a perinuclear vesicular compartment in PC12 cells,
but the perinuclear distribution of SNAP23, constituting
?3.5% of the protein, was completely unaffected by BoNT E
LC expression (Figure 1C, right, and D). These results elim-
inated the possibility that the BoNT E-induced redistribu-
tion of intracellular SNAP25 was mediated through cleavage
of SNAP23. The results indicate that SNAP23 does not func-
tion redundantly with SNAP25 in the endocytic pathway of
PC12 cells but rather that SNAP23 and SNAP25 traffic inde-
BoNT E Destabilizes an Endosomal SNARE Complex
and Disrupts Trafficking to the RE
The preceding studies indicated that BoNT E LC expression
promotes the redistribution of SNAP25 from a perinuclear RE
compartment into dispersed peripheral endosomes. To charac-
terize the peripheral endosomes containing SNAP25 in BoNT
E-expressing cells, we conducted colocalization studies for
Rab5, which is involved in primary endocytic vesicle and SE
fusion, and for Rab11, which is involved in RE trafficking, as
well as for the TGN marker TGN-38. Endogenous SNAP25
exhibited substantial overlap with Rab5- and Rab11-positive
endosomes as well as with TGN-38 (Figure 2, A and B). BoNT
E LC expression led to a redistribution of Rab5- and Rab11-
positive compartments to a peripheral location without alter-
ing the distribution of TGN-38 (Figure 2, A and B). SNAP25
maintained considerable colocalization with the Rab5- and
Rab11-positive endosomes but not with TGN-38–containing
compartments in the BoNT E LC-expressing cells (Figure 2, A
and B, arrowheads). Thus, cleavage by BoNT E causes SNAP25
to remain in peripheral endosomes that likely comprise the SE
compartment. This suggests that BoNT E cleavage inhibits
recycling endosome. (A) Representative Western blot shows the effect
of BoNT E LC expression on endogenous SNAP25 (marked End,
bottom) and on GFP-SNAP25 or GFP-SNAP25 D179K (top). (B) Rep-
resentative Western blot shows the effect of BoNT E LC expression on
endogenous SNAP25 (bottom) or rat or human GFP-SNAP23 (top)
detected with monoclonal GFP (top) or SNAP25 (bottom) antibodies.
Asterisks designate the cleaved product SNAP25 (1-180). GFP-D179K
E. (C) PC12 cells were transfected with plasmids encoding GFP-
pool was altered by coexpression of BoNT E (arrowheads). (D) The
fluorescence intensity of GFP-SNAP25 or GFP-SNAP23 was quanti-
tated in 50 randomly selected cells, and the percentage of perinu-
clear to total fluorescence was plotted as mean values ? SD; ***
and * indicate p ? 0.001 and p ? 0.05, respectively, for comparison
with control by t test. The perinuclear distribution of SNAP25 was
restored in cells expressing GFP-SNAP25 D179K.
BoNT E expression inhibits the transit of SNAP25 to the
Role of SNAP25 in Endosome Fusion
Vol. 17, May 20062115
trafficking of SNAP25 from the SE to the perinuclear RE com-
To further address this issue, we examined the distribu-
tion of syntaxin 13, a SNARE protein that localizes to pe-
ripheral SEs and to the perinuclear RE. Previous studies
showed that SNAP25 and syntaxin 13 coimmunoprecipitate
in a complex from cellular extracts (Prekeris et al., 1998;
Hirling et al., 2000; Aikawa et al., 2006). BoNT E LC expres-
sion led to a redistribution of myc-syntaxin 13 from the
perinuclear RE compartment to SEs, which also contained
SNAP25 (Figure 2C, top and middle rows, arrowheads).
Expression of the BoNT E-resistant SNAP25 D179K protein
restored the distribution of myc-syntaxin 13 to a perinuclear
RE distribution (Figure 2C, bottom row). The ?60% perinu-
clear distribution of myc-syntaxin 13 in control cells was
reduced to ?20% in cells expressing BoNT E LC, but it was
restored to ?55% in cells coexpressing BoNT E LC and
SNAP25 D179K (Figure 2E). Similar results were observed in
the cell bodies of primary hippocampal neurons (Figure 2D),
indicating a general role for SNAP25 in trafficking to the RE.
These results demonstrate that the endosomal trafficking of
syntaxin 13 is dependent upon SNAP25. Together, the re-
sults indicate that intact SNAP25 is required for the conver-
gence of Rab5-, Rab11-, and syntaxin 13-containing SEs into
the perinuclear RE compartment.
