Cell, Vol.
93, 423–432, May 1, 1998, Copyright 1998 by Cell Press
A Function for the AP3 Coat Complex
in Synaptic Vesicle Formation from Endosomes
Victor Faúndez,† Jim-Tong Horng,†
and Regis B. Kelly*
Department of Biochemistry and Biophysics
and Hormone Research Institute
University of California
San Francisco, California 94143-0534
Summary
Synaptic vesicles can be coated in vitro in a reaction
that is ARF-, ATP-, and temperature-dependent and
requires synaptic vesicle membrane proteins. The
coat is largely made up of the heterotetrameric com-
plex, adaptor protein 3, recently implicated in Golgi-
to-vacuole traffic in yeast. Depletion of AP3 from brain
cytosol inhibits small vesicle formation from PC12 en-
dosomes in vitro. Budding from washed membranes
can be reconstituted with purified AP3 and recombi-
nant ARF1. We conclude that AP3 coating is involved
in at least one pathway of small vesicle formation from
endosomes.
Introduction
Cargo proteins are primarily transported between intra-
cellular membrane compartments by carrier vesicles.
Formation of the carrier vesicles requires three steps:
segregation of cargo proteins from resident proteins of
the donor organelle, induction of membrane curvature,
and pinching-off of the nascent vesicle. These three
functions are usually attributed to a cytoplasmic coat
that is generated around the newly forming vesicles.
Budding from Golgi and budding from ER membranes by
COPI and COPII coats are relatively simple processes,
since budding can be reconstituted in vitro with only
coat heteroligomers and a small GTPase (Ostermann et
al., 1993; Bednarek et al., 1995; Rothman and Wieland,
1996; Schekman and Orci, 1996). Budding from the
plasma membrane and the trans–Golgi network requires
heterotetrameric coats, AP2 and AP1, respectively,
which show sequence homology to COPI. In addition,
however, AP2 and AP1-mediated budding both require
clathrin triskelions (Smythe et al., 1992; Stamnes and
Rothman, 1993; Traub et al., 1993; Kirchhausen et al.,
1997; Schmid, 1997). A fifth coat, AP3, has sequence
and structural homology to AP1 and AP2 and is found
on both Golgi and on endosomal membranes, by both
light and electron microscopy (Newman et al., 1995;
Simpson et al., 1996; Dell’Angelica et al., 1997a, 1997b,
1998), and is needed for an alternative Golgi-to-vacuolar
pathway in yeast (Cowles et al., 1997; Stepp et al., 1997).
The recruitment of four of the coat families to donor
membranes requires a small GTPase in its GTP-bound
form. For COPII, the GTPase is the sar1 protein (Kuge
et al., 1994; Oka and Nakano, 1994; Bednarek et al.,
* To whom correspondence should be addressed.
† These two authors contributed equally to this work.
1995); for COPI, AP1, and AP3, the GTPase is a member
of the ADP-ribosylation factor (ARF) family (Ostermann
et al., 1993; Stamnes and Rothman, 1993; Traub et al.,
1993; Bednarek et al., 1995; Simpson et al., 1996; Dell’
Angelica et al., 1997a). GTPgS can facilitate coat recruit-
ment (Donaldson et al., 1992; Helms and Rothman, 1992;
Robinson and Kreis, 1992; Simpson et al., 1996; Dell’
Angelica et al., 1997a) whereas hydrolysis of GTP to GDP
is linked to uncoating (Tanigawa et al., 1993). Indeed, it
has only been possible to isolate COPI- and COPII-
coated vesicles when GTP hydrolysis by sar1p or ARF
was inhibited (Oka and Nakano, 1994; Bednarek et al.,
1995; Orci et al., 1997).
Although the endosome functions as a sorting organ-
elle, little is known about which coats generate which
class of carrier vesicle. COPI, clathrin, and AP3 com-
plexes show ARF-sensitive association with endosomes
(Whitney et al., 1995; Aniento et al., 1996; Stoorvogel et
al., 1996; Dell’Angelica et al., 1997a). Only for COPI-
associated carrier vesicles do we know a function, namely
to carry cargo from early to late endosomes (Whitney et
al., 1995; Aniento et al., 1996). One endosomal trafficking
step that has received considerable attention is the gen-
eration of synaptic vesicles by an endocytosis-derived
mechanism in neurons and neuroendocrine cells (Takei
et al., 1996; Cremona and De Camilli, 1997; González-
Gáitan and Jäckle, 1997; Schmidt, 1997; Shupliakov et
al., 1997). It has been possible to reconstitute in a cell-
free system the formation of synaptic vesicles (SVs),
frequently called synaptic-like microvesicles (SLMVs),
using isolated endosomes from PC12 cells (Desnos et al.,
1995; Y. Lichtenstein, C. Desnos, V. F., L. Clift O’Grady,
and R. B. K., unpublished data). ARF1, ATP, and a high
molecular weight protein complex were sufficient for SV
formation and membrane protein sorting.
Using a back reaction in which coats are recruited to
the SVs themselves in the presence of GTPgS, we have
discovered that a crucial component of the high molecu-
lar weight complex is AP3.
The AP3 coat complex has four subunits, d, b3A/b3B
or b-NAP, m3A/m3B, and s3A/s3B (Simpson et al., 1996,
1997; Dell’Angelica et al., 1997a, 1997b; Ooi et al., 1997).
Two subunits of the complex (b3B and m3B) are neuro-
specific isoforms, widely expressed in the brain (Pev-
sner et al., 1994; Newman et al., 1995). AP3 is localized
in neurites of cerebellar granule neurons and in isolated
brain nerve terminals, consistent with a role in nerve
terminal membrane traffic (Newman et al., 1995; Simp-
son et al., 1996). We have successfully reconstituted in
vitro the membrane trafficking event requiring AP3.
