RESEARCH ARTICLE
353
Expression of auxilin or AP180 inhibits endocytosis
by mislocalizing clathrin: evidence for formation of
nascent pits containing AP1 or AP2 but not clathrin
Xiaohong Zhao1, Tsvika Greener1, Hadi Al-Hasani2, Samuel W Cushman2, Evan Eisenberg1 and
Lois E. Greene1,*
1Laboratory
of Cell Biology, NHLBI and 2Experimental Diabetes, Metabolism and Nutrition Section, NIDDK, NIH, Bethesda, MD, USA
*Author for correspondence (e-mail: [email protected])
Accepted 27 October 2000
Journal of Cell Science 114, 353-365 © The Company of Biologists Ltd
SUMMARY
Although uncoating of clathrin-coated vesicles is a key
event in clathrin-mediated endocytosis it is unclear what
prevents uncoating of clathrin-coated pits before they pinch
off to become clathrin-coated vesicles. We have shown that
the J-domain proteins auxilin and GAK are required for
uncoating by Hsc70 in vitro. In the present study, we
expressed auxilin in cultured cells to determine if this
would block endocytosis by causing premature uncoating
of clathrin-coated pits. We found that expression of auxilin
indeed inhibited endocytosis. However, expression of
auxilin with its J-domain mutated so that it no longer
interacted with Hsc70 also inhibited endocytosis as did
expression of the clathrin-assembly protein, AP180, or its
clathrin-binding domain. Accompanying this inhibition, we
observed a marked decrease in clathrin associated with the
plasma membrane and the trans-Golgi network, which
provided us with an opportunity to determine whether the
absence of clathrin from clathrin-coated pits affected the
distribution of the clathrin assembly proteins AP1 and
AP2. Surprisingly we found almost no change in the
association of AP2 and AP1 with the plasma membrane
and the trans-Golgi network, respectively. This was
particularly obvious when auxilin or GAK was expressed
with functional J-domains since, in these cases, almost all
of the clathrin was sequestered in granules that also
contained Hsc70 and auxilin or GAK. We conclude that
expression of clathrin-binding proteins inhibits clathrinmediated endocytosis by sequestering clathrin so that it is
no longer available to bind to nascent pits but that assembly
proteins bind to these pits independently of clathrin.
INTRODUCTION
in recruiting β-adrenergic receptors to clathrin-coated pits but
unlike APs it does not induce clathrin polymerization
(Goodman et al., 1996; Goodman et al., 1997).
In addition to APs a number of other proteins have been
discovered that are involved in the formation, invagination, and
pinching off of clathrin coated vesicles including dynamin,
amphiphysin, epsin, eps15, endophilin, syndapin I and the
small GTPase protein, Rab5-GDI (Van der Bliek et al., 1993;
Takei et al., 1995; David et al., 1996; Chen et al., 1998; Tebar
et al., 1996; McLauchlan et al., 1998; Ringstad et al., 1999;
Schmidt et al., 1999; Qualmann et al., 1999). In addition, rho,
rac, phospholipids, and actin are involved in clathrin coat
assembly and receptor recruitment (Rapoport et al., 1997;
Takei et al., 1998; Lamaze et al., 1996; Lamaze et al., 1997;
Munn et al., 1995; Gaidarov et al., 1999) as is
dephosphorylation of many of the proteins involved in
formation of clathrin-coated pits (Wilde and Brodsky, 1996;
Slepnev et al., 1998). Finally, after they pinch off, the clathrincoated vesicles are uncoated in an ATP dependent process by
Hsc70 and its partner proteins, auxilin or cyclin G-associated
kinase (GAK). These partner proteins not only assemble
clathrin but also have J-domains that enable them to interact
with Hsc70 (Prasad et al., 1993; Ungewickell et al., 1995; Jiang
et al., 1997; Greener et al., 2000). Recently, Cremona et al.
During receptor-mediated endocytosis, clathrin triskelions
polymerize and form clathrin-coated pits on the plasma
membrane that then invaginate into the cell to form clathrincoated vesicles (Keen, 1990; Pearse and Robinson, 1990).
Similar clathrin-coated pits also form on the trans-Golgi
network. In addition to clathrin and receptors, these pits
contain assembly proteins (APs) that catalyze the
polymerization of clathrin triskelions and in some cases bind
the receptors localized in the clathrin-coated pits. A number of
different APs have been described. AP1, AP2, AP3 and AP4
are multimeric subunit complexes of about 270 kDa (Keen,
1990; Robinson and Kreis, 1992; Simpson et al., 1997;
Dell’Angelica et al., 1999; Hirst et al., 1999); AP1 occurs on
the trans-Golgi network, AP2 on the plasma membrane, AP3
on both the trans-Golgi membrane and endosomes, and AP4 on
perinuclear structures. AP180 (Ungewickell and Oestergaard,
1989) and auxilin (Ahle and Ungewickell, 1990) are neuronal
specific APs that consist of single subunits of 92 kDa and 100
kDa, respectively; CALM and GAK, which have recently been
described, are the non-neuronal homologs of AP180 (Dreyling
et al., 1996) and auxilin (Kanaoka et al., 1997; Greener et al.,
2000), respectively. Finally β-arrestin is specifically involved
Key words: Auxilin, AP180, Endocytosis
354
JOURNAL OF CELL SCIENCE 114 (2)
(Cremona et al., 1999) have shown that hydrolysis of PIP2 by
synaptojanin is also important for uncoating in vivo.
