(Received for publication, October 7, 1994; and in revised form, December 12, 1994)
From the
The assembly of clathrin-coated buds on the Golgi requires the
recruitment of the heterotetrameric AP-1 adaptor complex, which is
dependent on both guanine nucleotides and the small GTP-binding protein
ADP-ribosylation factor (ARF). Here, we have investigated the
structural domains of the AP-1 complex necessary for ARF-mediated
translocation of the adaptor complex onto Golgi membranes and the
subsequent recruitment of clathrin onto the membrane. Controlled
proteolysis of purified AP-1, derived from bovine adrenal coated
vesicles, was used to generate AP-1 core fragments composed of the
amino-terminal trunk regions of the 1 and
subunits and
associated µ1 and
1 subunits, and lacking either the
1
subunit carboxyl-terminal appendage or both
1 and
subunit
appendages. On addition of these truncated fragments to AP-1-depleted
adrenal cytosol, both types of core fragments were efficiently
recruited onto Golgi membranes in the presence of GTP
S.
Recruitment of both core fragments was inhibited by the fungal
metabolite brefeldin A, indicative of an ARF-dependent process. Limited
tryptic digestion of recruited, intact cytosolic AP-1 resulted in the
quantitative release of the globular carboxyl-terminal appendage
domains of the
1 and
subunits. The adaptor core complex
remained associated with the Golgi membranes. Recruitment of cytosolic
clathrin onto the Golgi membranes was strictly dependent on the
presence of intact AP-1. Tryptic removal of the
1 subunit
appendage prevented subsequent clathrin recruitment. We conclude that
the structural determinants required for the ARF-mediated binding of
cytosolic AP-1 onto Golgi membranes are contained within the adaptor
core, and that the carboxyl-terminal appendage domains of the
1
and
subunits do not play any role in this process. Subsequent
recruitment of cytosolic clathrin, however, requires an intact
1
subunit.
The clathrin-coated vesicle exhibits a well defined structural
organization, and the major protein components have been purified and
characterized(1, 2, 3, 4) . In
addition to the principal coat protein clathrin, two major adaptor
complexes have been identified. One type is restricted to the trans-Golgi network (TGN) ()and Golgi-derived
coated vesicles and termed AP-1. The second, localized to the plasma
membrane and endocytic clathrin-coated vesicles, is termed AP-2.
Adaptors are heterotetrameric complexes, composed of one related and
one unique
100-kDa subunit, and two additional components, the
50-kDa µ and
20-kDa
subunits. Thus, the Golgi AP-1
adaptor complex is composed of
,
1, µ1, and
1
subunits, and the related AP-2 is composed of
,
2, µ2,
and
2 subunits. The
1 and
2 subunits are very similar
in structure(5, 6, 7, 8) , while the
and
subunits are the most distantly related adaptor
subunits(4, 9) . Electron microscopy has revealed that
the AP-2 adaptor has a large globular core domain and two exposed
globular head or appendage domains(
)(10) .
Controlled proteolysis of both AP-1 and AP-2 (11, 12, 13, 14) has shown that the
core is composed of the amino-terminal trunk regions of either the
and
2 or the
1 and
subunits as well as the intact
µ and
subunits, while the appendages represent the
30-40-kDa carboxyl-terminal regions of the large subunits.
Adaptors are believed to facilitate clathrin-coated vesicle
formation by combining a clathrin binding domain and a membrane
association domain within an oligomeric protein complex. When it became
apparent that the 1 and
2 subunits were highly related, it
was suggested that these subunits might contain a common domain capable
of binding to clathrin(5) . Indeed, the purified
2 subunit
interacts directly with preformed clathrin cages(15) , and,
more recently, it has been shown that recombinant
-type subunits
alone can induce clathrin polymerization(16) . Further attempts
to define the clathrin binding site more precisely indicated that
proteolytically generated cores can bind to preformed clathrin
cages(12) , suggesting that the clathrin binding site is
located in the amino-terminal trunk domain. However, in buffer
conditions that prevent adaptor self-association, tryptic removal of
the
subunit appendage results in the release of both appendage
and core fragments from clathrin-coated vesicles(14) . This
suggests that both the trunk and appendage domains of the
-type
subunits are required for high affinity clathrin
binding(14, 16) .