Results
ARF-Dependent Protein Recruitment
to Synaptic Vesicles
Budding of SVs from PC12 endosomes, labeled at 158C
with an antibody (125I-KT3) to an epitope-tagged form of
the synaptic vesicle protein VAMP (VAMP-TAg), requires
an ARF1-dependent recruitment of coats. To identify
Cell
424
coats involved in the PC12 endosome-derived SV bud-
ding, purified SVs were coated in an ARF1-dependent
“backward” coating reaction in which GTP hydrolysis
was inhibited.
Synaptic vesicles from either rat brain or from endo-
cytically labeled PC12 cells were purified by velocity
sedimentation and then incubated with rat brain cytosol,
an ATP-regenerating system, and myristoylated ARF1
mutants, either the GTP form (Q71L) or the GDP counter-
part (T31N). ARF-regulated recruitment of cytosolic pro-
teins to SVs increased the rate of vesicle sedimentation
into nonequilibrium sucrose gradients. Without coat re-
cruitment, purified rat brain and PC12 SVs were recov-
ered at 25%–26% and 22%–24% sucrose, respectively.
The sedimentation of both kinds of SVs was unaltered
by the addition of either rat brain cytosol or the ARF1
mutants alone (Figures 1A and 1B). In the presence of
both Q71L ARF1 and rat brain cytosol at 378C, synaptic
vesicles sediment to greater sucrose concentrations
than controls (32%–34% sucrose for PC12 SVs and
29%–30% sucrose for rat brain SVs) (Figures 1C and
1D). The GDP-form of ARF1 (T31N) did not increase the
sedimentation rate, consistent with a GTP-ARF-depen-
dent recruitment of protein to membranes (Faúndez et
al., 1997). When centrifuged for enough time to reach
equilibrium density, the coated PC12 vesicles migrated
as a single sharp peak at 39.7% 6 1.3% sucrose (n 5 4)
(r 5 1.17), whereas T31N-treated SVs equilibrated at
27.5% 6 0.5% sucrose (n 5 2), similar to untreated SVs,
which are recovered 27% 6 0.5% sucrose (n 5 2) (r 5
Figure 1. Cell-free Reconstitution of ARF-
Dependent Protein Recruitment to Synaptic
Vesicles
Size-purified synaptic vesicles from rat brain
(right) or from 125I-KT3 endocytically labeled
PC12N49A cells (left) were incubated with ei-
ther 30 mM Q71L ARF1 (open circles, trian-
gles, and squares) or T31N ARF1 mutant
(closed circles). Reaction mixtures were re-
constituted without cytosol (A and B) or in the
presence of 3 mg/ml rat brain cytosol (C–H,
circles). In (G) and (H), brain cytosol was re-
placed by either liver cytosol (triangles) or
yeast cytosol (squares). Mixtures were kept
at 378C for 30 min, except for (E) and (F) where
incubation was at 08C. Reactions were stopped
at 08C, and vesicles were resolved in 10%–
45% continuous sucrose gradients. Synaptic
vesicles migrated faster on sucrose gradients
(C and D, open circles) only in the presence
of rat brain cytosol and Q71L ARF1 mutant.
Synaptophysin in rat brain SV fractions was
detected with a specific antibody in an ELISA
assay; radioactivity in SVs from PC12 cells
was counted in a gamma counter.
1.11). The recruitment of coats to SVs was sensitive to
temperature. At 48C , or at 158C (data not shown), addi-
tion of the Q71L mutant in the presence of brain cytosol
did not modify SV migration (Figures 1E and 1F). Incuba-
tion of unlabeled brain SVs with labeled PC12 SVs de-
creased the Q71L-dependent shift of the labeled SVs
(data not shown). Thus, rat brain SVs are competing for
the same cytosolic factor(s) required for coating the
PC12 SVs.
ARF-dependent recruitment of coats to SVs was de-
pendent on the source of the cytosol. At equal protein
concentrations, neither rat liver nor yeast-derived cyto-
sol substituted for brain cytosol, implying that a neuron-
specific protein is recruited to the membranes (Figures
1G and 1H). Coat recruitment also required the addition
of a hydrolyzable form of ATP (data not shown).
GTP-ARF1 Mutant-Dependent Protein Recruitment
Is a Saturable Process that Requires
SV-Associated Proteins
Recruitment of cytosolic factors to SV is saturable. The
increase in sedimentation rate induced by cytosol and
the GTP-ARF1 mutant reached a plateau at 3 mg/ml
and 30 mM, respectively. Saturation of the density shift
occurred at 20 mM GTPgS. Samples were preincubated
at 378C with the dominant negative T31N mutant of ARF1
before the addition of GTPgS. The GDP-bound mutant
showed competitive inhibition, indicating that a limited
number of saturable ARF-binding sites are required for
AP3 and Vesicle Formation
425
Figure 2. SV Proteolysis Abrogates Q71L ARF1–Dependent Protein
Recruitment to SVs
Supernatants (27,000 3 g) enriched in 125I-KT3-labeled SVs were
prepared in the usual way. Supernatants were treated for 30 min at
08C with 25 mg/ml pronase either active or preinactivated with PMSF.
Proteolysis was stopped with 4 mM PMSF at 08C and SVs were
isolated in velocity glycerol gradients. Pronase-treated (squares) or
mock-digested (circles) vesicles were reconstituted with 3 mg/ml
rat brain cytosol in the presence of either 30 mM Q71L ARF1 mutant
(open symbols) or T31N ARF1 mutant (closed symbols). Reactions
were stopped and vesicle sedimentation was analyzed as previously
described. Proteolyzed vesicles did not shift to 33% sucrose on
sucrose gradients after addition of brain cytosol and Q71L ARF1.
the coating, and that the GTPgS effect is likely to involve
ARF (data not shown).