An important question regarding regulation of uncoating by
Hsc70 is why premature uncoating of clathrin-coated pits does
not occur before they pinch-off to form clathrin-coated
vesicles. Since the J-domain proteins auxilin and GAK are
critical for uncoating it seemed possible that the level of auxilin
or GAK present in the cell would not only affect the rate and
extent of uncoating of clathrin-coated vesicles but also whether
or not clathrin-coated pits were uncoated by Hsc70; if excess
auxilin or GAK caused premature uncoating of clathrin-coated
pits before they pinched off to form clathrin-coated vesicles, it
might markedly inhibit clathrin-mediated endocytosis. On the
other hand, quantitative western blot analysis showed that the
level of auxilin present in neuronal cells is almost 10 times the
level of GAK present in non-neuronal cells (Greener et al.,
2000), probably because recycling of synaptic vesicles requires
clathrin-mediated endocytosis to occur much more rapidly in
neuronal cells than in non-neuronal cells. Therefore, markedly
increasing the level of auxilin or GAK present in cultured cells
might actually increase the rate of uncoating of clathrin-coated
vesicles and thereby increase the rate of clathrin-mediated
endocytosis rather than inhibit it.
In the present study, we found that expression of auxilin or
GAK markedly decreased clathrin-mediated endocytosis in
HeLa and Cos cells and, at the same time, in many of the cells
led to the formation of clathrin-Hsc70-auxilin granules in the
cytosol and a decrease in clathrin associated with clathrincoated pits on the plasma membrane and the trans-Golgi
network. However, clathrin-mediated endocytosis was also
inhibited by auxilin with its J-domain mutated so that it no
longer supported uncoating by Hsc70 in vitro although in this
case the clathrin in the cytosol did not form granules but
appeared to become aggregated in the cytosol. A similar effect
occurred when AP180 or its clathrin-binding domain was
expressed. Surprisingly, however, in none of these cases was
localization of AP1 or AP2 affected despite the mislocalization
of clathrin suggesting first, that expression of clathrin-binding
proteins inhibits endocytosis by causing mislocalization of
clathrin away from nascent pits, and second, that the binding
of APs to these pits occurs independently of clathrin.
MATERIALS AND METHODS
Cell culture and transfection
HeLa and Cos cells were purchased form ATCC. Mouse
neuroblastoma N2A cells were a gift from Dr Y. Peng Loh (NICHD,
NIH). Cells were maintained in DMEM supplemented with 10%
fetal bovine serum, 2 mM glutamine, penicillin (100 unit/ml), and
streptomycin (100 unit/ml) in a humidified incubator with 5% CO2 at
37°C. All media and supplements were obtained from Biofluids, Inc.
(Rockville, MD, USA). For the purpose of immunofluorescence
studies calcium phosphate precipitation was used to transfect cell. In
order to get higher transfection efficiency in biochemical assays,
SuperFect (QIAGEN, Valencia, CA, USA) was used as transfection
reagent.
Antibodies
Monoclonal antibody M5 against Flag was obtained from Kodak
Scientific Imaging System (Rochester, NY USA). A rabbit antiserum
to Flag was from ZYMED Laboratories, Inc. (San Francisco, CA,
USA). Monoclonal anti-HA antibody (HA. 11) was purchased from
Berkeley Antibody Co. (Richmond, CA, USA). Rabbit antibody
against Golgi β coatomer (β-COP), monoclonal anti-clathrin heavy
chain (X22), monoclonal anti-α-adaptin (AP.6) are from Affinity
BioReagents, Inc. (Golden, CO, USA). Monoclonal antibody against
γ-adaptin of AP1 (100/3) was obtained from Sigma (St Louis, MO,
USA). Monoclonal mouse anti-human transferrin receptor antibody
was purchased from Biomeda Corp. (Foster City, CA, USA). A rabbit
antiserum to human transferrin was obtained from Boehringer
Mannhein (Indianapolis, IN, USA). Monoclonal and polyclonal
antibodies against Hsc70 were purchased from Stressgen
Biotechnologies Corp. (Victoria, BC, Canada). Fluorescenceconjugated
secondary
antibodies
were
from
Jackson
ImmunoResearch laboratories, Inc. (West Grove, PA, USA). 125Isheep anti-mouse antiserum was from Amersham Pharmacia Biotech
(Piscataway, NJ, USA).
Plasmid construction
Auxilin and AP180 and their truncated mutants were prepared as Flag
fusion proteins (Fig. 1A-B) using a pFlag-CMV-2 expression vector
from Kodak Scientific Imaging System (Rochester, NY, USA).
Auxilin and AP180 cDNA were subcloned to give pTG176 and
pTG135 expressing wild-type AP180 and wild-type auxilin,
respectively. This wild-type construct of auxilin was later used to
make pTG177 expressing mutated auxilin contains a non-active Jdomain, where the HPDK conserved motif was changed to AAAK.