BoNT E cleavage of SNAP25 destabilizes exocytic SNARE
complexes consisting of SNAP25, syntaxin 1A, and VAMP 2
(Hayashi et al., 1994). We determined whether BoNT E cleav-
age has a similar effect on endosomal SNARE complexes. An
endosomal SNARE complex consisting of SNAP25, syntaxin
13, and VAMP 2 has been isolated previously (Prekeris et al.,
1998; Hirling et al., 2000). We found that expressed myc-syn-
taxin 13 could be isolated with immobilized SNAP25 antibody
from PC12 cell detergent extracts, indicating an association of
the two proteins (Figure 3A). BoNT E LC expression led to a
strong decrease in myc-syntaxin 13 associated with SNAP25,
indicating that the C terminus of SNAP25 was essential for this
association (Figure 3A). We also determined the effect of BoNT
E LC expression on the association of GFP-VAMP 2 with
myc-syntaxin 13 (Figure 3B). GFP-VAMP 2 was coimmunoiso-
lated with myc-syntaxin 13 in control cells, but this complex
was strongly reduced in BoNT E LC-expressing cells (Figure
3B). Coexpression of SNAP25 D179K, and to a much lesser
extent rat SNAP23, restored the association of GFP-VAMP 2
with myc-syntaxin 13 in BoNT E LC-expressing cells (Figure
3B). The results indicate that the association of syntaxin 13 with
BoNT E cleavage destabilizes an endosomal SNARE complex
that contains SNAP25.
SNAP25-containing primary endocytic vesicles merge with
syntaxin 13 in SEs before the delivery of SNAP25 to the RE
(Aikawa et al., 2006; Figure 8). To determine the possible site at
which SNAP25/syntaxin 13-containing endosomes encounter
VAMP 2 for formation of an endosomal SNARE complex, we
determined the distribution of VAMP 2 (Figure 3C). VAMP 2
in PC12 cells has previously been reported to localize to the RE
transit of syntaxin 13 to the VAMP 2-positive
recycling endosome. (A and B) PC12 cells
transfected with plasmids encoding GFP-
Rab5, GFP-Rab11, or BoNT E LC as indicated
were stained with rabbit SNAP25 and mouse
TGN38 antibodies. Arrowheads indicate ex-
amples of overlap of SNAP25 (red) with Rab5
(green) or Rab11 (green) in cells expressing
BoNT E. (C) PC12 cells transfected with
plasmids encoding myc-syntaxin 13, GFP-
SNAP25, or GFP-SNAP25 D179K and BoNT E
LC as indicated were stained with monoclo-
nal myc antibody. Arrowheads indicate ex-
amples of colocalization of syntaxin 13 (red)
with SNAP25 (green). The perinuclear distri-
bution of syntaxin 13 disrupted by BoNT E
LC was restored by expression of SNAP25
D179K. (D) Hippocampal neurons transfected
with plasmids encoding syntaxin 13-GFP
(green) and BoNT E LC as indicated were
stained with monoclonal SNAP25 antibody
(red). (E) The immunofluorescence intensity
of syntaxin 13 in images similar to those in C
was quantitated in 50 randomly selected cells,
and the percentage of perinuclear to total flu-
orescence was plotted as mean values ? SD;
*** corresponds to p ? 0.001.
BoNT E expression inhibits the
Y. Aikawa et al.
Molecular Biology of the Cell 2116
(de Wit et al., 1999; Martinez-Arca et al., 2003). Consistent with
this, we found that VAMP 2 exhibited substantial colocaliza-
tion with syntaxin 13 in a perinuclear compartment in control
cells (Figure 3C, top). Whereas BoNT E LC expression mark-
edly shifted the distribution of syntaxin 13 to peripheral SEs, it
had no effect on the localization of VAMP 2 in the RE (Figure
3C, bottom). These results indicate that SNAP25 transit to the
RE is not blocked in BoNT E LC-expressing cells due to alter-
ations in the morphology of the RE. Instead, the results suggest
that SNAP25/syntaxin 13-containing SEs encounter VAMP 2
upon entry into the RE. SNAP25 cleavage by BoNT E blocks SE
convergence into the RE (Figure 2, A and C) and inhibits
endosomal SNARE complex formation (Figure 3, A and B).
Together, the results suggest that BoNT E blocks the trafficking
of SNAP25 in the SE compartment to the perinuclear RE com-
partment by destabilizing endosomal SNARE complexes.
SNAP25 Is Required for General Endosome
Trafficking to the RE
The preceding studies indicated a requirement for SNAP25 in
its own endosomal transit from SEs to the RE. Because previ-
ous studies (Aikawa et al., 2006) showed that SNAP25-contain-
ing primary endocytic vesicles merge with those derived from
the clathrin-dependent pathway at an earlier stage, we deter-
mined whether the endosomal trafficking of Tf was also de-
pendent on SNAP25 function. We incubated control and BoNT
E LC-expressing cells with Alexa 568-conjugated Tf for 5, 15,
and 30 min. Although SNAP25-containing primary endocytic
vesicles do not colocalize with Tf-labeled primary endocytic
vesicles (Aikawa et al., 2006), expressed syntaxin 13-GFP exhib-
ited substantial colocalization with Tf in 5-min incubations
(Figure 4A, arrowheads) as reported previously (Prekeris et al.,
1998). In 15- and 30-min incubations, Tf entered the perinuclear
RE in control cells as reported previously (Daro et al., 1996;
Sheff et al., 1999), which contained syntaxin 13-GFP (Figure 4A,
arrowheads). However, in BoNT E LC-expressing cells, Tf was
not transported into the perinuclear RE but remained associ-
ated with peripherally distributed SEs containing syntaxin 13-
GFP (Figure 4A). BoNT E LC expression reduced Tf transit into
the perinuclear RE by ?40% (Figure 4B). Similar Tf uptake
studies in cells coexpressing GFP-SNAP25 and BoNT E LC
showed that the peripherally distributed SEs into which Tf was
delivered in 15- and 30-min incubations also contained GFP-
SNAP25 (Figure 4C, arrowheads). These results reveal that Tf
trafficking to the RE is blocked by SNAP25 cleavage. The
inhibition of endosomal trafficking by BoNT E seems to be at a
step beyond the merger of Tf-, SNAP25-, and syntaxin 13 in the
SE and before transit to the RE. This implies that BoNT E may
generally inhibit cargo traffic to the RE compartment.