Before clathrin binding and polymerization can occur, however, the adaptors must associate stably with the appropriate intracellular membrane compartment. According to the simplest model for the interaction of adaptors with membranes(1, 3, 4, 17, 18) , it was assumed that they attach directly to the cytoplasmic portions of selected receptors. Indeed, there is evidence that adaptors can interact directly, albeit weakly, with the cytoplasmic regions of certain proteins sorted into clathrin-coated vesicles(17, 18, 19, 20, 21, 22) . This simple model, while attractive, failed to explain the restricted localization of AP-1 within the cell and why AP-1 does not bind to cytoplasmically oriented trafficking motifs in receptors transiting through other intracellular compartments(2, 3, 4) . Furthermore, the dramatic effect of brefeldin A (BFA) on the intracellular localization of AP-1 also suggested that clathrin coat formation is highly regulated (23, 24) .
Recently, the recruitment of cytosolic AP-1 onto purified Golgi membranes was reconstituted in vitro(25, 26) . Adaptor binding was found to be dependent on GTP and antagonized by BFA. ARF, a small GTP-binding protein, was identified as the GTP-requiring component(25, 26) . Coatomer-coated vesicle formation also proceeds by the initial recruitment of ARF(27, 28) , explaining the sensitivity of both clathrin- and coatomer-coated vesicle formation to BFA. However, since no clear ARF specificity has yet been discerned(25, 26) , we proposed that ARF may be required to ensure the selective interaction of AP-1 with a specific docking protein in the context of the TGN membrane. While our model overcomes the limitations of the direct association model, the structural determinants on the AP-1 complex that are recognized by the putative docking protein remain to be identified.
The role of the
and
subunit appendages has been probed using a
chimera-based approach(29) . Swapping the
and
subunit head and/or hinge regions did not appear to affect either the
assembly or targeting of the AP-1 or AP-2 chimeric complexes. Here, we
have taken a different approach to directly assess the structural
features of the AP-1 heterotetramer required for Golgi membrane
association and subsequent clathrin recruitment. Defined tryptic
fragments were produced by controlled
proteolysis(8, 14) , and this has enabled us to follow
the ARF-dependent recruitment of the different fragments onto Golgi
membranes. We have found the translocation of the AP-1 core complex to
be indistinguishable from intact AP-1. This precludes the globular
carboxyl terminus and hinge regions from controlling this process. In
contrast, clathrin binding to Golgi membranes was found to be strictly
dependent on an intact
1 subunit.
Adrenal or rat liver cytosol
was depleted of clathrin by gel filtration on Sepharose 4B. 50-60
ml of cytosol was rapidly thawed, centrifuged at 150,000 g for 60 min to remove aggregated material, and loaded at 1 ml/min
onto a Sepharose 4B column (5.0
70 cm) equilibrated in 25
mM Hepes-KOH, pH 7.3, 125 mM potassium acetate, 5
mM magnesium acetate, and 0.05% sodium azide. Fractions of 20
ml were collected, and the elution positions of clathrin and AP-1 and
AP-2 adaptors was determined on immunoblots using mAb TD.1 and mAb
100/1. The clathrin triskelia eluted well ahead of the adaptors, and
fractions after the clathrin peak were pooled, concentrated by
precipitation with 60% ammonium sulfate, and dialyzed against 25 mM Hepes-KOH, pH 7.0, 125 mM potassium acetate, 5 mM magnesium acetate, and 1 mM dithiothreitol (Buffer C).
After centrifugation at 12,000
g for 15 min, the
clathrin-depleted cytosol was aliquotted, quick frozen in dry ice, and
stored at -80 °C. The clathrin-containing fractions were
pooled separately and concentrated approximately 10-fold by
ultrafiltration using an Amicon YM 30 filter. After centrifugation at
12,000
g for 15 min, the clathrin-enriched fraction
was stored at 4 °C.
Endogenous AP-1 was removed from the
clathrin-depleted adrenal cytosol by immunodepletion(26) .
Briefly, cytosol was passed over an immobilized mAb 100/3 column
equilibrated in buffer C on ice, and the loading effluent was reapplied
to the column several times, before similar passages over a second mAb
100/3 column equilibrated in buffer C on ice. The resulting
AP-1-depleted cytosol was analyzed on immunoblots using
affinity-purified AE/1, and the extent of depletion was more than 95%.
Clathrin-coated vesicles and coated vesicle-derived AP-1 were purified
from frozen bovine adrenal glands as described in detail
previously(8) . Protein concentrations were estimated using
either the Coomassie Blue method (35) with bovine serum albumin
as a standard or, for the purified AP-1 preparations, by A, using an E
value of 6.0(14) .
