University of Cambridge, Department of Clinical Biochemistry, Cambridge CB2 2QR, United Kingdom
AP-1 and AP-2 adaptors are recruited onto
the TGN and plasma membrane, respectively. GTPS
stimulates the recruitment of AP-1 onto the TGN but
causes AP-2 to bind to an endosomal compartment (Seaman, M.N.J., C.L. Ball, and M.S. Robinson. 1993. J. Cell Biol. 123:1093-1105). We have used subcellular
fractionation followed by Western blotting, as well as
immunofluorescence and immunogold electron microscopy, to investigate both the recruitment of AP-2 adaptors onto the plasma membrane and their targeting to
endosomes, and we have also examined the recruitment
of AP-1 under the same conditions. Two lines of evidence indicate that the GTP
S-induced targeting of
AP-2 to endosomes is mediated by ADP-ribosylation factor-1 (ARF1). First, GTP
S loses its effect when
added to ARF-depleted cytosol, but this effect is restored by the addition of recombinant myristoylated
ARF1. Second, adding constitutively active Q71L ARF1 to the cytosol has the same effect as adding
GTP
S. The endosomal membranes that recruit AP-2
adaptors have little ARF1 or any of the other ARFs associated with them, suggesting that ARF may be acting
catalytically. The ARFs have been shown to activate
phospholipase D (PLD), and we find that addition of
exogenous PLD has the same effect as GTP
S or Q71L
ARF1. Neomycin, which inhibits endogenous PLD by
binding to its cofactor phosphatidylinositol 4,5-bisphosphate, prevents the recruitment of AP-2 not only onto
endosomes but also onto the plasma membrane, suggesting that both events are mediated by PLD. Surprisingly, however, neither PLD nor neomycin has any effect on the recruitment of AP-1 adaptors onto the
TGN, even though AP-1 recruitment is ARF mediated.
These results indicate that different mechanisms are
used for the recruitment of AP-1 and AP-2.
PROTEINS are transported from one membrane compartment of the cell to another by means of carrier
vesicles. The first step in the formation of these vesicles is the recruitment of cytosolic proteins onto a "donor" membrane compartment, where they assemble into a
coat. This coat may serve two purposes: to deform the
membrane into a budding vesicle and to select the vesicle
cargo by interacting with the cytoplasmic domains of some
of the proteins in the donor membrane (Schekman and
Orci, 1996 The process of coat recruitment is still not well understood, although it is thought that there are specific docking
sites on the membrane for coat proteins. In addition, in
most cases a small GTP-binding protein has been shown to
be involved in coat recruitment, somehow priming the
membrane for the subsequent binding of coat proteins.
Thus, the coatomer or COPI coat, which is recruited onto
the membranes of the Golgi stack and intermediate compartment, requires ADP-ribosylation factor (ARF)1 for its
membrane association, and studies making use of purified components have implicated the most abundant of the
ARF isoforms, ARF1, in this event (Donaldson et al.,
1992 There is one other well-characterized type of coat in the
cell, which mediates the formation of endocytic-coated
vesicles at the plasma membrane and which consists of
clathrin and AP-2 adaptors. The subunits of the AP-2
adaptor complex are closely related to those of the AP-1
complex and more distantly related to those of the AP-3
complex. However, unlike AP-1 and AP-3, AP-2 recruitment does not appear to be dependent on a conventional ARF. Thus, the drug brefeldin A (BFA), which prevents
the nucleotide exchange of most ARFs, causes AP-1, AP-3,
and coatomer to redistribute to the cytoplasm when added
to living cells, while the distribution of AP-2 remains unchanged (Donaldson et al., 1990 So far, five ARFs have been identified in humans (Price
et al., 1988 The mechanism by which ARF promotes the recruitment of cytosolic coat components onto the membrane is
still not understood. One possibility is that ARF may interact directly with coat proteins, an idea supported by the
finding that ARF1 can bind to one of the coatomer subunits
(Zhao et al., 1997 Here we use an in vitro system to investigate both the
recruitment of AP-2 adaptors onto the plasma membrane
and their alternative targeting to the endosomal compartment. As a comparison, we have also used our in vitro system to study the recruitment of AP-1 adaptors. We have
raised a panel of monospecific anti-ARF antibodies and
have used these and recombinant ARF to investigate the
potential role of ARF(s) in adaptor recruitment. We find
that ARF1 can mediate not only the recruitment of AP-1
adaptors onto the TGN, but also the GTP Production of Anti-ARF Antibodies
The full-length coding sequences for ARFs 1, 4, 5, and 6 were amplified by
PCR from human brain cDNA (CLONTECH Laboratories, Palo Alto,
CA) and ligated into pBluescript. DNA manipulations were carried out
essentially as described by Sambrook et al. (1989) MC1061 cells were transformed with the plasmids and expression of
the GST fusion proteins was induced with 0.1 mM IPTG. The fusion proteins were affinity purified on glutathione-Sepharose (Pharmacia LKB