Since coat and ARF1 can be recruited to protein-free
lipid interfaces (Franco et al., 1993; Helms et al., 1993;
Ktistakis et al., 1996), we asked if the SV protein recruit-
ment was due to a lipid binding process or to cyto-
plasmic domains of proteins. Controlled protease di-
gestions were performed using endocytically labeled,
PC12-derived synaptic vesicles. Under the conditions
tested, the protease treatment did not change the SV
migration on glycerol velocity gradients nor reduce the
luminal content of labeled anti-TAg antibody (data not
shown). Control reactions were identical except that
protease inhibitors were added to pronase prior to the
addition of membranes. Vesicles treated with pronase
lost their ability to migrate faster in the presence of the
Q71L ARF1 mutant and rat brain cytosol (Figure 2, open
squares). Controls where pronase incubation was in the
presence of protease inhibitors showed the normal
change in sedimentation rate (Figure 2, open circles).
No shift was observed when T31N ARF was used, with
or without pronase activity (Figure 2, closed symbols).
These results show that the GTP-ARF1–triggered pro-
tein recruitment to SV is a saturable process that re-
quires protein acceptor site(s) on the vesicles.
AP3 Coat Complex Recruitment to Synaptic
Vesicles Is ARF-Dependent
In order to identify the putative coat, we performed pro-
tein recruitment reactions using a PC12 SV affinity
column. Synaptic vesicles were immunoimmobilized to
protein G-Sepharose beads that were coated with anti-
body (SY38) to the synaptophysin cytosolic domain.
These antibodies did not inhibit the change in sedimen-
tation rate on sucrose gradients (data not shown). After
incubating the immobilized vesicles in the presence of
rat brain cytosol and GTPgS at 378C, two prominent
bands became associated with the vesicles, one of ap-
proximately 150 kDa and the other migrating around 21
kDa (Figure 3A, compare lanes 3 and 4). Recruitment
of these proteins to the beads required that SVs were
attached (Figure 3A, compare lanes 2 and 4). The GTP-
bound form of ARF recruited the same proteins to the
vesicles whether it was the Q71L ARF mutant or wild-
type ARF bound to nonhydrolyzable GTPgS (Figure 3A,
compare lanes 4 and 8). The bands of about 21 kDa
found in the absence of SVs (Figure 3A, lane 6) corre-
sponded to nonspecific binding of the mutant ARF to
the matrix (data not shown).
The identity of the 150 kDa band was addressed by
immunoblot. Two different antibodies against the hydro-
philic portion of b-NAP/b3B (Newman et al., 1995; Dell’
Angelica et al., 1997a) strongly recognized the band
(Figure 3B, lane 4; Figure 3C, lanes 4 and 8; data not
shown) whether the coat was recruited by GTPgS or by
the addition of the Q71L GTP-ARF1 mutant (Figure 3C,
lanes 4 and 8, respectively). In agreement with the pro-
tein stain (Figure 3A), no b3 subunit recruitment was
detected in the absence of vesicles (Figure 3B, lane 2;
Figure 3C, lanes 2 and 6) or the activated ARF protein
(Figure 3B, lane 3; Figure 3C, lanes 3 and 7). Other
subunits of the AP3 coat complex were recruited con-
comitantly with the b3 subunit. The bands around 21
kDa were identified as the subunits A and B of the s3
protein (Figure 3B, lane 12; 3C, lanes 4 and 8), and also,
ARF proteins recruited to the vesicles (Figure 3B, lane
16). In addition, although it was not clearly distinguish-
able by total protein staining, m3/p47 was also recruited
to the vesicles, in parallel with the other subunits of the
complex (Figure 3B, lane 8 ).
The AP3 complex was also recruited to rat brain–
derived SVs in the presence of GTPgS. Rat brain synap-
tic vesicles were incubated in the presence of cytosol
either in the absence or presence of 20 mM GTPgS and
analyzed on sucrose gradients. After the addition of
GTPgS, the vesicles were shifted from 24% to 29% su-
crose (Figures 4A and 4B). Concomitantly, ARF (Figures
4G and 4H), and the AP3 subunits, b3 and s3, became
associated with the vesicles (Figures 4C and 4D, and
4E and 4F, respectively). These results show that the
AP3 complex binds to SVs in an ARF-dependent way
regardless of the source of synaptic vesicles.
To determine whether AP3 was exclusively recruited
to SVs, we assayed for other coats whose binding to
membranes is regulated by small GTP-binding pro-
teins (Figure 5). Neither AP1, assessed by antibodies to
g-adaptin, nor COPI, assessed by antibodies to b- and
e-COP (data not shown), nor COPII, assessed by anti-
bodies to mSec13p, was recruited to PC12 SVs after
the addition of GTPgS. Similarly, neither clathrin nor
AP180 was detected on PC12 SVs after the addition of
GTPgS. However, a-adaptin and dynamin were recruited
to membranes in the presence of nucleotides. These
interactions with SVs were not detected by protein stain
(Figure 3A), suggesting a relatively low abundance of
these proteins.
Weak interaction of AP2 with rat brain SVs was ob-
served. Coat complexes recruited to rat brain SVs by
GTPgS were analyzed by sucrose gradient centrifuga-
tion. The nucleotides induced a detectable increase in
the amount of a-adaptin in the position of the shifted
SVs (Figures 4I and 4J). These results show that the AP3
Cell
426

Figure 3. AP3 Coat Complex Is Recruited to PC12 SVs by an ARF-Dependent Mechanism

PC12-derived SVs were immobilized on an anti-synaptophysin (SY38) antibody–protein G-Sepharose column. Control reactions were performed
using columns containing murine IgG. Unbound vesicles were washed away, and columns were incubated with 3 mg/ml rat brain cytosol in
the absence (odd lanes) or presence (even lanes) of either 20 mM GTPgS or 50 mM Q71L ARF1. Incubations were performed at 378C for 40
min with continuous agitation. Reactions were stopped at 08C and columns washed extensively at 08C. Complexes were eluted from the
columns and resolved in 8%–18% gradient PAGE-SDS gels. Proteins that bound were analyzed either by fluorescent protein stain (SYPRO
Orange) (A) or by immunoblot (B and C) using antibodies against the proteins specified in the figure. Only in the presence of SVs and GTPgS
(B, lanes 4, 8, 12; C, lane 4) or Q71L ARF1 (C, lane 8) were the subunits of the AP3 coat complex bound to the column. ARF1 binding was
also observed in the presence of GTPgS (B, lane 16). The b3 subunit could also be detected in the SYPRO orange–stained gel (asterisk)
indicating that it was a major protein. The other bands are antibody subunits (double asterisk) or BSA (ampersand).