N- and C-terminal fragments of auxilin were made as follows. The
first 1,224 base pairs of auxilin cDNA were subcloned to give pTG168
expressing the 45 kDa N-terminal fragment of auxilin which contains
the tensin domain. The last 1,521 base pairs of auxilin cDNA were
subcloned to give pTG197 expressing the 56 kDa C-terminal fragment
of auxilin which contains the clathrin binding and J-domains. N- and
C-terminal fragments of AP180 were made as follows. The first 1,674
base pairs of AP180 cDNA were subcloned to give pTG190
expressing the 61 kDa N-terminal domain of AP180 containing the
domain which interacts with phospholipase D. The last 1,791 base
pairs of AP180 cDNA were subcloned to give pTG192 expressing the
65 kDa C-terminal domain of AP180 containing its clathrin binding
domain (Lee et al., 1997). GAK was subcloned into GFP vector
(Kioka et al., 1999) with the epitope tag at the N-terminal of GAK.
Endocytosis assays
For immunofluorescence microscopy studies, cells were grown on
glass coverslips and transfected 24-48 hours before the assay. Cells
were washed three times with PBS and incubated with DMEM
containing 0. 5% BSA for 30 minutes at 37°C. Human transferrin was
then added to the media at the final concentration of 30 µg/ml.
Incubation was continued at 37°C for 5 to 10 minutes. To measure
bulk fluid-phase endocytosis, 1 mg/ml lysine-fixable FITC-dextran
(70,000) from Molecular Probes was added to the cells in DMEM
containing 0.5% BSA for 1 hour at 37°C. Cells were washed quickly
with PBS, fixed in 2% formaldehyde and processed for
immunofluorescence microscopy.
To measured transferrin internalization biochemically, a modified
biotinylated transferrin uptake assay (Smythe et al., 1992) was used.
Briefly, Cos cells grown in 6-well plates were first depleted of
endogenous transferrin. Biotinylated transferrin was then added and
incubated with cells for minutes. After removing free biotinylated
transferrin by washing, avidin was added to the plates to mask surfacebound biotinylated transferrin. Internalized biotin activity in cell
lysates were then assayed quantitatively using streptavidinhorseradish peroxidase in an ELISA plate coated with a rabbit
antibody against transferrin. Experiments were performed in
triplicate.
Immunofluorescence microscopy
Cells were treated as indicated in each experiment, fixed in 2%
Expression of auxilin or AP180 inhibits endocytosis
355
Fig. 1. Auxilin and AP180 DNA constructs
used in the study. (A) Auxilin; (B) AP180.
All constructs are N-terminal Flag-tagged.
formaldehyde at room temperature for 15
minutes. After washing the cells three time
with PBS containing 10% FBS, cells
were incubated with primary antibodies for
1 hour at room temperature. Cells were
washed again three times and incubated
with fluorescence-conjugated secondary
antibodies.
GLUT4 glucose transporter
endocytosis
Rat adipose cells from male rats were
prepared and GLUT4 endocytosis assays
were conducted as previously described (AlHasani et al., 1998). Briefly, cells were
transfected by electroporation with HAGLUT4 alone or cotransfected with HAGLUT4 and Flag-AP180, auxilin or their
mutants as indicated. Cell surface GLUT4 in
absence or presence of insulin (1×104
microunits/ml) were measured by the
binding of monoclonal anti-HA antibody to
cell surface HA-GLUT4 followed by the
addition of 125I-sheep anti-mouse antibody.
Cell surface associated radioactivity was
counted in a γ-counter. Unless stated
otherwise, the values obtained from
transfected cells were subtracted from all
other values to correct nonspecific antibody binding. Antibody
binding assays were routinely performed in duplicate, but
occasionally were done in quadruplicate.
RESULTS
Effect of auxilin on transferrin endocytosis
To determine the effect of expression of auxilin in cultured
cells, we first expressed auxilin with a N-terminal Flag epitope
in Cos and HeLa cells. Fig. 2A shows the fluorescence
obtained when Cos cells were stained with an anti-Flag
antibody to detect the expressed auxilin. Transferrin uptake in
the same cells was also imaged by immunofluorescence
microscopy as shown in Fig. 2B. Comparison of Fig. 2A and
B shows that, in the non-transfected cells, the transferrin was
mainly localized to the recycling endosome, whereas the cells
expressing auxilin showed marked inhibition of transferrin
uptake. Likewise, in HeLa cells, expression of auxilin
markedly inhibited transferrin uptake (Fig. 2C,D).
Quantification of this effect (Table 1) showed that 10% of the
control cells had little or no transferrin uptake compared to
65% of the transfected cells. Interestingly, while in both
transfected HeLa and Cos cells the expressed auxilin was
cytosolic, its distribution did not appear to be uniform.
Although the expressed auxilin varied from a grainy
appearance to obvious speckles which ranged in size from tiny
particles to large granules, its cellular appearance did not seem
to be related to the inhibition of transferrin uptake. In contrast,
the uptake of FITC-dextran, a marker for bulk fluid-phase
endocytosis, was comparable in auxilin transfected and control
cells (Fig. 2E,F). These results establish that expression of
auxilin specifically affects clathrin-dependent receptor
mediated endocytosis in transfected cells, but not clathrinindependent fluid phase uptake.