The goal of this study was to identify the structural regions
of the heterotetrameric AP-1 adaptor complex which are involved in the
ARF-dependent recruitment onto Golgi membranes and the subsequent
recruitment of cytosolic clathrin. To distinguish between the
endogenous rat liver Golgi-associated AP-1 and exogenously added
cytosolic AP-1, using a species-specific mAb directed against the
subunit, we had to modify our existing AP-1 binding assay by
substituting bovine adrenal gland cytosol for rat liver cytosol. To
limit our analysis to the initial events in clathrin-coated vesicle
assembly, clathrin-depleted adrenal cytosol (26) has been used
for several of the experiments described below.
Figure 1:
GTP-dependent association of ARF and
AP-1 with Golgi membranes. Tubes containing 50 µg/ml rat liver
Golgi, 5 mg/ml clathrin-depleted adrenal cytosol, 100 µM GTPS, and 50 µg/ml BFA in a final volume of 400 µl
were prepared on ice as indicated in the figure. The asterisk denotes addition after 10 min of incubation at 37 °C. All
reactions were terminated after 20 min, and the membrane fractions were
analyzed on immunoblots with mAb 100/3 (upper portion) or by
[
-
P]GTP overlay (lower portion).
The
80-kDa band detected with mAb 100/3 represents an endogenous
proteolytic degradation product of the
subunit of the adrenal
AP-1. In the lower panel, the
22- to
30-kDa
GTP-binding proteins represent an uncharacterized group of low
molecular mass GTP-binding proteins while the band migrating slightly
ahead of the 20-kDa standard is ARF, based on both co-migration with
purified ARF and sensitivity to BFA. The upper and lower portions
originate from a single blot, and the position of the molecular size
markers is indicated on the left.
Figure 2:
Controlled tryptic digestion of adrenal
AP-1 recruited onto Golgi membranes. 50 µg/ml rat liver Golgi
membranes, 5 mg/ml clathrin-depleted adrenal cytosol, and 100
µM GTPS was incubated at 37 °C for 15 min, and
the Golgi membranes were recovered. Following resuspension in buffer C,
equal aliquots were removed and incubated with increasing
concentrations of trypsin and Triton X-100 as indicated in the figure.
After digestion at 37 °C for 10 min, the samples were analyzed by
immunoblotting with mAb 100/1 (panel A), AE/1 (panel
B), and anti-
-mannosidase II (panel C). Note that
the
68-kDa doublet recognized by the anti-
subunit antibodies
represents endogenous proteolytic degradation
products.
To ascertain whether the AP-1 appendages,
the core, or both remained membrane-associated, the Golgi fraction was
sedimented following digestion, and the resulting supernatant and
pellet fractions were analyzed (Fig. 3). On untreated membranes
or membranes that contained excess trypsin inhibitor prior to addition
of trypsin, the recruited AP-1 remained membrane-associated during the
10-min digestion at 37 °C (lanes b and h). This
is in contrast to endogenous Golgi-associated AP-1, which dissociates
from the membranes on incubation at 37 °C(26) . The
stability of the recruited exogenous AP-1 is probably a consequence of
the GTPS, which we believe traps the AP-1 irreversibly on the
membranes. In the presence of low concentrations of trypsin, the bulk
of both the generated
1 trunk and the undigested intact
subunit pelleted with the Golgi membranes (Fig. 3, panels A and B, lane d). However, all of the
40-kDa
1 subunit appendage fragment formed was observed exclusively in
the supernatant fractions (panel A, lane c). The
40-kDa adrenal
1 subunit appendage fragment is smaller than
the
44-kDa brain AP-1
1 subunit appendage (14) since
a 14-amino acid neuron-specific insert is absent(7) . At higher
trypsin concentrations, all the detectable 32-kDa
subunit
appendage fragment generated was also found only in the supernatants (panel B, lane e).
Figure 3:
Membrane association of the AP-1 core
fragment following tryptic digestion. AP-1-containing Golgi membranes
were prepared with adrenal cytosol and GTPS and then incubated
with 0 (lanes a and b), 10 (lanes c and d), or 50 (lanes e and f) µg/ml trypsin
or excess soybean trypsin inhibitor and 50 µg/ml trypsin (lanes
g and h) at 37 °C for 10 min. Reactions were
terminated on ice, excess trypsin inhibitor was added, and the Golgi
pellet and supernatant fractions were obtained by centrifugation. Equal
volumes of each supernatant (S, lanes a, c, e, and g) and pellet (P, lanes b, d, f, and h) fractions were analyzed by
immunoblotting with either a mixture of anti-
subunit mAb 100/1
and GD/1 (panel A) or anti-
subunit AE/1 (panel
B).