complex is preferentially recruited into SVs, but that
some AP2 recruitment may also be occurring.
The AP3 Complex Is Required for SV
Budding from PC12 Endosomes
We used the cell-free SV budding assay (Desnos et
al., 1995) to examine which adaptor complexes were
required. The AP3 coat complex from bovine brain cyto-
sol could be enriched approximately 60-fold and sepa-
rated from AP2 by a combination of ammonium sulfate
precipitation, DEAE Sepharose, Superose 6, and Mono
Q chromatography (Figure 6A). The AP3-containing frac-
tions were essentially devoid of the AP2 complex and
ARF1 by Western blot (data not shown). When labeled

Figure 4. AP3 Coat Complex Is Recruited to

Rat Brain SVs by an ARF-Dependent Mech-
anism
Rat brain SVs were incubated at 378C for 30
min in the presence of rat brain cytosol (3 mg/
ml) either in the absence (left panels) or pres-
ence (right panels) of GTPgS (20 mM). Vesicles
were sedimented in continuous 10%–45%
sucrose gradients. Fractions were collected
from the bottom, and their proteins were
resolved in 8%–18% gradient PAGE-SDS
gels and analyzed by immunoblot. Depicted
are immnunoblots performed with antibodies
against synaptophysin (A and B), b3 (C and
D), s3A-B (E and F), ARF1 (G and H), and
a-adaptin (I and J). Soluble fractions from the
top of the gradients (right) are not included.
AP3 and Vesicle Formation
427
Figure 5. SVs Preferentially Recruit the AP3 Coat Complex
Experiments were performed as described in Figure 3 except that
immunoblots were probed with antibodies against subunits of differ-
ent coats. Positive controls were either purified clathrin-coated vesi-
cles (clathrin and AP2), GTPgS and cytosol treated high-speed mem-
brane pellets from PC12 cells (b-COP, msec13p, g-adaptin, and
dynamin), or AP180 immunoprecipitated out of cytosol (AP180). By
Western blot, only a-adaptin and dynamin were significantly re-
cruited to the vesicles after addition of GTPgS.
PC12 donor membranes were incubated with ARF1-,
ATP-, and AP3-containing fractions, SV budding was
observed (Figure 6B). In a second set of experiments,
rat brain cytosol was immunodepleted using polyclonal
anti-s3 antibodies that recognize the whole AP3 com-
plex in its native state (Dell’Angelica et al., 1997a). These
antibodies removed more than 95% of the s3 subunits
from brain cytosol (Figure 6C, insert). Cell-free SV forma-
tion was analyzed using s3-depleted cytosol and donor
membranes from cells labeled either at 158C or in the
presence of BFA (5 mg/ml) (Figure 6D). Results were
normalized against control reactions performed with
mock-depleted rat brain cytosol. Both donor membranes
decreased their capacity to bud SVs by 42% 6 7% (n 5
7) from 158C-labeled donor membranes and 39% 6 4%
(n 5 5) from the BFA-labeled ones (Figure 6B). The effect
of depleting the s3 subunit was abrogated by adding
back the Mono Q AP3-containing fractions (n 5 4) (Fig-
ure 6D), showing that the s3 depletion effect upon vesi-
cle formation was reversible and linked to the removal
of AP3 complex.
These indications that AP3 was involved in coating
and budding motivated us to determine if AP3 and ARF1
were the only cytosolic requirements for budding to
purify the AP3 to homogeneity. The budding activity
continued to coelute with AP3 after additional chroma-
tography steps on hydroxyapatite and Mono S columns.
At this point, the preparation had a specific activity in
the budding assay that was about 500-fold higher than
the starting material. Analysis of the preparation by SDS-
PAGE revealed that the preparation contained only five
bands (Figure 6E) that were shown to be the d, b3, m3,
s3A, and s3B subunits by immunoblotting with specific
antibodies. To examine cytosol dependence, PC12 mem-
branes were washed free of cytosol through a Percoll
gradient then incubated under standard assay condi-
tions using a concentration of brain cytosol that gave a
maximum response. Replacing cytosol with pure AP3
and recombinant ARF1 allowed SV budding to an extent
even greater than that achieved with saturating levels
of brain cytosol. Neither AP3 nor ARF1 alone reconsti-
tuted the budding activity (9% 6 7% and 33% 6 3%,
respectively). Thus, the AP3 coat complex and ARF1
are sufficient for the formation of SVs from endosomal
donors.
Discussion
Coated intermediates involved in membrane traffic have
been identified by forming them from donor compart-
ments and preventing their uncoating. Here we describe
the formation of a coated intermediate by a reverse
reaction, incubating purified carrier vesicles, in this case
synaptic vesicles, under conditions that favor their coat-
ing. AP3 and ARF1 were shown to be recruited to both
PC12 and brain synaptic vesicles. We then asked if the
forward reaction involved AP3 by measuring SV forma-
tion in vitro in the presence or absence of AP3 and ARF1.
ARF1 and AP3 were sufficient for reconstituting SV gen-
eration in vitro and are the only cytoplasmic components
that appear to be required for the vesiculation of mem-
brane from endosomes into synaptic vesicles (Desnos
et al., 1995).
ATP and temperature-dependent synaptic vesicle coat-
ing were not reported in earlier studies (Zhang et al.,
1994). One possibility is that the recently described AP3
coats self-assemble much more readily than the other
more fully described adaptors. It is also possible that
earlier studies were more biased toward spontaneous
binding reactions occurring at 48C. The coating reaction
described here requires temperatures above 158C, and
ATP, suggesting an enzymatically driven or regulated
assembly.