To further verify that auxilin inhibits transferrin uptake, we
Table 1. Percentage of cells with reduced transferrin uptake
Expressed protein
Cells
HeLa
Cos
None
Auxilin
Auxilin-C
Auxilin-N
J-domain
auxilin mutant
AP180
AP180-C
AP180-N
10
(n=199)
10
(n=151)
69
(n=86)
67
(n=39)
50
(n=195)
55
(n=87)
19
(n=203)
7
(n=41)
65
(n=57)
66
(n=70)
94
(n=112)
98
(n=56)
91
(n=160)
98
(n=48)
22
(n=117)
13
(n=77)
Transfection, transferrin uptake and immunofluorescence microscopy were performed on cells as described in Materials and Methods. The total number of
transfected cells on the slides were counted as well as the number of transfected cells that showed significantly reduced transferrin uptake. The results are
expressed as the percentage of transfected cells that displayed reduced transferrin internalization. About 10% of control cells show very little uptake of transferrin
in a typical experiment under our experimental conditions.
356
JOURNAL OF CELL SCIENCE 114 (2)
Fig. 2. Effect of auxilin expression on
transferrin and fluid phase uptake. Cells
grown on coverslips were transiently
transfected with Flag-tagged auxilin (A-F),
auxilin C-terminal fragment (G,H), or full
length auxilin with a nonfunctional Jdomain mutant (I,J). Transferrin uptake
assay and fluid phase uptake using FITClabeled dextran were performed as
described in Materials and Methods.
(A,C,E,G,I) indicate transfected cells which
was detected using mAb M5 anti-Flag
antibody. (B,D,H,J) Transferrin
internalization; (F) FITC-labeled dextran.
(A,B), Cos cells; (C-J), HeLa cells.
biochemically compared the uptake of
biotinylated transferrin in mock and
auxilin transfected Cos cells. Using Cos
cells in which about 30% of the
population was transfected with auxilin,
we found that these cells took up
about 30% less transferrin than the
mock-transfected cells in good
agreement with the 30% transfection
efficiency (data not shown). Therefore,
both biochemical and fluorescence
microscopy studies showed that
transient expression of auxilin markedly
inhibits clathrin-mediated endocytosis.
Since auxilin contains three domains,
we next investigated which of these
domains is required for inhibition of
endocytosis. We expressed either the Nterminal tensin domain, the C-terminal
portion of auxilin lacking the tensin
domain but containing the clathrinbinding domain and the J-domain, or
full-length auxilin with the critical
residues, HPDK, of the J-domain (Sell
et al., 1990; Tsai and Douglas, 1996)
mutated to AAAK, so that, in vitro, we
found that the mutated auxilin no longer
supported uncoating (data not shown).
As shown in Table 1, expression of the
N-terminal tensin domain of auxilin in
both HeLa and Cos cells had no
significant effect on transferrin uptake,
whereas, the C-terminal portion of
auxilin, which acts like intact auxilin in
vitro in uncoating clathrin coated
vesicles, also acted like intact auxilin in
vivo, inhibiting endocytosis and
forming auxilin granules in the cytosol
(Fig. 2G,H). We next tested whether the
expressed auxilin had to interact with
Hsc70 in order to inhibit endocytosis by
expressing auxilin with its J-domain
mutated so that it could no longer
interact with Hsc70 in vitro. Unexpectedly, we found that
expression of this mutated auxilin inhibited transferrin uptake
just like expression of intact auxilin (Fig. 2I,J; Table 1), raising
the possibility that inhibition of endocytosis by expression of
Expression of auxilin or AP180 inhibits endocytosis
357
Fig. 3. Effect of AP180 expression on
transferrin and fluid phase uptake.
Transferrein internalization was
measured in Cos cells (A,B) and HeLa
cells (C-D) transfected with AP180.
(A,C) Transfected cells; (B,D)
transferrin internalization. (E,F) Fluid
phase uptake of HeLa cells transfected
with AP180 stained for AP180(E) or
FITC-labeled dextran (F).
(G,H) Transferrin receptor distribution
in HeLa cells expressing AP180.
(G) Transfected cells using a rabbit
polyclonal anti Flag antibody;
(H) transferrin receptor localization.
(I,J) Transferrin internalization in
AP180 transfected N2A cells.
(I) Transfected cells; (J) transferrin
internalization.
auxilin is not related to its
involvement in uncoating of clathrin
but rather to its activity as an
assembly protein. Interestingly,
however, in contrast to what is
observed with expression of intact
auxilin or the C-terminal portion of
auxilin with an intact J-domain,
there
was
a
morphological
difference in that none of the cells
expressing auxilin with a mutated Jdomain showed formation of auxilin
granules.
Effect of AP180 on transferrin
endocytosis
The observation that expression
of auxilin inhibits endocytosis is
intriguing because expression of
numerous other intact proteins
involved in endocytosis such as
Eps15, dynamin, amphiphysin, rho,
rac, and β-arrestin do not inhibit
endocytosis (Benmerah et al., 1998;
Damke et al., 1994; Lamaze et al.,
1996; Goodman et al., 1996; Wigge
et al., 1997); endocytosis is inhibited
only by expression of domains or
mutants of these proteins that
interfere with the function of the
parent proteins (Damke et al., 1994;
Wigge et al, 1997; Benmerah et
al., 1998; Benmerah et al., 1999;
Owen et al., 1999; Nesterov et
al., 1999). It therefore seems possible
that expression of auxilin inhibits
endocytosis by overwhelming the
regulatory mechanisms in place to
prevent inappropriate polymerization
of clathrin in the cytosol. If so, inhibition of endocytosis by
expression of auxilin may be a general phenomenon that not only
occurs with auxilin but with other clathrin-binding proteins as
well. To investigate this question we determined whether
endocytosis is inhibited by expression of the nerve-specific
AP180, which, like auxilin, is monomeric.