Figure 4:
Recruitment of purified coated
vesicle-derived AP-1 or AP-1 tryptic fragments onto Golgi membranes.
Tubes containing either 5 mg/ml clathrin-depleted (Control)
adrenal cytosol (lanes b and c) or 5 mg/ml clathrin-
and AP-1-depleted (Depleted) adrenal cytosol (lanes d and e) or 5 mg/ml depleted cytosol plus 20 µg/ml
purified AP-1 (lanes f and g) or 5 mg/ml depleted
cytosol plus 20 µg/ml truncated AP-1 with appendageless 1
subunits (lanes h-k) were prepared together with 20
µg/ml Golgi membranes and 50 µg/ml BFA as indicated in the
figure. All the tubes contained 100 µM GTP
S, added
either at the beginning of the assay, or, when BFA was present, after
10 min at 37 °C. Reactions were terminated after 20 min at 37
°C, and the Golgi membrane pellets were recovered by
centrifugation. Aliquots corresponding to 1/80 of each supernatant (panels A and B) and 1/2 of each pellet (panels
C-F) fraction were analyzed on duplicate immunoblots using
either anti-
subunit mAb 100/1 (panels A and C)
or GD/1 (panel E) or anti-
subunit mAb 100/3 (panels
B and D) or AE/1 (panel
F).
From a comparison of the
supernatant fractions from incubations containing either control or
AP-1-depleted cytosol, it is apparent that the immunodepletion had
quantitatively removed AP-1 (Fig. 4, panels A and B, lanes b and c compared to lanes d and e). Consequently, no recruitment of adrenal AP-1, as
judged by the presence of an exogenous subunit, was evident in
the pellet fractions (panel D, lane e) of incubations
containing the depleted adrenal cytosol. However, when probed with
either mAb 100/1 (panel C) or GD/1 (panel E), two
closely migrating
-type subunits were observed on the Golgi
membranes incubated with the AP-1-depleted cytosol and GTP
S (panels C and E, lane e). The slower
migrating
form corresponds to the
1 subunit of residual
endogenous rat liver Golgi-associated AP-1, which has not completely
dissociated from the membrane during the course of the
assay(26) . The partner
subunit can be seen in panel
F (lanes a and e). Because this
subunit
was detected with the AE/1 antibody (panel F) but not the
species-specific mAb 100/3 (panel D), this membrane-associated
AP-1 must be of rat origin. Note also that more of this endogenous AP-1
was observed on the membranes incubated together with the AP-1-depleted
adrenal cytosol than on the Golgi membranes incubated without cytosol (panels C, E, and F, lane e compared to lane a). This demonstrates that in the
presence of AP-1-depleted cytosol, the endogenous rat AP-1 that had
dissociated from the Golgi can rebind to the membranes in an
ARF-dependent manner. The lower
-type band remains to be
identified definitively. Additional experiments have shown that no
subunit had been recruited, excluding the possibility that the
lower band was a
2 subunit. The unidentified band might be a
degradation product of the endogenous Golgi-associated
1 subunit
or, less likely, a free monomeric
subunit.
When purified
coated vesicle-derived AP-1 was added into the depleted cytosol to
levels approximating 1 to 5 times the estimated endogenous
concentration, the level of exogenous AP-1 recruited onto the membrane
was similar to that of the cytosolic form of AP-1 (Fig. 4, panels C and D, lane g compared to lane
c). In both cases, however, the majority of the AP-1 adaptors
remained in the supernatants (panels A and B), which
is consistent with our previous observation that another cytosolic
component limits the extent of AP-1 recruitment(26) . When the
truncated AP-1 tryptic fragment, lacking the 1 subunit appendage,
was added to the depleted cytosol together with GTP
S, the
resulting Golgi membranes showed that the
60-kDa
1 trunk was
membrane-associated (panel C, lane i). On a duplicate
blot probed either with mAb 100/3 (panel D) or AE/1 (panel
F), the intact
subunit was also seen to be membrane-bound (lane i), indicating that the tryptic
1 fragment was
indeed part of a heterotetrameric complex. Again, the bulk of the added
fragments remained in the supernatant fraction (panels A and C). BFA effectively antagonized the translocation of the
1-appendageless core in the presence of GTP
S (panels
C-F, lane k). Analysis of the blots with GD/1, an
antiserum raised against a conserved peptide epitope in the
subunit hinge, that recognizes the
1 appendage (Fig. 3),
revealed that the free
1 subunit appendage had not been recruited
onto the membrane (panel E, lane i). Thus, the
translocation of AP-1 onto the TGN appeared to proceed normally in the
absence of the
1 subunit head and hinge regions.