We examined the endocytic trafficking of CTxB, which binds
plasma membrane GM1 glycosphingolipids and is trafficked
into the Golgi by a clathrin-independent endocytic pathway
(Nichols et al., 2001). In cells that expressed the constitutive
Rab5Q79Lmutant, Alexa 643-conjugated CTxB accumulated in
the expanded SE vacuole in a 10-min incubation, which indi-
cates that CTxB internalization in PC12 cells is mediated via
trol cells, the transit of Alexa 643-conjugated CTxB to the pe-
rinuclear RE and Golgi was observed after 15-min incubations
(Figure 4D, bottom row, arrowheads). In contrast, in cells ex-
pressing BoNT E LC, Alexa 643-conjugated CTxB was retained
in peripheral SEs that contained SNAP25 (Figure 4E, top row).
Accumulation of CTxB in the perinuclear region was reduced
by 45% in BoNT E LC-expressing cells (Figure 4F). Expression
of the BoNT E-resistant SNAP25 D179K mutant, however,
restored the trafficking of Alexa 643-conjugated-CTxB to the
perinuclear RE-TGN compartment (Figure 4E, bottom row).
These results complement those obtained for Tf uptake and
indicate that SNAP25 functions generally in the endocytic
pathway at a point beyond the convergence of multiple endo-
cytic pathways in the SE compartment. SNAP25 function
seems to be required for transit of cargo from SEs to the
perinuclear RE compartment.
SNAP25 Down-Regulation Similarly Disrupts
The preceding studies indicate that BoNT E cleavage of
SNAP25 inhibits endosome trafficking from the SE-to-RE com-
partments. Inhibition of trafficking might result from a loss-of-
function of SNAP25 such as the inability to participate in the
formation of SNARE complexes involving syntaxin 13 on SEs
complex. (A) PC12 cells were transfected with the indicated plas-
mids and SNAP25 immunoprecipitates were prepared from deter-
gent lysates for analysis by Western blotting for myc-syntaxin 13.
myc-Syntaxin 13 in SNAP25 immunoprecipitates decreased in cells
expressing BoNT E LC. (B) PC12 cells were transfected with the
indicated plasmids and myc-syntaxin 13 immunoprecipitates were
prepared from detergent lysates for analysis by Western blotting for
VAMP 2-GFP. VAMP 2-GFP in myc-syntaxin 13 immunoprecipi-
tates was decreased in BoNT E LC-expressing cells but was restored
in cells coexpressing GFP-SNAP25 D179K. (C) PC12 cells trans-
fected with plasmids encoding VAMP 2-GFP, myc-syntaxin 13, and
BoNT E LC as indicated were fixed for staining with myc and
SNAP25 antibodies. VAMP 2 (green) colocalized with syntaxin 13
(red) and SNAP25 (cyan) in the perinuclear RE in control cells (top),
whereas syntaxin 13 was dispersed to a peripheral location away
from perinuclear VAMP 2 in BoNT E LC-expressing cells (bottom).
BoNT E expression destabilizes an endosomal SNARE
Role of SNAP25 in Endosome Fusion
Vol. 17, May 20062117
and VAMP 2 on the RE. Alternatively, the BoNT E LC cleavage
fragments of SNAP25 might act in some manner as dominant
inhibitors of endosome trafficking. To assess these alterna-
tive explanations and to independently determine whether
SNAP25 is required for endosome trafficking, we tested the
impact of down-regulating SNAP25 in PC12 cells. One of
several siRNA-expressing plasmids (shRNA 2) was found to
largely eliminate the expression of GFP-SNAP25 from a
cotransfected plasmid and to down-regulate endogenous
SNAP25 in proportion to transfection efficiency (Figure 5, A
and B). Cells transfected with the shRNA 2 plasmid exhib-
ited no immunoreactive SNAP25 and syntaxin 13 was dis-
persed from an RE to peripheral SEs (Figure 5C) similar to
the effect of BoNT E expression (Figure 3C). The effective
down-regulation of SNAP25 protein levels was accompa-
nied by the inhibition of Tf uptake into the perinuclear RE
compartment (Figure 5D). Instead, Tf remained associated
with the peripheral SEs containing syntaxin 13 (Figure 5D).