JOURNAL OF CELL SCIENCE 114 (2)
Fig. 4. GLUT4 endocytosis in transfected cells. Primary
culture of rat adipocytes were co-transfected with HA-tagged
GLUT4 and various DNA constructs of Flag-tagged AP180,
auxilin, their fragments and mutant as indicated. Noted as G4,
control cells were transfected with HA-GLUT4 only. Cell
surface GLUT4 in absence (basal) and presence of insulin
were measured using mAb anti-HA antibody followed by the
incubation with 125I-sheep anti-mouse antibody.
Cell Surface HA-Glut4 (% max. control)
358
120
100
As we observed with expression of auxilin, expression of
AP180 markedly inhibited transferrin uptake in both Cos (Fig.
3A,B) and HeLa cells (Fig. 3C,D), although like auxilin with
a mutated J-domain, the expressed AP180 did not form
granules. Quantification of the inhibition of transferrin uptake
(Table 1) showed that expression of AP180 reduced transferrin
uptake in about 95% of the transfected population of Cos and
HeLa cells, which shows that regardless of the level of
expression of AP180, it causes a marked reduction in clathrin
mediated endocytosis. This is a greater inhibition of transferrin
uptake than we observed with auxilin and, in agreement with
this observation, we found that in the transfected Cos cells
much of the transferrin was localized on the plasma membrane
(Fig. 3A,B), an effect that we did not observe with expression
of auxilin. Similarly, expression of AP180 in HeLa cells may
also have caused much of the transferrin to accumulate on the
plasma membrane as shown by the diffuse staining of the
transferrin in the transfected cells (Fig. 3C,D). By acid washing
the cells briefly in 0.5% acetic acid/0.5 M NaCl, pH 2.4, to
remove cell surface-bound transferrin, the transferrin
associated with the AP180 transfected HeLa cells was
completely removed (data not shown). This establishes that the
transferrin is associated with the plasma membrane. Similar to
the results obtained in cells expressing auxilin, the expression
of AP180 was specific to clathrin-mediated endocytosis since
fluid phase uptake, as measured by uptake of FITC-dextran,
was unaffected by expression of AP180 (Fig. 3E,F).
The association of transferrin with the plasma membrane of
the AP180 transfected cells predicts that there should be an
increase in transferrin receptor on the plasma membrane in
transfected cells expressing AP180. Fig. 3G,H show that
the transferrin receptors in non-transfected HeLa cells are
localized to coated pits and endosomal compartments, while in
the AP180 transfected cells much of the receptor appears to be
localized diffusely on the plasma membrane. Therefore, our
data strongly suggest that expression of AP180 is inhibiting
internalization of the transferrin receptor rather than a later step
in endocytosis.
Since neither Cos nor HeLa cells normally express AP180,
we carried out a similar experiment using mouse neuro-2A
cells after first demonstrating by western blot analysis, using
an antibody specific for AP180, that these cells indeed
express AP180 (data not shown). As we observed for Cos and
HeLa cells, the transfected neuro-2A cells expressing AP180
showed markedly reduced transferrin uptake (Fig. 3I,J).
Western blot experiments showed that the transfected neuro2A cells produced, after correcting for transfection efficiency,
80
60
40
20
0
G4
Auxilin-N
Auxilin
Auxilin-C
Basal
AP180-N
AP180
AP180-C
Insulin
about 20-fold more AP180 than normal neuro-2A cells (data
not shown). Therefore, even in cells that normally express
AP180, over-expression of this protein markedly inhibits
transferrin uptake.
We next examined whether it is, in fact, the clathrin binding
domain of AP180 that is causing inhibition of transferrin
uptake or whether this inhibition is due to the ability of AP180
to inhibit phospholipase D activity (Lee et al., 1997). The
latter activity is localized to the N-terminal fragment of AP180
(Lee et al., 1997), while the C-terminal fragment has the
clathrin assembly activity (Ye and Lafer, 1995). Like
expression of intact AP180, expression of the C-terminal
fragment of AP180 inhibited transferrin uptake in HeLa cells,
while expression of the N-terminal fragment of AP180 had no
effect. Table 1 quantifies the effect of the C- and N-terminal
fragments of AP180 on transferrin uptake in a large population
of transfected HeLa and Cos cells. These results clearly show
that it is the clathrin-assembly activity of AP180 that is
responsible for inhibiting transferrin uptake, not its ability to
inhibit phospholipase D activity.
Effect of AP180 and auxilin on GLUT4 glucose
transporter endocytosis
If expression of AP180 and auxilin or their clathrin binding
domains indeed inhibits transferrin uptake by inhibiting
endocytosis non-specifically, uptake of proteins other than
transferrin receptor should also be affected by this expression.