Biotech., Piscataway, NJ), using the method of Smith and Johnson (1988) To test the specificity of the antibodies, 50 µg of each full-length ARF-
GST fusion protein was digested with 500 ng activated Factor X according
to Smith and Johnson (1988) Expression and Purification of Recombinant ARF1
Recombinant myristoylated wild-type and mutant ARF1 were prepared
from BL21 Escherichia coli that had been transformed with plasmids encoding N-myristoyl transferase and either wild-type ARF1, Q71L ARF1,
or T31N ARF1 (Dascher and Balch, 1994 Rat Liver Fractionation
Fractionation of rat liver membranes was carried out essentially as described by Branch et al. (1987) Preparation of Cytosol
Pig brain cytosol was prepared in cytosol buffer (25 mM Hepes-KOH, pH
7.0, 125 mM potassium acetate, 2.5 mM magnesium acetate, 1 mg/ml glucose, 1 mM DTT) as described by Seaman et al. (1993) For ARF depletion, 4 ml of cytosol was fractionated at 30 ml/h on a Superose 6 column (45 × 1.6 cm) equilibrated in cytosol buffer containing
0.2 mM PMSF. Fractions containing ARFs were identified by Western
blotting, and the remaining ARF-depleted fractions pooled and concentrated to the original volume using a Centricon-10 concentrator (Amicon,
Beverly, MA). ARF immunoreactivity was undetectable in the ARF-depleted fractions.
In Vitro Recruitment
Recruitment experiments were carried out both on enriched rat liver
membrane fractions and on permeabilized normal rat kidney (NRK) cells.
400-µl aliquots of the membrane fractions, prepared as above, were diluted threefold with STM and collected by pelleting at 90,000 g for 15 min
in a rotor (model TL100.2; Beckman Instruments). Membrane pellets were
resuspended in 50 µl of cytosol containing 1 mM ATP, an ATP-regenerating system (5 mM creatine phosphate, 80 µg/ml creatine phosphokinase),
and 100 µM EGTA and incubated at 37°C for 10 min. GTP In some experiments, recombinant ARF1, bacterial PLD, and/or neomycin were added. Recombinant ARF1 was added to a final concentration of between 20 µg/ml and 200 µg/ml, depending on the experiment. Endogenous ARF1 in our cytosol preparations was estimated to be ~10
µg/ml by Western blotting. Bacterial PLD (type VII from Streptomyces species) was obtained from Sigma Chemical Co. It had a specific activity
of 1,200 U/mg, 1 U being defined as the amount of enzyme required to liberate 1 µmol of choline per hour at pH 5.6 at 30°C. It was added to a final
concentration of between 0.5 and 37.5 µg/ml, depending on the experiment. As a comparison, in a recent study by Ktistakis et al. (1996) Methods for recruitment onto permeabilized NRK cells have been previously described (Robinson and Kreis, 1992 Assay for PLD Activity
PLD activity was determined essentially as described by Bi et al. (1997) Electron Microscopy
For immunogold localization of newly recruited AP-2 adaptors, NRK
cells grown in 60-mm dishes were permeabilized by immersion into liquid
nitrogen before incubation with cytosol, as previously described (Seaman
et al., 1993 Recruitment of AP-2 Adaptors onto Rat
Liver Membranes
Previous studies have shown that the recruitment of adaptor complexes onto membranes can be reconstituted in
vitro using permeabilized NRK cells as a source of target
membranes and pig brain cytosol as an exogenous source
of adaptors. After incubation at 37°C in the presence of an
ATP-regenerating system, newly recruited adaptors can
be detected by immunofluorescence using species- or tissue-specific antibodies that do not recognize the endogenous adaptors (Robinson and Kreis, 1992
To generate target membranes for adaptor recruitment,
rat liver postmitochondrial supernatants (5 ml) were fractionated on a 34-ml Nycodenz gradient, and 1-ml fractions
were incubated with pig brain cytosol in the presence of an
ATP-regenerating system, either with or without GTP In the absence of GTP To confirm that the AP-2 detected in this assay was in
fact membrane associated, fractions were fixed after incubation with pig brain cytosol and processed for immunoelectron microscopy (Fig. 1 c), using the brain-specific
anti- Role of ARF1 in the Binding of AP-2 Adaptors
To determine whether low levels of a conventional ARF,
such as ARF1, might be involved in the binding of AP-2 to
endosomes, we generated ARF-depleted cytosol by gel filtration and then added back defined components. Fig. 2
(first three lanes) shows an experiment in which the ARF-depleted cytosol was mixed with low molecular weight,
ARF-containing fractions to generate reconstituted cytosol and then added to membranes in the presence or absence of GTP
Because GTP
To confirm that Q71L ARF1 causes AP-2 adaptors to
bind to the perinuclear endosomal compartment, a recruitment experiment was carried out using permeabilized
NRK cells, and the cells were labeled for immunofluorescence using the tissue-specific Thus, these data indicate that ARF1 can mediate the
binding of AP-2 adaptors to the endosomal compartment,
even though there is relatively little ARF1 associated with
this compartment.