Figure 5:
Subcellular localization of recruited
exogenous AP-1 and tryptic core fragment. Permeabilized NIH 3T3 cells
were incubated with rat liver cytosol supplemented with purified
adrenal AP-1 (a and b), purified adrenal AP-1 and 100
µM GTPS (c and d), or
1
subunit appendageless adrenal AP-1 plus 100 µM GTP
S (e and f) at 37 °C for 15 min. The cells were
then prepared for immunofluorescence analysis using polyclonal
anti-clathrin light chain (lanes a, c, and e) and anti-
subunit mAb 100/3 (b, d,
and f) antibodies.
The effect of removing the 1 appendage
on the binding of the exogenous AP-1 complex added to rat liver cytosol
with GTP
S is shown in Fig. 5f. Strong perinuclear
staining was observed, and the recruitment of the trypsinized AP-1
complex was indistinguishable from the intact coated vesicle-derived
AP-1. Taken together, our data indicate that tryptic removal of the
1 subunit appendage had no effect on AP-1 recruitment onto the
Golgi membrane fraction.
Having established that the globular
carboxyl-terminal and intact hinge regions of the 1 subunit were
not required for TGN recruitment, we next examined any role played by
the
subunit appendage in this process. A tryptic AP-1 core
fragment, lacking both
1 and
subunit appendages, was
generated by trypsinization and removal of remaining intact
subunit-containing adaptors by immunoadsorption on mAb 100/3 and then
added to the AP-1-depleted cytosol. The appendageless AP-1 core
prepared in this way remains as an assembled
heterotetramer(14) , but since removal of the
subunit
appendage abolishes reactivity with both mAb 100/3 and AE/1, we were
only able to follow the fate of the
subunit trunks. Nevertheless,
our experiments showed that the appendageless AP-1 core was recruited
onto the Golgi membranes as efficiently as the intact AP-1 complex (Fig. 6, panel B, lane g compared to lane
c). Again, recruitment of the core fragment was inhibited by BFA (panel B, lane i). Similar levels of recruitment of
the different cores were observed over a range of concentrations ( Fig. 4and Fig. 6), so major differences in affinity
between intact AP-1 and the core fragments appears unlikely. Taken
together, these experiments show that neither appendage domain is
essential for either recruitment or membrane association in the
presence of GTP
S. These results are also consistent with the
behavior of the endogenously generated proteolytic degradation products
found in our adrenal cytosol preparations. Endogenous core fragments
lacking portions of the
subunit appendage or the
1 subunit
appendage were seen to associate with Golgi membranes in the
experiments presented in Fig. 1, Fig. 2, and Fig. 3.
Figure 6:
Recruitment of appendageless AP-1 core
complex onto Golgi membranes. Tubes containing either 5 mg/ml
clathrin-depleted (Control) adrenal cytosol (lanes b and c), 5 mg/ml clathrin- and AP-1-depleted (Depleted) adrenal cytosol (lanes d and e),
or 5 mg/ml depleted cytosol plus 5 µg/ml truncated adrenal AP-1
lacking both 1 and
appendages (lanes f-i)
were prepared together with 20 µg/ml Golgi membranes and 50
µg/ml BFA as indicated in the figure. All the tubes contained 100
µM GTP
S, added either at the beginning of the assay,
or, when BFA was present, after 10 min at 37 °C. Reactions were
terminated after 20 min at 37 °C, and the Golgi membrane pellets
were recovered by centrifugation. Aliquots corresponding to 1/80 of
each supernatant (panel A) and 1/2 of each pellet (panel
B) fraction were analyzed on immunoblots using anti-
subunit
mAb 100/1.
Figure 7:
Recruitment of cytosolic clathrin onto
Golgi membranes. Tubes containing 5 mg/ml whole rat liver (Control) cytosol (lanes b, c, e,
and f) or 5 mg/ml clathrin-depleted rat liver (Depleted) cytosol (lanes h, i, k,
and l) were prepared together with 50 µg/ml Golgi
membranes and 50 µg/ml BFA as indicated in the figure. All the
tubes contained 100 µM GTPS, added either at the
beginning of the assay, or, when BFA was present, after 10 min at 37
°C. Reactions were terminated after 20 min, and the Golgi membrane
pellets were recovered by centrifugation. Aliquots corresponding to
1/80 of each supernatant (panel A) and 1/2 of each pellet (panel B) fraction were analyzed on blots using either a
mixture of anti-clathrin mAb TD.1 and anti-
subunit mAb 100/1 (panels A and B, upper portion) or
[
-
P]GTP overlay (panel B, lower portion).