To test this prediction we investigated the effect of expression
of AP180, auxilin and their clathrin-binding domains on the
level of GLUT4 glucose transporter present on the plasma
membrane of adipocytes. In its cycle between the plasma
membrane and an intracellular compartment, the GLUT4
glucose transporter is thought to be internalized by clathrinmediated endocytosis (Robinson et al., 1992; Chakrabarti et al.,
1994; Volchuk et al., 1998). Therefore if expression of AP180
and auxilin inhibits internalization of GLUT4 in a primary
culture of rat adipocytes, transfected cells should display a
higher level of GLUT4 on the cell surface in the absence of
insulin. Fig. 4 shows that this is indeed the case. As we
observed for internalization of transferrin, expression of
AP180 had a somewhat greater effect than expression of
auxilin. In fact, expression of the clathrin binding portion of
AP180 brought the basal level of GLUT4 glucose transporters
on the plasma membrane up to the level observed in the
presence of insulin while expression of the N-terminal
fragments of AP180 and auxilin had almost no effect. These
data confirm that, for uptake of GLUT4 transporter as well as
Expression of auxilin or AP180 inhibits endocytosis
transferrin receptor, expression of the APs auxilin or AP180
interferes with clathrin-mediated endocytosis.
Effect of AP180 on the distributions of clathrin and
APs
We next investigated whether
expression of AP180 affects the
distribution of clathrin in HeLa cells
since our data strongly suggested
that it is the clathrin-binding ability
of AP180 that is required for
inhibition of clathrin-mediated
endocytosis. Fig. 5 shows the
localization of clathrin in HeLa cells
expressing either intact AP180 (Fig.
5A,B) or the C-terminal fragment of
AP180 (Fig. 5C,D). Normally
clathrin is associated with both the
plasma membrane and the transGolgi network but in the transfected
cells, there seemed to be a loss of
clathrin from the trans-Golgi
network and an appearance of
aggregated clathrin in the cytosol. In
agreement with the observed loss of
clathrin from the trans-Golgi
network, using a chimeric protein,
the IL-2 receptor α chain (Tac)
containing a signal localization
sequence to the lysosome (Marks et
al., 1995), we found that overexpression of AP180 or its Cterminal fragment increased plasma
membrane association of the
chimeric Tac, indicating inhibition
of transport of this fusion protein
from the trans-Golgi network to the
lysosome (data not shown). On the
other hand, expression of the Nterminal fragment of AP180 had no
effect on the distribution of clathrin
(Fig. 5E,F).
The observation that expression of
AP180 or its clathrin binding
domain removed clathrin from the
trans-Golgi network allowed us to
investigate whether this affected the
distribution of AP1 on the transGolgi network. Strikingly, despite
the decrease in clathrin associated
Fig. 5. Effect of AP180 expression on
localization of clathrin, AP1 and βCOP
in HeLa cells. HeLa cells were
transfected with Flag-tagged AP180
(A,B and G-J), Flag-tagged AP180 Cterminal fragment (C,D), or Flagtagged AP180 N-terminal fragment
(E,F), fixed, and stained for Flag using
a rabbit polyclonal antibody (A,C,E,G)
or mAb M5 (I), clathrin (B,D,F), AP1
(H), and βCOP (J).
359
with the trans-Golgi network in the cells expressing AP180,
there was no change in the distribution of the γ chain of AP1;
it remained bound to the trans-Golgi network even in the
absence of clathrin (Fig. 5G,H). As expected, the Golgi coat
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JOURNAL OF CELL SCIENCE 114 (2)
Fig. 6. (A-C) Confocal microscopic photograph of clathrin localization in cells transfected with Flag-tagged AP180. (A) The field where z cut
was performed. (B,C) z cut photographs show clathrin distribution. Green, AP180; Red, clathrin. Only clathrin is shown in B and C. Arrows
indicate transfected cells. (D-G) AP2 localization in cells expressing AP180. (D,F) Transfected cell; (E,G) AP2 localization.
Expression of auxilin or AP180 inhibits endocytosis
Fig. 7. Effect of auxilin
expression on localization of
clathrin, Hsc70, AP2 and AP1 in
HeLa cells. The transfected cells,
labeled by either M5 antibody (C)
or a rabbit antiserum against Flag
(A,E,G,I), were stained for
clathrin (B), Hsc70 (D), AP2
(F,H) and AP1 (J).
protein
β-coatomer
also
appeared normal in AP180
transfected cells (Fig. 5I,J).
We also investigated whether
AP180 affects the localization
of clathrin and AP2 on
the plasma membrane. The
presence of aggregated clathrin
in the cytosol partially
obscured the amount of
clathrin associated with the
plasma membrane, but confocal
microscopy suggested that
there was indeed less clathrin
associated with the plasma
membrane of HeLa cells
expressing AP180 than with
the plasma membrane of
untransfected cells (Fig. 6AC). Furthermore, in agreement
with our observation that AP1
localization on the trans-Golgi
network is unaffected by
expression of AP180, we
observed
no
significant
difference in the amount of
AP2 associated with the
plasma membranes of the
transfected and untransfected
cells (Fig. 6D-G).
This lack of effect of AP180
on AP2 localization was
further
confirmed
by
comparing the fluorescence
intensity per unit area in
control and AP180 expressing
cells. Using the Metamorph
imaging computer program,
we found that the fluorescence
intensity was 18.01±4.90 and
18.68±4.20 in control and
AP180
expressing
cells,
respectively. These results
suggest that the AP2 pit
density was not significantly
affected due to expression of
AP180. Therefore, in a result
that has important implications
for the mechanism of clathrincoated pit formation, the
sequestration of clathrin does
361
362
JOURNAL OF CELL SCIENCE 114 (2)
Fig. 8. Effect of GFP-GAK expression
in HeLa cells on transferrin
internalization and localization of
clathrin, Hsc70, AP2, and AP1.