The assay we utilize measures budding from the endo-
some. It cannot detect vesicle budding from biosyn-
thetic compartments such as endoplasmic reticulum or
Golgi membranes since the synaptic vesicles were la-
beled via antibody endocytosis. The precursor is not the
plasma membrane since more than 95% of the plasma
membrane can be removed from the donor membranes
without eliminating the budding activity (Y. Lichtenstein,
C. Desnos, V. F., L. Clift-O’Grady, and R. B. K., unpub-
lished data). Furthermore, it is known both by light and
electron microscopy that AP3 can be bound to endo-
somes (Dell’Angelica et al., 1997a, 1998). Endosomes
in PC12 cells that are labeled by internalizing markers
at 158C can also be shown to have AP3 coats by immu-
noelectron microscopy (J. Klumperman, personal com-
munication). If AP3 is mediating vesicle budding from
PC12 endosomes, then two issues arise. How can we
relate these observations to the data that AP2 and
clathrin are involved in SV formation and to the more
recent evidence implicating AP3 in biosynthetic rather
than endocytotic pathways?
Cell
428
Figure 6. The AP3 Coat Complex Is Required for SV Budding In
Vitro
(A) Enrichment of the AP3 coat complex from bovine brain cytosol.
Sequential fractionation of bovine brain cytosol (BBC) through
DEAE-Sepharose, Superose 6, and Mono Q. Fractions from the
different fractionation steps were resolved in PAGE-SDS gels and
analyzed by immunoblot for the presence of b3 subunit and s3A-B.
A and B represent pooled fractions from the Mono Q column that
eluted before and after the AP3-containing peak.
(B) The AP3-enriched fraction can support an ARF1-dependent SV
budding from PC12 cell endosomes. PC12 N49A cells were labeled
with 125I-KT3 at 158C. Cell homogenates were sedimented through
a Percoll gradient. Labeled membranes were reconstituted at 48C
with bovine brain cytosol or Mono Q fractions supplemented with
50 mM ARF1. Budding reactions were performed at 378C for 30 min,
and the amount of SVs generated was assessed in glycerol velocity
gradients. The AP3-containing fractions were substantially enriched
in budding activity compared to bovine brain cytosol (BBC) and
pools A and B. Gray bars represent the protein concentration of the
different fractions in the budding assay. Data represent an average 6
standard error of the mean (n 5 3).
(C) Cytosol depletion of the AP3 complex decreases the SV budding
activity. PC12N49A cells were labeled at 158C. Washed membranes
were reconstituted with s3-depleted cytosol (insert, letter D) or
mock-depleted (insert, M). Budding reactions were performed as
described in (B). A representative glycerol gradient profile from in
vitro reactions performed with mock-depleted cytosol (closed cir-
cles) and s3-depleted cytosol (open circles) is depicted.
(D) Combined data from in vitro budding experiments from PC12N49A
membranes labeled either at 158C or 378C in the presence of 5 mg/
ml brefeldin A (BFA) to prevent SV formation in vivo (Faúndez et al.,
1997). s3 depletion substantially reduces the SV budding activity
of both properties of donor membranes. The final column (Mono Q)
describes experiments in which the Mono Q AP3-enriched fractions
were added back to the s3-depleted cytosol, and the budding was
performed from membranes labeled at 158C. The SV budding
Considerable evidence indicates that synaptic vesi-
cles can be formed by a pathway that involves AP2,
clathrin, and dynamin (van de Goor et al., 1995; Koenig
and Ikeda, 1996; Takei et al., 1996; Cremona and De
Camilli, 1997; González-Gaitán and Jäckle, 1997; Shupli-
akov et al., 1997). Elegant electron microscopic studies
have shown that steps mediated by clathrin and dy-
namin appear to occur at the plasma membrane al-
though some budding may take place from internalized
membranes (Takei et al., 1996). One plausible explana-
tion is that nerve terminals use two modes of vesicle
formation, one that generates synaptic vesicles from the
plasma membrane using clathrin and dynamin and a
second that uses AP3 and ARF1 to generate synaptic
vesicles from endosomes. Direct experimental support
for two pathways of biogenesis has recently been ob-
tained in PC12 cells. In addition to the AP3-requiring,
clathrin-independent pathway from the endosomes de-
scribed here, PC12 cells have a second pathway that
is AP3 independent but requires AP2 and clathrin (G. Shi,
V. F., J. Roos, and R. B. K., unpublished data). One
possible function for the AP3 pathway in neurons is
the generation of synaptic vesicles from the extensive
invaginations that form in nerve terminals after intense
stimulation (Heuser and Reese, 1973).
It is also necessary to reconcile AP3-mediated bud-
ding from endosomes with recent data from yeast, Dro-
sophila, and mice that implicate AP3 in a biosynthetic
pathway to lysosomes and a subclass of storage vesi-
cles. Deletion of AP3 subunits inhibits the delivery of
newly synthesized alkaline phosphatase and a t-SNARE,
Vam3p, to the yeast vacuole (Cowles et al., 1997; Stepp
et al., 1997), which implies that AP3 may facilitate bud-
ding from Golgi membranes. Similarly, Drosophila gar-
net mutants, defective in a protein homologous to the
delta subunit of AP3, have a reduced number of pigment
granules in their eyes and other tissues (Ooi et al., 1997;
Simpson et al., 1997). Since pigment granules can be
considered as modified lysosomes, AP3 would again
appear to be involved in a specialized Golgi-to-lyso-
somal pathway, in this case to a pigment storage vesicle.