Controlled proteolysis of the Golgi membrane-AP-1-clathrin complex
is shown in Fig. 8. To obviate the problem of background
clathrin, clathrin-depleted cytosol was used to generate the
membrane-associated complex. As we have shown above for the
Golgi-associated adrenal AP-1, 2 µg/ml trypsin similarly cleaved
the rat liver AP-1 1 subunit into the
60-kDa trunk fragment
while leaving the
subunit intact (panels A and B, lane d). The quantitative recovery of the clathrin
heavy chain in the supernatant fraction under these conditions (panel A, lane c) demonstrates that the
clathrin-membrane association is dependent on an intact
1 subunit.
Proteolytic degradation of the
subunit required higher
concentrations of trypsin (panel B, lane e) and was
therefore not correlated with release of clathrin from the membrane.
Figure 8:
Dissociation of membrane-bound clathrin
following tryptic removal of the 1 subunit appendage. Clathrin-
and AP-1-containing Golgi membranes were prepared using
clathrin-depleted rat liver cytosol and GTP
S and incubated with 0
µg/ml (lanes a and b), 2 µg/ml (lanes c and d), 10 µg/ml (lanes e and f)
trypsin, or excess soybean trypsin inhibitor and 10 mg/ml trypsin (lanes g and h) at 37 °C for 10 min. Reactions
were terminated on ice, excess trypsin inhibitor was added, and the
Golgi pellet and supernatant fractions were obtained by centrifugation.
Equal volumes of each supernatant (S, lanes a, c, e, and g) and pellet (P, lanes b, d, f, and h) fractions
were analyzed by immunoblotting with either a mixture of anti-clathrin
mAb TD.1 and anti-
subunit mAb 100/1 (panel A) or
anti-
subunit AE/1 (panel B).
Finally, we found that prior tryptic removal of the recruited 1
subunit appendage inhibited subsequent clathrin binding in a two-step
assay (Fig. 9). AP-1 was first recruited onto Golgi membranes
from clathrin-depleted rat liver cytosol in the presence of GTP
S.
The membranes were recovered, trypsinized, and then added to a second
incubation containing a cytosolic clathrin-enriched fraction. Any
subsequent ARF and AP-1 recruitment was prevented in the second step
incubations by the addition of 20 µM BFA(40) .
Under these conditions, recruitment of cytosolic clathrin was strictly
dependent on the presence of Golgi-associated AP-1 (Fig. 9, panel A, lane d compared to lane a).
Controlled tryptic removal of the
1 subunit appendage was
correlated with an inability to accumulate clathrin on the Golgi
membranes during the second step incubation (panel A, lane
g). Only background levels of clathrin (lanes a, c, and f) were observed. No degradation of the
subunit was observed under these conditions (panel B),
illustrating that a Golgi-associated AP-1 heterotetramer, lacking only
the
1 head and part of the hinge domain, was unable to recruit
clathrin from a soluble pool.
Figure 9:
Clathrin recruitment is prevented
following tryptic removal of the 1 subunit appendage domain of
Golgi-associated AP-1. AP-1-containing Golgi membranes were prepared
using clathrin-depleted rat liver cytosol and GTP
S, recovered by
centrifugation and then incubated at 37 °C with either excess
soybean trypsin inhibitor and 2 µg/ml trypsin (lanes
b-d) or 2 µg/ml trypsin (lanes e-g).
After 10 min, the membranes were again recovered and then used for the
second step reaction. Tubes containing untreated Golgi (lane
a), mock-digested Golgi (lanes b and d), or
trypsinized Golgi (lanes e and g) membranes and 0.1
mg/ml Sepharose 4B clathrin-enriched fraction (lanes a, c, d, f, and g) were prepared on
ice. 20 µg/ml BFA was added to each tube followed by incubation at
37 °C for 15 min. The membranes were pelleted and resolved on
duplicate 12.5% SDS-polyacrylamide gels, transferred to nitrocellulose,
and analyzed by immunoblotting with a mixture of anti-clathrin mAb TD.1
and anti-
subunit mAb 100/1 (panel A) or anti-
subunit AE/1 (panel B).