(A,C,E,G,I) Transfected cells and the
corresponding panels show transferrin
internalization (B), clathrin (D), Hsc70
(F), AP2 (H) and AP1 (J).
not
significantly
affect
the
localization of the key APs
involved in receptor recruitment
and clathrin polymerization at the
plasma membrane and trans-Golgi
network suggesting that these APs
bind to nascent pits independently
of clathrin.
Effect of auxilin and GAK on
distributions of clathrin and
APs
To investigate further whether
expression of clathrin APs affect
clathrin
distribution
without
affecting the localization of AP2
and AP1, we investigated the
effect of auxilin on the localization
of clathrin and the APs. Strikingly,
we found that, in both Cos cells
(data not shown) and HeLa cells,
auxilin granules always contained
clathrin (Fig. 7A,B) and Hsc70
(Fig. 7C,D). Using colocalization
with a Lamp-1 antibody, we
determined that these proteins are
not localized in the lysosomes
(data not shown). The association
of clathrin with the auxilin
granules was accompanied by a
marked decrease of clathrin in the
cytosol, which, in turn, made it
easier to discern than in the cells
expressing AP180, that there was a
marked decrease in clathrin
associated with the clathrin-coated
pits on the plasma membrane as
well as on the trans-Golgi network.
On the other hand, there was no
apparent association of either AP2
or AP1 with the granules. And
consistent with this observation,
we did not observe significant
redistribution of either AP2 (Fig.
7E-H) or AP1 (Fig. 7I,J) in these
cells confirming that, as in cells
expressing AP180, nascent pits
containing APs form on the
plasma membrane and the transGolgi network of these cells in the absence of clathrin.
Further support that nascent pits containing APs can form
on the plasma membrane and the trans-Golgi network of cells
independent of clathrin binding comes from studies with the
auxilin homolog, GAK, which, in contrast to auxilin, is an
endogenous protein in HeLa cells. Cells transfected with GFP-
Expression of auxilin or AP180 inhibits endocytosis
GAK showed decreased transferrin uptake, an effect that was
particularly dramatic in the cells showing formation of GAK
granules (Fig. 8A,B). Furthermore, as with the auxilin
granules, clathrin was associated with the GAK granules (Fig.
8C,D), and in cells with GAK granules, there was a marked
decrease in clathrin associated with the trans-Golgi network
and clathrin-coated pits on the plasma membrane. In fact, in
some cases, almost all of the clathrin in the cell was associated
with the GAK granules making it particularly clear that,
compared to the dramatic changes in clathrin distribution, there
was no significant change in the distribution of AP1 and AP2.
Specifically, the AP1 was still associated with the trans-Golgi
network (Fig. 8I,J), while the AP2 retained its punctate
appearance on the plasma membrane although some cytosolic
AP2 appeared to be associated with the GAK-clathrin granules
in the cytosol (Fig. 8G,H). Interestingly, when the cells
transfected with either auxilin or GAK were stained for Hsc70,
we found that the granules that contained clathrin and auxilin
or GAK also contained Hsc70 (Fig. 7C,D and Fig. 8E,F),
which explains why we did not observe these granules in cells
expressing auxilin with a mutated J-domain that could not
interact with Hsc70. Therefore, in the cells expressing GAK as
well as auxilin, nascent pits containing APs form on the plasma
membrane and the trans-Golgi network even though clathrin is
not associated with these pits.
DISCUSSION
There is strong evidence that Hsc70, acting with the J-domain
proteins auxilin or GAK, plays a major role in uncoating
clathrin-coated vesicles both in vitro and in vivo, but does not
uncoat clathrin-coated pits (Heuser and Steer, 1989). We were,
therefore, interested in whether expression of auxilin or GAK
in cultured cells increased or decreased clathrin-mediated
endocytosis. In the present study we found that expression of
either auxilin or over-expression of GAK inhibited clathrinmediated endocytosis in Cos and HeLa cells. However, even
expression of auxilin with a mutated J-domain inhibited
clathrin-mediated endocytosis. Furthermore, expression of the
clathrin assembly protein AP180 also inhibited endocytosis
and here too it was the clathrin-binding domain of AP180 that
was responsible for this inhibition. Since this work was
completed, Tebar et al. (Tebar et al., 1999) showed that CALM,
a homolog of AP180 expressed in non-neuronal cells, also
inhibited clathrin-mediated endocytosis when it was expressed
in Cos cells where it is normally present, again supporting the
view that expression of proteins or domains of proteins that act
as clathrin assembly proteins inhibit clathrin-mediated
endocytosis.