The inhibition of Tf and syntaxin 13 trafficking to the RE by
shRNA 2 was highly significant (Figure 5, E and F). These
results indicate that abolishing SNAP25 expression has an
impact on endosomal trafficking similar to that of BoNT E
expression. Thus, it is the loss of SNAP25 function rather
than the dominant inhibitory effects of cleavage fragments
that mediates BoNT E inhibition of endosome trafficking.
Overall, the results strongly support the conclusion that
SNAP25 is required for SE-to-RE trafficking.
SNAP25 Is Not Required for Rab5-Dependent Homotypic
Early Endosome Fusion
Recent in vitro studies suggested that SNAP25 was required
for homotypic early endosome fusion in HeLa cell mem-
brane fractions (Sun et al., 2003). However, SNAP25 was not
found to colocalize with primary endocytic vesicles loaded
with Tf in 5-min incubations in PC12 cells (Aikawa et al.,
2006). Moreover, BoNT A, a neurotoxin protease that also
cleaves SNAP25, did not inhibit homotypic early endosome
fusion in membrane preparations from PC12 cells (Holroyd
et al., 1999). Thus, it was unclear whether SNAP25 was
required for homotypic early endosome fusion events in
CTxB to the recycling endosome. (A) PC12 cells
expressing syntaxin 13-GFP, human Tf recep-
tor, and BoNT E LC where indicated were in-
cubated with Alexa 568-conjugated Tf for 5, 15,
and 30 min. Extensive colocalization of syntaxin
13-GFP (green) with Tf (red) was observed (ar-
rowheads). In cells expressing BoNT E LC, Tf
and syntaxin 13-GFP were retained in periph-
eral endosomes. (B) The fluorescence intensity
of Alexa568-conjugated Tf was quantitated in
50 randomly selected cells, and the percentage
in the perinuclear RE to total fluorescence was
plotted as mean values ? SD; *** denotes p ?
0.001 and *p ? 0.05 for comparison with control
by t test. (C) PC12 cells expressing GFP-
SNAP25, human Tf receptor, and BoNT E LC
were incubated with Alexa568-conjugated Tf.
of Tf (red) with GFP-SNAP25 (green). (D and E)
PC12 cells expressing GFP-Rab5Q79Lor GFP-
SNAP25 or GFP-SNAP25 D179K were incu-
bated with Alexa643-conjugated CTxB on ice
for 30 min, washed, and incubated at 37°C for
15 min to internalize the CTxB. CTxB localizes
to the vacuolar SE compartment in Rab5Q79L-
expressing cells (D, top). CTxB uptake into a
perinuclear RE/TGN compartment is inhibited
in BoNT E LC-expressing cells (E, top) and is
restoredin cells expressing
D179K (E, bottom). Arrowheads indicate exam-
ples of colocalization of SNAP25-GFP (green)
and CTxB (cyan). (E) The fluorescence intensity
of CTxB was quantitated in 50 randomly se-
SD, with *** denoting p ? 0.001 for BoNT E-
expressing versus control cells.
BoNT E inhibits the transit of Tf and
Y. Aikawa et al.
Molecular Biology of the Cell2118
PC12 cells in vivo. To address this, we determined whether
BoNT E inhibits the homotypic early endosome fusion that
is promoted by expression of the constitutive Rab5Q79Lmu-
tant, which results in the formation of enlarged SE vacuoles
(Stenmark et al., 1994; Roberts et al., 1999). Although BoNT E
LC effectively inhibited the interaction of syntaxin 13 with
VAMP 2 in Rab5Q79L-expressing cells (Figure 6A), it did not
prevent the formation of syntaxin 13-containing SE vacuoles
induced by Rab5Q79Lexpression (Figure 6B). These results
indicate that intact SNAP25 is not required for Rab5-depen-
dent homotypic early endosome fusion.
A second approach for assessing the role of SNAP25 in
endosomal trafficking would be to prevent its internalization
from the plasma membrane or its trafficking to the SE com-
partment. To do so, we took advantage of our finding that
ARF6 regulates SNAP25 endocytosis (Aikawa et al., 2006).
Overexpression of the constitutive-active ARF6Q67Lmutant
promotes extensive SNAP25 internalization into a vacuolar
compartment but inhibits SNAP25 transit out of this compart-
ment to syntaxin 13-containing SEs (Aikawa et al., 2006). In the
current studies, we found that the vacuolar accumulation of
SNAP25 induced by ARF6Q67Lexpression was not affected by
BoNT E LC coexpression (Figure 6C, arrowheads), which in-
dicates that ARF6Q67Larrests SNAP25 trafficking at an early
stage after internalization. Expression of ARF6Q67Lwas found
to prevent Tf accumulation in the RE compartment (Figure 6E,
arrowheads), which is similar to the effect of BoNT E LC
expression (Figure 4A). However, ARF6Q67Lexpression failed
to affect Rab5Q79L-induced SE vacuole formation (Figure 6D,
middle row). These results also indicate that SNAP25 is not
required for Rab5-dependent homotypic early endosome
Previous studies showed that the dominant-negative
ARF6T27Nmutant interfered with SNAP25 internalization
from the plasma membrane (Aikawa et al., 2006), and this
was confirmed in the current work (Figure 6D, bottom row).