Pigment dilution mutants in mice also affect biogenesis
of melanosomes and of platelet dense granules and
lysosomes (Erickson, 1997; Seymour et al., 1997). Two
of the coat color mutants, the mocha and pearl mice,
have defects in AP3 (P. Kantheti and M. Burmeister,
personal communication; Seymour et al., 1997). One
obvious explanation is that AP3 facilitates vesicle forma-
tion at two sites, one at the Golgi complex and the other
at endosomes.
inhibition obtained by s3 depletion is reversed by this fraction. Data
represent an average 6 standard error of the mean.
(E) Purified brain AP3 and ARF1 are sufficient to reconstitute the
SV budding activity. Membranes from cell homogenates of N49A
cells, labeled at 158C, were Percoll washed. Budding reactions were
performed at 378C for 30 min in the presence of either 1.5 mg/ml
of bovine brain cytosol (BBC, n 5 3), 8.5 mg/ml of purified bovine
brain AP3 coat complex (AP3, n 5 2), 40 mM recombinant wild-type
ARF1 (ARF1, n 5 2), or the combined addition of ARF1 and AP3
complex (40 mM and 8.5 mg/ml, respectively; AP3/ARF1, n 5 3). In
vitro budding reactions performed in the presence of pure AP3
coat complex or ARF1 alone generated small amounts of vesicles
whereas both components together were synergistic in vesicle gen-
eration.
AP3 and Vesicle Formation
429
An alternative explanation is that AP3 is needed to
form this class of storage vesicle from endosomal inter-
mediates. Some storage granules in the cytoplasm can
be labeled by the uptake of material from outside the
cell, which indicates an overlap between the biosyn-
thetic and endocytotic pathways. Thus, melanosomes
can be labeled with internalized melanocyte-stimulating
hormone by pinocytosis (Lerner et al., 1979) or by the
phagocytotic internalization of latex or gold beads (Le-
Poole et al., 1993; Schraermeyer and Stieve, 1994). Stor-
age granules of cells of hematopoietic origin can also
be labeled by extracellular markers (Handagama et al.,
1989, 1993). Perhaps AP3 will be found to affect the
biogenesis of all storage vesicles whose formation, like
the formation of SVs described here, involves an endo-
somal intermediate.
The genetic data from the pigment dilution mice imply
that AP3 has an important but not essential pathway in
the brain. In the mocha mouse, both the neuronal and
the nonneuronal forms of AP3 are missing (P. Kantheti
and M. Burmeister, personal communication). The mo-
cha mice are viable but have strong neurological symp-
toms (Noebels and Sidman, 1989). The neurological
symptoms probably cannot be attributed to defects in
lysosomal function because pearl mice, defective only
in nonneuronal AP3, do not have them. Although there
are possible explanations of the neurological symptoms
other than defects in an endosomal pathway of synaptic
vesicle biogenesis, it is certainly an option worth ex-
ploring.
Experimental Procedures
Reagents
[125I]Na and ECL reagents were obtained from Amersham Corp (Ar-
lington Heights, IL). All the nucleotides, creatine phosphate, creatine
kinase, and Sephadex G25 were purchased from Boehringer Mann-
heim Corp (Indianapolis, IN). DEAE Sepharose, Protein G-Sepharose
4 Fast Flow, Superose 6, and the Mono Q and Mono S FPLC columns
were obtained from Pharmacia Biotech (Uppsala, Sweden). Brefel-
din A was purchased from Epicenter Technologies (Madison, WI).
Cell culture media and reagents were obtained from the University
of California Cell Culture Facility (San Francisco, CA). Geniticin
(G418) and IPTG were obtained from GIBCO BRL (Gaithersburg,
MD). Percoll and all the other reagent grade chemicals were pur-
chased from either Sigma (St. Louis, MO), Fisher Chemical (Fairlawn,
NJ), or Calbiochem (San Diego, CA). Female Sprague-Dawley rats
were from Bantin and Kingman (Fremont, CA).
Cell Culture
The PC12 cell line stably transfected with the mutant VAMP N49A
was grown and induced with 6 mM sodium butyrate as described
(Grote et al., 1995).
Antibodies
Monoclonal antibodies against synaptophysin (SY38) were pur-
chased from Boehringer Mannheim (Indianapolis, IN). Polyclonal
antibodies against b-NAP647–796 that recognize b3A and b3B, against
delta, against m3/p47, and against s3 were a generous gift of Drs.
E. Dell’Angelica and J. Bonifacino (NIH, Bethesda, MD) (Ooi et al.,
1997; Dell’Angelica et al., 1997a). The polyclonal antibody Nb di-
rected against b-NAP was a gift of Dr. R. Darnell (Rockefeller Univer-
sity, NY) (Newman et al., 1995). Anti-ARF antibodies used in this
study were either polyclonal antibodies raised in rabbits immunized
with myristoylated recombinant human mutant ARF1 Q71L or the
monoclonal antibody 1D9 (Cavenagh et al., 1996) (a generous gift
of Dr. R. Kahn, Emory University, Atlanta, GA). Polyclonal antibodies
directed to the peptide 496–513 of b-COP were obtained from Affin-
ity Bioreagents (Golden, CO), and anti-eCOP antibodies (Hara-Kuge
et al., 1994) were a generous gift of Dr. J. Rothman (Memorial Sloan-
Kettering Cancer Center, NY). Polyclonal antibodies against mam-
malian sec13p were provided by Dr. C. Kaiser (MIT) (Shaywitz et
al., 1995). TD1 anti-clathrin monoclonal antibody (Näthke et al., 1992)
was a gift of Dr. Frances Brodsky (U. of California, San Francisco,
CA). Monoclonal antibodies against a-adaptin clone 8 and 100/2
were purchased from Transduction Lab (Lexington, KY) and Sigma
Chemical (St. Louis, MO), respectively. Monoclonal anti-g-adaptin
clone 88 was obtained from Transduction Lab. Polyclonal antibodies
against dynamin (2072) were a gift of Dr. J. B. Roos (U. of California,
San Francisco, CA) (Estes et al., 1996). Monoclonal antibodies
against AP180 clone AP180-I were purchased from Sigma (St. Louis,
MO). Monoclonal antibody 69.1 anti-VAMP2/synaptobrevin 2 was a
generous gift of Dr. R. Jahn (Yale University) (Baumert et al., 1989).