Studies on localization of clathrin provided an explanation
for the inhibition of clathrin-mediated endocytosis by overexpression of clathrin-binding proteins. In cells expressing
auxilin, GAK, AP180, or their clathrin-binding domains,
clathrin was either aggregated in the cytosol or, in the case of
GAK or auxilin with an intact J-domain, in GAK- or auxilinclathrin-Hsc70 granules. At the same time there was a decrease
in the level of clathrin associated with the trans-Golgi network
and the plasma membrane. This led to an opportunity to
determine whether the absence of clathrin from clathrin-coated
pits affected the distribution of the clathrin assembly proteins
363
AP1 and AP2. Strikingly, we did not observe a decrease in the
level of AP1 associated with the trans-Golgi network, nor did
we observe a change in the distribution of AP2 on the plasma
membrane. Interestingly, when Tebar et al. (Tebar et al., 1999)
expressed CALM, they observed a similar depletion of clathrin
from the trans-Golgi network with no effect on AP1
distribution, but did not observe a decrease in clathrin at the
plasma membrane. Therefore, our results show for the first
time that, even in the absence of clathrin binding, AP2
apparently forms nascent pits on the plasma membrane.
Both APs and clathrin are present in the cytosol as well as
on cellular membranes and therefore, when clathrin-coated pits
form, both APs and clathrin must be recruited to the
membrane. There has been speculation that formation of
clathrin-coated pits involves co-assembly of clathrin, AP2 and
receptors on the plasma membrane (Pearse and Crowther,
1987) but our data suggest that AP recruitment is independent
of clathrin recruitment. In this regard, there is strong evidence
that AP1 is recruited to the trans-Golgi network by the binding
of ARF1, which then dissociates once the AP1 and clathrin are
bound (Zhu et al., 1998), but it is not yet understood what
causes recruitment of AP2 to the plasma membrane. In any
case our results strongly suggest that nascent pits containing
APs can form in the absence of clathrin binding. These data
are consistent with the observation that when clathrin is
removed from existing coated pits by potassium depletion or
treatment with hypertonic solution, AP2 remains behind
(Hansen et al., 1993; Brown et al., 1999). They are also
consistent with the finding of Hannan et al. (Hanna et al., 1998)
who found that clathrin and AP2 are independently uncoated
from clathrin-coated vesicles. Finally, they are consistent with
the results of Liu et al. (Liu et al., 1998) who found that, when
they over-expressed clathrin hubs, not only was endocytosis
inhibited but, in addition, there was increased clathrin heavy
chain associated with the plasma membrane. Yet despite this
increase, there was no change in the distribution of AP2.
Since many of the proteins involved in endocytosis shuttle
between cytosolic and membrane-bound pools, a key question
in the regulation of endocytosis is what keeps these proteins
from polymerizing in the cytosol. There is evidence that
phosphorylation may regulate clathrin polymerization in the
cell (Wilde and Brodsky, 1996) and there is also evidence that
Hsc70 acting as a chaperone may form a complex with clathrin
triskelions and APs that prevent them from polymerizing in the
cytosol (Eisenberg and Greene, 1998; Black et al., 1991). In
this regard, our observation that GAK- or auxilin-clathrinHsc70 granules form in cells expressing auxilin or overexpressing GAK provides the first direct evidence that clathrin,
Hsc70, and auxilin indeed form a complex in vivo as well as
in vitro (Jiang et al., 2000), although we could only
demonstrate complex formation in cells over-expressing
auxilin or GAK. The data presented in this paper also show
that the mechanisms that prevent clathrin from polymerizing
in the cytosol can be overwhelmed by increasing the levels of
APs in the cell suggesting that polymerization of clathrin in
clathrin-coated pits rather than in the cytosol depends on
multiple regulatory factors including the concentration of APs
present in the cell.
The observation that expression of AP180 or auxilin inhibits
clathrin-mediated endocytosis provides a simple method of
testing whether a given process in the cell involves clathrin-
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JOURNAL OF CELL SCIENCE 114 (2)
mediated endocytosis. Using this method we confirmed that the
GLUT4 glucose transporters are internalized from the plasma
membrane by clathrin-mediated endocytosis and, at the same
time, demonstrated that expression of AP180 and auxilin not
only inhibited endocytosis in immortalized cells but also in
primary tissue culture cells. Our studies on the GLUT4
transporter show that the strongest inhibition of clathrinmediated endocytosis occurred with the clathrin-binding
domain of AP180. In fact, expression of this domain inhibited
clathrin-mediated endocytosis so strongly that the basal level
of the GLUT4 transporter on the plasma membrane of
transfected adipocytes almost reached the same level as in cells
treated with insulin. On the other hand, although it has been
reported that the GLUT4 glucose transporter interacts with
AP1 and AP3 (Gillingham et al., 1999), over-expression of
AP180 or its clathrin binding domain had no effect on the
transport of GLUT4 glucose transporters to the plasma
membrane in the presence of insulin suggesting that clathrin is
not involved in this transport. Therefore, in future studies,
expression of the clathrin-binding domain of AP180 should
provide a simple method of determining whether clathrinmediated endocytosis is involved in various processes in the
cell, a method that will compliment the use of clathrin hubs to
inhibit endocytosis (Liu et al., 1998). Since one method
decreases the level of clathrin heavy chain associated with the
plasma membrane while the other increases it, agreement
between the effects of these two methods will strengthen the
conclusion that clathrin-mediated endocytosis is required for a
particular process.
We thank Drs Julie Donaldson and Harish Radhakrishna for their
many helpful discussions, Dr Kenneth Yamada for the GFP-vector, Dr
Ivan Bonifacino for the Tac construct, and Dr Xufeng Wu for her
valuable help with the confocal microscopy work.
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