Adaptors are thought to play a pivotal role in the assembly
of clathrin-coated vesicles by determining the intracellular site of
coat nucleation and by promoting assembly of the growing lattice. To
perform this regulatory role, recruited cytosolic adaptors must
establish multiple contacts with components within the membrane and
with the cytosolic clathrin, and it is likely that this is the
underlying reason for the structural complexity of the adaptor
heterotetramer. In this study, we have delineated the regions required
for the ARF-dependent membrane association of AP-1 and the subsequent
recruitment of cytosolic clathrin triskelia onto the TGN. We found that
an AP-1 adaptor core, lacking both the 1 and
subunit
appendages, was fully competent to bind to Golgi membranes. Binding of
the cores was dependent on ARF
GTP and strongly inhibited by BFA,
as also observed for intact AP-1 complex(25, 26) .
This suggests that the structural determinants required for the
ARF-dependent recruitment of AP-1 onto the Golgi membranes reside
within the core fragment.
While this work was in progress, the
results of a study examining the intracellular distribution of chimeric
adaptor complexes in which the and
subunit heads were
either switched or deleted were published(29) .
Immunofluorescence analysis of cells transfected with these various
constructs showed that the chimeric or headless adaptors associated
with the appropriate intracellular membranes in spite of the
modifications. It was concluded that the
subunit appendage has,
at most, a passive role in determining the intracellular targeting of
AP-1 (29) . Our work now provides independent biochemical
confirmation of this conclusion and, in addition, establishes that the
appendage of the
1 subunit is not required for the ARF-dependent
membrane association of AP-1.
Although we have not yet established
whether GTPS inhibits the complete assembly of a coated vesicle or
the budding process, clearly the analog does not interfere with the
recruitment of cytosolic clathrin triskelia. Our observation that an
intact
1 subunit is required for this recruitment of clathrin is a
strong argument for the selective association of this subunit with
clathrin. This idea was initially proposed when it was determined that
the
-type subunits of AP-1 and AP-2 were highly related, both by
peptide mapping and immunological criteria (5) and by
sequencing(6, 7, 8) . Our data are also
consistent with the finding that under conditions that reduce
aggregation of adaptors, cleavage of the
1 subunit in the hinge
region was sufficient to release the truncated AP-1 core, together with
the appendage, from purified coated vesicles(14) . Furthermore,
a
1-like subunit, purified from bacteria expressing the
recombinant protein, induces clathrin coat assembly in
vitro(16) . Together, these observations all implicate the
1 subunit in clathrin association.
The 2 subunit of AP-2
also interacts directly with clathrin (14, 15, 16) . Weaker interactions between
the AP-2 adaptor core and clathrin cages have been reported, but these
interactions failed to induce clathrin
assembly(11, 12, 13, 14) . The
subunit of AP-2 also appears to bind to
clathrin(41, 42) , and it has been proposed that this
is the first contact that is established between these two
molecules(41) . By contrast, we found that clathrin could not
be recruited onto Golgi-associated AP-1 lacking only the
1
appendage. Therefore, if the
subunit of AP-1 binds to cytosolic
clathrin triskelia first, the affinity of this initial interaction is
not sufficient to retain clathrin on the TGN in the absence of an
intact
1 subunit. Thus, while it is possible that the adaptor
cores contain a second clathrin binding site, which is independent of
the one that involves the
appendage, it seems to play a secondary
role in the early recruitment steps of clathrin onto TGN-associated
AP-1.
Our data on the requirement of an intact 1 subunit for
the recruitment of clathrin differ from the results of a recent study
showing that clathrin could assemble onto elastase-generated, plasma
membrane-associated, AP-2 cores(43) . The most obvious
difference between the two systems is the adaptor population being
studied. AP-1 and AP-2 adaptors might utilize different domains for
clathrin recruitment. However, given the high degree of sequence and
structural homology between the
1,
2, and
1-like
subunits(5, 6, 7, 8) , this appears
unlikely. The differences may rather be related to the fact that, under
our experimental conditions, we have analyzed de novo formation of a coated bud on the TGN, while the AP-2 binding
system is more likely to reflect rebinding of clathrin to pre-existing
coated pit structures that had been previously disrupted by
nonphysiological manipulation of pH and salt conditions. This makes
these two systems difficult to compare.