Expression of the ARF6T27Nmutant also inhibited Tf accu-
mulation in the RE compartment (Figure 6E, bottom row)
consistent with its inhibition of SNAP25 internalization.
However, ARF6T27Nexpression did not affect Rab5Q79L-
induced SE vacuole formation (Figure 6D, bottom row).
Collectively, the results indicate that SNAP25 is not required
for Rab5-dependent homotypic early endosome fusion and
are consistent with the conclusion that SNAP25 is required
for a later step in the endosome recycling pathway.
Recycling SNAP25 Is Required for Neurite Outgrowth
In addition to the well characterized role of SNAP25 in regu-
lated vesicle exocytosis, SNAP25 has been shown to be essen-
tial for neurite outgrowth in PC12 cells (Osen-Sand et al., 1993,
1996). The basis for a SNAP25 requirement in neurite out-
growth remains unclear. Although it was suggested that
SNAP25 might mediate constitutive vesicle exocytosis and
membrane addition to the plasma membrane (Tang, 2001), we
found no effect of BoNT E LC expression on the constitutive
trafficking of a VSV-G-GFP fusion protein to the plasma mem-
brane in PC12 cells (our unpublished data). Our results show-
ing that SNAP25 is essential for a step in the endosome recy-
cling pathway suggested the alternative explanation that
neurite outgrowth may use membrane recycled through the
SNAP25-dependent endosome recycling pathway. To address
this possibility, we determined the effect of altering endosomal
SNAP25 levels on NGF-induced neurite outgrowth in PC12
We confirmed a requirement for SNAP25 in NGF-induced
neurite outgrowth by measuring the total length of neuritic
processes in BoNT E LC-expressing compared with control
cells. Neurite lengths were measured and grouped into three
categories: 1) very short (?0.5 cell body diameter), 2) inter-
mediate length (0.5–1 cell body diameter), and 3) long (?1
cell body diameter). BoNT E LC expression was found to
decrease the number of cells with long neurites (27 versus
56% for control, p ? 0.001) and to increase the number of
cells with short neurites (58 versus 28% for control, p ? 0.01)
(Figure 7A). The results were similar to those reported pre-
PC12 cells. (A) PC12 cells transfected with GFP-SNAP25 and control or
SNAP25 shRNA plasmids 1 or 2 were subjected to Western blotting
with GFP, SNAP25, or Rab 3A antibodies as indicated. SNAP25
shRNA 2 expression extensively down-regulated GFP-SNAP25 and
down-regulated endogenous SNAP25 levels in proportion to transfec-
tion efficiencies without affecting Rab 3A levels. (B) Densitometry was
used to quantitate relative levels of endogenous (end) SNAP25 and
GFP-SNAP25 in A. (C) PC12 cells transfected with SNAP25 shRNA 2
plus plasmid encoding syntaxin 13-GFP were stained with SNAP25
antibody. Syntaxin 13-GFP was dispersed into peripheral endosomes
SNAP25 shRNA 2 plasmid and with plasmids encoding syntaxin
13-GFP (green) and transferrin receptor. Cells were imaged after a
30-min incubation with Alexa568-conjugated Tf (red). Bars, 10 ?m. (E)
The fluorescence intensity of Tf was quantitated in 50 randomly se-
lected cells (similar to D), and the percentage of perinuclear to total
fluorescence was plotted as mean values ? SD, with *** denoting p ?
0.001 for cells transfected with SNAP25 shRNA 2 versus control plas-
mid. (F) The fluorescence intensity of syntaxin 13-GFP was quantitated
in 50 randomly selected cells (similar to D), and the percentage of
perinuclear to total fluorescence was plotted as mean values ? SD,
with *** denoting p ? 0.001 for cells transfected with SNAP25 shRNA
2 versus control plasmid.
Endosome trafficking is inhibited in SNAP25-deficient
Role of SNAP25 in Endosome Fusion
Vol. 17, May 20062119
viously using BoNT E (Martinez-Arca et al., 2000) or BoNT A
(Osen-Sand et al., 1996; Grosse et al., 1999; Morihara et al.,
1999) to inactivate SNAP25.