Mouse IgG was obtained from Jackson Immunoresearch Laboratory
(West Grove, PA). Affinity-purified peroxidase conjugated secondary
antibodies were purchased from Cappel, ICN.
Expression and Purification of Recombinant Proteins
Wild-type, Q71L, and T31N mutant human ARF1 cDNAs, kindly pro-
vided by Dr. D. Shields (Albert Einstein College of Medicine, Bronx,
NY), were used to generate mutant proteins as described (Faúndez
et al., 1997). GST-VAMP2 fusion protein was purified following the
manufacturer’s instructions. The recombinant protein was concen-
trated in Centriprep 30 (Amicon, Beverly, MA) to 1 mg/ml.
SV Isolation and Quantification
SVs were isolated from rat brain essentially as described by Clift-
O’Grady et al. (1990), except that the last step of size purification
of the SVs was done in glycerol velocity gradients, 5%–25% in
intracellular buffer (38 mM potassium aspartate, 38 mM potassium
glutamate, 38 mM potassium gluconate, 20 mM potassium MOPS
[pH 7.2], 5 mM reduced glutathione, 5 mM sodium carbonate, 2.5
mM magnesium sulfate), spun at 218,000 3 g for 75 min in an
SW55 rotor (Beckman Instruments, Palo Alto, CA). The SV peak,
determined either by immunoblot or an ELISA-based assay using
the SY38 antibody (Wagner et al., 1978), was pooled and flash frozen
in liquid nitrogen and stored at 2808C until use. Similar results were
obtained using frozen or freshly prepared vesicles.
PC12 SVs were isolated from the PC12/N49A cell line transfected
with an epitope-tagged version of the synaptic vesicle protein
VAMP2, mutant N49A (Grote et al., 1995). Cells were labeled at 158C
with iodinated anti-TAg antibodies washed and homogenized as
described (Desnos et al., 1995; Faúndez et al., 1997). Labeled SVs
were isolated on glycerol velocity gradients as described for rat
brain vesicles.
The synaptic vesicle content was determined semiquantitatively
by immunoblot, using the monoclonal antibody 69.1 anti-VAMP2
and GST-VAMP2 as standard. Several dilutions of GST-VAMP stan-
dard (115 ng to 1.15 mg) were resolved in 15% PAGE-SDS gels
together with several dilutions of SVs and immunoblotted with the
69.1 monoclonal antibody. The immune complexes were detected
by ECL. The SV content was determined after quantitating different
time exposures with the NIH Image 1.60 program (Faúndez et al.,
1997).
Cytosol Preparations
Either rat or bovine brain cytosol and rat liver cytosol were prepared
as described (Desnos et al., 1995). Saccharomyces cerevisiae cyto-
sol (strain CTY182 Mata, ura 3–52, his 3–200, lys 2–801) was ob-
tained as described (Baker et al., 1988) (a gift of Dr. A. E. Cleves,
MetaXen, Hayward, CA).
SV Coating Assay
Cell-free synaptic vesicle coating assays were performed in 250 ml
total volume in intracellular buffer, using PC12/N49A 125I-KT3 labeled
vesicles, 100–500 ng of VAMP2 content per assay or rat brain synap-
tic vesicles, 300–1400 ng of VAMP2 content per assay; and in the
absence or presence of rat brain cytosol, ATP regenerating system
and either T31N ARF1 or Q71L ARF1 mutants or GTPgS. Reconsti-
tuted mixtures were kept at 48C for 15 min. Coating reactions were
Cell
430
started by warming to 378C. Reactions were stopped at 48C for 10
min and loaded on the top of continuous 10%–45% sucrose gradi-
ents buffered in 20 mM MOPS-KOH (pH 7.4), 0.5 mM MgCl2 . Sucrose
gradients were centrifuged at 183,000 3 g for 150 min in an SW55
rotor. Fractions (27–28) were collected from the bottom of the gradi-
ent and either counted in a gamma counter or resolved in PAGE-
SDS gels and immunoblotted with different antibodies.
Protease Treatment
Homogenates containing 125I-KT3 labeled SVs were prepared as
before except that protease inhibitors were omitted from the buffer.
A postnuclear supernatant (S1; 1000 3 g for 5 min) was sedimented
at 27,000 3 g for 35 min, and the supernatant generated (S2) was
used for controlled proteolysis. S2s (1 mg) were treated with 25
mg/ml of pronase either inhibited or not with 4 mM PMSF. Digestions
were performed for 30 min at 08C, and reactions were stopped by
the addition of 4 mM PMSF. Mock digested and digested S2 were
loaded onto 5%–25% glycerol gradients prepared in intracellular
buffer over a 50% sucrose cushion, then spun at 218,000 3 g for
75 min in an SW55 rotor (Beckman Instruments, Palo Alto, CA).
Fractions (17–18) were collected from the bottom. The SV peak from
both conditions was identified by counting. No differences in the
sedimentation properties and intravesicular 125I-KT3 content were
detected under these conditions. Complete proteolysis was con-
firmed by blotting vesicles with antibodies to synaptophysin and
VAMP2. SVs were used immediately for coating reactions as already
described.
SV Affinity Chromatography
Antibodies against the cytosolic tail of synaptophysin (SY38, 2.5 mg)
were bound to 25 ml of protein G Sepharose. SVs were bound to
the matrix for 3 hr at 48C, and the unbound vesicles were washed
away in intracellular buffer supplemented with 0.1% ovalbumin. Re-
action mixtures containing 3 mg/ml rat brain cytosol, in the absence
or presence of either 50 mM Q71L ARF1 mutant or 20 mM GTPgS,
were incubated for 15 min at 48C followed by warming to 378C for
40 min with periodic resuspension of the beads. After arresting the
reactions at 48C, the matrix was washed in intracellular buffer, and
the retained proteins were eluted with Laemmli sample buffer and
resolved in 8%–18% gradient PAGE-SDS gels prior to immu-
noblotting.