At present, the available
evidence supports the notion that AP-1 initially associates with the
Golgi by binding to a TGN-specific docking protein in an ARF-regulated
manner(23, 24, 25, 26) . The results
of our controlled proteolysis experiments indicate that this
association of AP-1 with the putative docking protein occurs through
the core domain. We have also shown that the 1 subunit is involved
in clathrin recruitment. What then is the function of the
subunit
appendage? The considerable amino acid sequence divergence between the
appendages of the
,
, and
subunits has prompted the
suggestion that the appendages might encompass a receptor binding
domain(6, 7, 12, 13, 29) .
This hypothesis is appealing because the proline- and glycine-rich
hinge regions, separating the globular heads from the core complex, may
be flexible and accommodate sorting motifs located at varied positions
within the cytoplasmic domains of different
receptors(6, 7) . One attractive possibility is that
the
subunit appendage interacts with the cytoplasmic domains of
membrane receptors which are preferentially sorted into Golgi-derived
clathrin-coated vesicles. In fact, the analogous subunit of the AP-2
adaptor, the
subunit, can bind to plasma membranes after
dissociation from the heterotetrameric complex (20) . The
binding is partially inhibited by peptides corresponding to the
cytoplasmic domains of two receptors endocytosed within clathrin-coated
vesicles(20) . One interpretation of the partial nature of the
inhibition is that the
subunit recognizes both a plasma membrane
docking protein (43, 44) and sorting motifs on select
membrane proteins, and only the latter interaction can be inhibited by
receptor peptides(20) . Because AP-2 cores can compete with
intact AP-2 for plasma membrane binding(43) , it seems
reasonable to propose that like AP-1, AP-2 associates with a specific
docking protein on the plasma membrane via the core domain.
The
experiments presented here allow us to expand our model for the
association of AP-1 with the cytoplasmic surface of the
Golgi(26) . We have deduced that when AP-1 is recruited onto
the TGN, the bound adaptor would lie parallel to the plane of the
membrane, associated with the docking protein via the core domain (Fig. 10). Note that our experiments do not preclude a role for
the µ1 or the 1 subunit in the docking process. Anchored by a
docking protein, a growing adaptor-containing coat structure might trap
select TGN membrane proteins through multiple weak interactions between
the
subunit appendages and the cytoplasmic portions of the
selected proteins. These interactions would restrict the lateral
diffusion of the cargo, resulting in their concentration and
preferential inclusion within a coated pit. At some point during the
assembly of the coated vesicle, AP-1 may be released from the putative
docking protein, perhaps regulated by nucleotide hydrolysis of
ARF
GTP. Several lines of evidence, including the data shown here,
support the notion that the clathrin-AP-1 interaction would be
maintained by the
1 subunit (14, 15, 16) . Polygonally arranged clathrin
would remain membrane-associated due to multiple, cooperative low
affinity adaptor-receptor interactions, docking protein-AP-1
interactions at the growing edges of the lattice, and perhaps
adaptor-adaptor interactions, as exemplified by AP-2.
Figure 10:
Schematic representation of the
AP-1-clathrin complex assembled on the TGN. A possible orientation for
AP-1 and clathrin relative to the TGN membrane is shown. AP-1 membrane
association is ARF-dependent and occurs by the interaction of the core
domain of the adaptor complex with the proposed docking protein.
Clathrin recruitment follows and requires an intact 1 subunit. For
clarity, the orientation of the
1 subunit is shown to indicate the
interaction with clathrin. Our data do not preclude that the
subunit trunk might also play a role in AP-1 membrane association. The
µ1 and
1 subunits are positioned within the AP-1 core,
reflecting the resistance of these subunits to proteolysis. A sorting
motif within the cytoplasmic domain of a receptor to be included in the
growing coated bud is indicated in black. Proteins are not
drawn to scale, and although the putative docking protein is drawn as a
transmembrane protein, no direct evidence for this is available at
present.
Upon budding and removal of the clathrin coat by the uncoating ATPase Hsc70(3, 45, 46) , the interaction between individual adaptors and receptors may not be sufficiently strong to maintain adaptors on the membrane. If this were the case, upon removal of the clathrin, the remaining coat components would be released from the budded vesicle surface without necessitating additional uncoating factors. However, the multivalency of the proposed adaptor-membrane association may result in only slow release of the adaptors (47, 48) and could be regulated by specific cytoplasmic factors(49) . We have not yet examined what requirements the clathrin has to meet in order to be recruited by membrane-bound AP-1 and if there is any difference between cytosolic and coated vesicle-derived clathrin as has been observed for AP-2-mediated clathrin recruitment(50) . These and other questions concerning the role of light chains and clathrin domains can now be studied using our membrane binding assay.