To alter the endosomal distribution of SNAP25, we con-
ducted similar studies in cells expressing wild-type ARF6,
ARF6Q67L, or ARF6T27N. Expression of wild-type ARF6, which
colocalized with SNAP25 in NGF-treated cells in the cell body
and in neuritic growth cones (Figure 7C, top row, arrowheads),
had no effect on neurite outgrowth (our unpublished data). In
contrast, the expression of ARF6T27N, which inhibits SNAP25
internalization, resulted in fewer cells with long neurites and
more cells with short neurites (Figure 7B). Similar, but stronger
effects, on neurite outgrowth were observed in cells expressing
ARF6Q67L(Figure 7B), which colocalized with SNAP25 in en-
dosomal compartments and in cytoplasmic vesicles in growth
cones (Figure 7C, middle row) that lacked syntaxin 13 (our
unpublished data). High levels of ARF6Q67Lexpression traps
SNAP25 in an endosomal vacuole before its merge with syn-
taxin 13-containing SEs (Aikawa et al., 2006). Cells that ex-
pressed high levels of ARF6Q67Lexhibited SNAP25-containing
endosomal vacuoles that were devoid of syntaxin 13 (our un-
published data) and exhibited very short neurites (Figure 7C,
bottom row). These results indicate that blocking the normal
endosomal trafficking of SNAP25 by expressing ARF6 mutants
has a strong inhibitory effect on NGF-induced neurite out-
growth. The results are consistent with the proposal that neu-
rite outgrowth relies upon a recycling pool of membrane
that is dependent upon the function of SNAP25 in endo-
SNAP25 Functions in Endosome Fusion
The key conclusion of this study is that SNAP25, a SNARE
protein required for regulated secretory vesicle exocytosis at
the plasma membrane in neural and neuroendocrine cells,
has a second SNARE role in the endosome recycling path-
way. The site at which SNAP25 is required for endosome
fusion corresponds to the trafficking of endocytosed cargo
from SEs to the RE (Figure 8). The finding that SNAP25
functions in the endosome recycling pathway provides a
rationale for understanding the dynamic trafficking itinerary
of SNAP25 (Aikawa et al., 2006) and clarifies the multiple
cellular effects of botulinum neurotoxins that target SNAP25
SNAP25 is internalized by a dynamin-independent, ARF6-
regulated endocytic pathway that maintains the intracellular
pool of SNAP25 (Aikawa et al., 2006). SNAP25-containing pri-
mary endocytic vesicles merge with those derived from clath-
rin-dependent endocytosis to enter an early endosomal com-
partment referred to as the SE (Figure 8). In the SE, SNAP25
colocalizes with syntaxin 13, which functions in trafficking
early endosome fusion. (A) Myc antibody immunoprecipitates were
prepared from detergent lysates of cells transfected with indicated
plasmids and analyzed by Western blotting for VAMP 2-GFP. Coim-
munoprecipitation of VAMP 2-GFP with myc-syntaxin 13 was de-
creased by BoNT E LC expression even in Rab5Q79L-expressing cells.
(B) PC12 cells transfected with plasmids encoding myc-syntaxin 13,
GFP-Rab5Q79Lwithout (top) or with (bottom) BoNT E LC (bottom)
were stained with monoclonal myc antibody. Syntaxin 13 (red) local-
ized to Rab5Q79L-promoted SE vacuoles (green) in control and BoNT E
SNAP25 is not required for Rab5-dependent homotypic
LC-expressing cells (arrowheads). (C) PC12 cells transfected with plas-
mids encoding ARF6Q67Land BoNT E LC were stained with SNAP25
antibody. SNAP25 localization (green) to ARF6Q67L-promoted vacu-
oles (red) was not prevented in cells expressing BoNT E LC. (D) PC12
cells transfected with plasmids encoding GFP-Rab5Q79L(green) with
and stained with monoclonal SNAP25 antibody (red). Arrowheads
indicate the Rab5Q79L-induced SE vacuole. (E) PC12 cells transfected
with plasmids encoding GFP-SNAP25 (green), human transferrin re-
ceptor and ARF6Q67Lor ARF6T27Nas indicated were incubated with
Alexa 568-conjugated Tf (red) for 30 min. Alexa 568-Tf accumulated in
the perinuclear RE in control cells but not in cells expressing ARF6Q67L
Y. Aikawa et al.
Molecular Biology of the Cell2120
within the endosome recycling pathway (Prekeris et al., 1998;
Hirling et al., 2000). The exact relationship between SEs and the
RE compartment in cells has not been established. It is thought
that the tubulovesicular SE compartment sorts vacuolar cargo
to late endosomes, whereas tubular portions deliver recycling
membrane cargo to the RE (Maxfield and McGraw, 2004). It is
unclear whether tubular SE elements give rise to the RE com-
partment or transit to and fuse with a preexisting RE compart-
ment (Sheff et al., 2002). Our results are compatible with either
possibility but indicate that SNAP25 function is required for
this trafficking step in the endosomal recycling pathway (Fig-
ure 8). BoNT E LC expression inhibited the transit of SNAP25/
syntaxin 13-containing SEs and internalized cargo (Tf and
CTxB) to the perinuclear RE compartment. VAMP 2, which is
predominantly located in the RE compartment, coisolates with
SNAP25/syntaxin 13-containing SNARE complexes (Prekeris
et al., 1998; Hirling et al., 2000; Sun et al., 2003; Aikawa et al.,
2006). BoNT E LC expression disrupted this endosomal
SNARE complex. Such complexes isolated from PC12 cell de-
tergent extracts may represent accumulated cis-SNARE com-
plexes in the RE that arise from trans-complexes that mediate
fusion of SEs into the RE (Figure 8). That BoNT E LC expres-
sion resulted in the destabilization of these SNARE complexes
is consistent with an essential role for SNAP25 at this traffick-
ing step for fusion events involving SNAP25/syntaxin 13 on
SEs and VAMP 2 in the RE (Figure 8).