Cytosol Fractionation and Immunodepletion
Size fractionation of the rat brain cytosol in a Superose 6 column
was performed as previously described (Faúndez et al., 1997).
To obtain fractions enriched in AP3 complex, liquid nitrogen flash-
frozen bovine brains (Pel-Freeze, Biologicals, Rogas, AR) were
thawed then homogenized using a Waring blender at a ratio 1:2
(w:v) in buffer A (25 mM Tris-HCl [pH 8.0]; 500 mM KCl, 2 mM
EGTA, 1 mM DTT in the presence of a proteinase inhibitory mix).
Homogenates were sedimented at 10,400 3 g for 1 hr in a GSA
rotor, and supernatants were dialyzed for 2 hr against 6 liters of
buffer B (25 mM Tris-HCl [pH 8.0]; 5 mM MgCl2 , 1 mM DTT) and
then overnight in 6 liters of the same buffer. After sedimenting the
dialyzed material at 11,750 3 g for 1 hr in a GSA rotor, the superna-
tants were salted out by 35% (NH4)2SO4. Precipitated proteins were
recovered by spinning at 10,400 3 g for 20 min. Pellets were resus-
pended in buffer B and applied to a DEAE-Sepharose CL-6B column
preequilibrated with buffer B containing 100 mM NaCl and 10%
glycerol (v:v). Unbound proteins were washed out with equilibrium
buffer. Bound proteins were eluted by a linear gradient of 100–600
mM NaCl in buffer B. b-NAP and s3 were detected by immunoblot,
and the fractions containing both of them were pooled and applied
to a Superose 6 column preequilibrated in intracellular buffer. Peak
fractions containing both b-NAP and s3 were directly applied to a
Mono Q 5-5 column preequilibrated in 150 mM NaCl in buffer B and
eluted by a linear gradient of 100–600 mM NaCl in buffer B. The
Mono Q peak containing both b-NAP and s3, and the fractions
upstream (A) or downstream (B) of it were pooled, concentrated,
and extensively dialyzed against intracellular buffer before being
used in the cell-free synaptic vesicle assay. The AP3 complex eluted
from the Mono Q column at 320 mM NaCl. These fractions were
devoid of AP2. This procedure enriched the AP3 complex 60 times
over homogenate levels. AP3 was further purified from Mono Q
fractions by subsequent hydroxyapatite and Mono S chromatogra-
phy. AP3-containing fractions from the Mono Q column were supple-
mented with phosphate buffer to a final concentration of 150 mM
and loaded onto a hydroxyapatite column (CHT5-I, Bio-Rad). Pro-
teins were eluted with 150–550 mM potassium phosphate buffer
(containing 5 mM MgCl2, 1 mM DTT, and 10% glycerol). AP3-con-
taining fractions were dialyzed against buffer C (25 mM MES-KOH
[pH 6.0], 5 mM MgCl2, 1 mM DTT, and 10% glycerol). The sample
was absorbed to a Mono S column (Pharmacia, Uppsala, Sweden)
equilibrated with buffer C. The column was washed with buffer C
and subsequently eluted with 0–500 mM NaCl in buffer C. The AP3-
containing fractions were pooled and dialyzed in intracellular buffer.
Immunodepletions were performed with preimmune or anti-s3
antibodies prebound to protein G-Sepharose essentially as de-
scribed (Faúndez et al., 1997)
Cell-Free SV Biogenesis Assay
PC12 N49A cells were labeled at 158C with iodinated anti-TAg anti-
bodies as described (Desnos et al., 1995; Faúndez et al., 1997).
After washing the unbound 125I-KT3 antibody, cells were homoge-
nized either immediately or after an incubation at 378C for 15 min
in the presence of 5 mg/ml brefeldin A in media DME-H21 supple-
mented with 10 mM HEPES at pH 7.4. To remove endogenous PC12
cytosol, cell homogenates were loaded on the top of a discontinuous
gradient of 3 ml 50% Percoll and 9 ml 10% Percoll prepared in
intracellular buffer. Samples were sedimented at 27,000 3 g for 45
min in an SS34 rotor. Membranes were recovered from the Percoll
interface to be used in the cell-free assays.
Percoll-washed membranes (250–500 mg per assay) prepared
from either 158C or brefeldin A-treated cells were reconstituted in
the presence of an ATP regenerating system (1 mM ATP, 8 mM
creatine phosphate, 5 mg/ml creatine kinase) either with 2.5 mg/ml
of cytosol either mock-depleted or depleted with antibodies against
s3, or Mono Q fractions supplemented with 30 mM wild-type ARF1.
Reactions were kept at 48C for 15 min to be initiated at 378C for 30
min. The vesicle budding was stopped at 48C. All the fractionation
procedures, glycerol velocity gradients, and vesicle budding activity
quantitations were performed following the procedures described
in Faúndez et al. (1997).
Other Procedures
KT3 monoclonal antibodies were iodinated by chloramine-T (Faún-
dez et al., 1992). Protein assays were performed using the Bio-Rad
Protein Assay Dye Reagent (Bio-Rad, Richmond, CA) using BSA as
standard. Gel protein staining was performed using the fluorescent
dye SYPRO Orange (Bio-Rad, Richmond, CA) following the manu-
facturer’s instructions.
Acknowledgments
We wish to thank Drs. Darnell, Rothman, Kahn, Bonifacino, Brodsky,
Kaiser, Jahn, and Shields for gifts of antibodies or DNA. Comments
on the manuscript by Drs. Brodsky, Mostov, and Herman were much
appreciated. The research was supported by NIH grants (NS09878,
NS15927, and DA10154), by an NIH Fogarty Fellowship to Dr. Faún-
dez, and an equipment grant from the Academic Senate, UCSF.
Received December 8, 1997; revised March 25, 1998.
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