The involvement of SNAP25 in intracellular membrane
fusion events in the endosomal pathway had not previously
been established in vivo. Previous in vitro studies in HeLa
neurite extension. (A) PC12 cells expressing GFP (white) or GFP
plus BoNT E LC (black) were treated with NGF for 2 d to induce
neurite outgrowth. Neurite length in transfected cells was measured
and grouped into the indicated categories. (B) PC12 cells expressing
ARF6WT(white), ARF6Q67L(black), or ARF6T27N(gray) were treated
with NGF for 2 d to induce neurite outgrowth. (C) PC12 cells
expressing ARF6WTor ARF6Q67Lwere treated with NGF for 2 d to
induce neurite outgrowth. SNAP25 (green) and ARF6 (red) were
localized by immunofluorescence. Arrowheads indicate examples
of ARF6 colocalization with SNAP25. Cells expressing high levels of
ARF6Q67L(bottom row) exhibited vacuoles containing SNAP25 and
very short neurites.
Role of endosome recycling pathway in NGF-induced
is internalized into tubulovesicular endosomes from the plasma
membrane by a dynamin-independent, ARF6-regulated pathway
(right), which is distinct from the clathrin-dependent pathway of
endocytosis (left) (Aikawa et al., 2006). Expression of ARF6T27N
blocks SNAP25 internalization, whereas ARF6Q67Lexpression en-
hances SNAP25 internalization but blocks merger of SNAP25 endo-
somes into SEs. SNAP25 endosomes lack syntaxin 1 but merge with
syntaxin 13 in the SE (Aikawa et al., 2006). Tubular elements derived
from the SE either coalesce to form the RE compartment or fuse with
a preexisting RE compartment. The trafficking of cargo from periph-
eral SEs to the perinuclear RE requires intact SNAP25 and is inhib-
ited by BoNT E. Syntaxin 13/SNAP25-containing SE elements en-
counter VAMP 2 in the RE to form SNARE complexes consisting of
SNAP25/syntaxin 13/VAMP 2.
Summary of the trafficking itinerary of SNAP25. SNAP25
Role of SNAP25 in Endosome Fusion
Vol. 17, May 20062121
cell homogenates showed that BoNT E inhibited early en-
dosome fusion (Sun et al., 2003; Yan et al., 2004). Homotypic
early endosome fusion requires Rab5 and the Rab5 effectors
EEA1 and Rabaptin-5, which interact with syntaxin 13
(McBride et al., 1999). To determine whether SNAP25 is
essential for Rab5-dependent homotypic early endosome
fusion in PC12 cells, we expressed the Rab5Q79Lmutant,
which induces the formation of an enlarged SE vacuole
(Stenmark et al., 1994; Roberts et al., 1999). In PC12 cells, this
enlarged SE compartment contained SNAP25 and syntaxin
13 as expected. However, coexpression of BoNT E LC failed
to alter the Rab5Q79L-induced enlargement of the SE com-
partment, indicating that SNAP25 is not essential for Rab5-
dependent homotypic early endosome fusion. The results
are consistent with a primary role for SNAP25 at a later step
in the endosome recycling pathway.
Multiple Roles for SNAP25 as a SNARE Protein
SNAP25 is an essential participant in at least two membrane
fusion steps in neuroendocrine cells: in regulated secretory
vesicle exocytosis and in SE transit to the RE in the endo-
some recycling pathway. This is consistent with the isolation
of SNAP25 in two distinct SNARE complexes containing
syntaxin 1A or syntaxin 13 with VAMP 2 (Prekeris et al.,
1998; Sutton et al., 1998). These results provide additional
evidence for the concept that SNARE proteins are multifunc-
tional and can engage in several combinatorial interactions
that mediate distinct membrane-specific fusion reactions
(Nichols and Pelham, 1998; Hay, 2001).
Sorting steps during membrane budding reactions allow
distinct combinations of SNARE proteins to distribute into
separate membrane domains where they participate in fusion
reactions characteristic of the individual donor and acceptor
membranes. SNAP25 endocytosis segregates it from syntaxin
1A but SNAP25-containing primary endocytic vesicles merge
(Aikawa et al., 2006). Whereas homotypic SE fusion seems to
involve syntaxin 13 (McBride et al., 1999), the present as well as
previous work (Holroyd et al., 1999 but see Sun et al., 2003)
indicates that homotypic SE fusion does not involve SNAP25.
Instead, SNAP25 is involved in later possibly heterotypic fu-
sion steps in the recycling pathway where SNAP25/syntaxin
13-containing tubular elements from SEs encounter VAMP 2 in
the RE compartment. Syntaxin 13 also seems to function in the
recycling from the RE back to the plasma membrane, whereas
SNAP25 does not (Prekeris et al., 1998). Overall, the sorting and
trafficking itinerary of an individual SNARE protein would
confer its ability to adopt multiple roles in several distinct
donor–acceptor membrane interactions.
The siRNA-mediated down-regulation of SNAP25 inhib-
ited endosomal trafficking in a manner similar to BoNT E LC
expression. These results indicate that the loss-of-function in
SNAP25 results in endosome trafficking deficits. Is it possi-
ble that the disruption of endosomal trafficking in BoNT E
LC-expressing or SNAP25-deficient cells is secondary to the
inhibition of Ca2?-dependent vesicle exocytosis? This is un-
likely for a number of reasons. First, we observed endosome
trafficking deficits in cells that were not stimulated to exhibit
regulated vesicle exocytosis. Constitutive vesicle exocytosis
does not seem to involve SNAP25. Second, the expression of
tetanus toxin LC, which inhibits regulated vesicle exocyto-
sis, did not result in altered endosome trafficking (our un-
published data). Third, PC12 cells that are incapable of
regulated exocytosis due to synaptotagmin depletion do not
exhibit altered endosomal SE to RE trafficking (K. Lynch and
T. Martin, unpublished findings). These findings indicate
that the inhibition of regulated vesicle exocytosis per se does
not secondarily affect endosomal trafficking to the RE.
Consequences for Understanding Botulinum
BoNTs E and A are highly specific proteases for SNAP25 that
inhibit Ca2?-triggered vesicle exocytosis in neurons and neu-
roendocrine cells (Blasi et al., 1993; Schiavo et al., 1993). How-
ever, treatment by these toxins has other effects, which include
the inhibition of neurite outgrowth (Osen-Sand et al., 1996;
Grosse et al., 1999; Morihara et al., 1999; Martinez-Arca et al.,
2000) and receptor trafficking (Lan et al., 2001; Washbourne et
al., 2004). These effects of neurotoxins had been interpreted to
indicate a role for SNAP25 in constitutive vesicle exocytosis at
the plasma membrane (Tang, 2001), but we were unable to
detect any inhibitory effects of BoNT E LC expression on con-
stitutive VSV-G trafficking to the plasma membrane. Our find-
ing that SNAP25 function in the endosome recycling pathway
is inhibited by BoNT E cleavage provides an alternative inter-
pretation that some effects of BoNT E or BoNT A result from
the inhibition of membrane recycling. We found that BoNT E
VAMP 2). Conversely, the overexpression of syntaxin 13 or
SNAP25, but not other SNARE proteins, was reported to en-
hance neurite outgrowth in PC12 cells (Tang, 2001).
To assess the role of endosomal SNAP25 in neurite out-
growth, we altered the internalization of SNAP25 or its
transit into the SE by expressing ARF6 mutants (Aikawa et
al., 2006). Blocking SNAP25 internalization or its transit to
the SE strongly decreased neurite outgrowth in PC12 cells
similar to a recent study on retinal neurons (Albertinazzi et
al., 2003). Although the results are consistent with a pro-
posed key role for endosomal SNAP25 in facilitating mem-
brane recycling for neurite outgrowth, it is also likely that
ARF6 mutants act on a variety of effectors that contribute to
this outcome. ARF6 proteins regulate recycling in the endo-
cytic pathway to deliver membrane to regions of plasma
membrane remodeling (Radhakrishna and Donaldson, 1997;
D’Souza-Schorey et al., 1998; Prigent et al., 2003). Overex-
pression of the Sec10 subunit of the exocyst complex, re-
cently reported to be an effector of ARF6 (Prigent et al., 2003),
was found to block neurite outgrowth in PC12 cells (Vega
and Hsu, 2001). These studies delineate a role for ARF6 in
the polarized membrane insertion of recycling surface com-
ponents internalized via the endocytic pathway and indicate
a central role for recycling membrane in neurite outgrowth.
Although our studies indicated that BoNT E inhibits traffick-
ing to the RE compartment in hippocampal neurons, the con-
sequences of this for neuronal trafficking need further investi-
gation. Development of the nervous system, including axon
and dendrite extension, is normal in the SNAP25?/?mouse
(Washbourne et al., 2002). It may be the case that SNAP23
functions redundantly with SNAP25 in neurons to compensate
for some functions in the SNAP25?/?mouse, although this
was not found to be the case for PC12 cells where SNAP23
exhibited a distinct intracellular trafficking itinerary.
This work was supported by U.S. Public Health Service Grants DK-25861 and
DK-40428 to T.F.J.M. and by a Ruth L. Kirschstein National Research Service
Award predoctoral fellowship to K.L.L.
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