Phospholipase D Can Stimulate AP-2 Recruitment
How is ARF1 acting to promote the recruitment of AP-2
adaptors onto endosomes? The relatively low levels of
ARF1 associated with the endosomal compartment suggest that it may be acting catalytically. One possibility is
that it may be acting via PLD since previous reports have
shown that ARF is an effective activator of this enzyme
(Brown et al., 1993 Fig. 4 a shows an experiment assessing the recruitment
of cytosolic AP-2 adaptors onto rat liver membranes in the
presence of varying amounts of bacterial PLD. Although
EGTA is normally added to the incubation mixture, most
of these incubations were carried out in the absence of
EGTA since bacterial PLD is reported to require Ca2+ for
maximum activity (Imamura and Horiuti, 1979
Perhaps surprisingly, since its membrane association is
believed to be ARF1 mediated, the recruitment of AP-1
adaptors was not appreciably enhanced by PLD, even at
the highest concentrations used (Fig. 4 b). Thus, although
GTP The effect of PLD was also examined using permeabilized NRK cells. For these experiments, 12.5 µg/ml PLD
plus 100 µM EGTA were used to avoid any Ca2+-induced
mistargeting (see Seaman et al., 1993 Neomycin Inhibits Both Endosomal and Plasma
Membrane AP-2 Recruitment
The effect of exogenous bacterial PLD on AP-2 recruitment suggests that GTP When 1 mM neomycin was added, the amount of newly
recruited AP-2 was significantly reduced both in the absence and in the presence of GTP
These observations were confirmed by immunofluorescence labeling of permeabilized cells after adaptor recruitment (Fig. 6). The normal punctate plasma membrane recruitment of AP-2 adaptors seen in the absence of GTP
To compare the effect of neomycin on AP-2 recruitment
with its effect on PLD activity, NRK cells were grown
overnight with [3H]palmitic acid to label membrane lipids.
They were then permeabilized and incubated for 20 min
with increasing concentrations of neomycin plus 1% butanol so that PLD activity could be measured by assaying for the production of phosphatidyl butanol. At the end of
the incubation, lipids were extracted and analyzed by
TLC. A similar experiment was carried out on unlabeled
NRK cells to assess the effect of the same concentrations
of neomycin on AP-2 recruitment. Fig. 7 shows the results
of the two experiments.
Because whole cells were used, the effect of GTP Characterization of AP-2-binding Membranes
The immunofluorescence results presented so far demonstrate that GTP Rat liver membrane pools from Nycodenz gradients
were incubated with cytosol in the presence of GTP
To investigate the localization of the AP-2 adaptors at
an ultrastructural level, we carried out a recruitment experiment using permeabilized NRK cells incubated with
cytosol in the presence of GTP
Previous studies have shown that the recruitment of both
AP-1 and AP-2 adaptors in vitro is affected by GTP In this study, we began by focusing on the effect of
GTP Further evidence for the involvement of PLD in AP-2
recruitment comes from our studies on the effect of neomycin. Neomycin inhibits PLD indirectly by binding to
PIP2, a cofactor for optimum enzyme activity (Hammond
et al., 1995 There are several observations that suggest that PIP2
may be at least as important as PA. First, AP-2 adaptors
have been shown to bind with high affinity to inositol
phosphates and inositol phospholipids (Beck and Keen,
1991 Fig. 10 depicts a model for the recruitment of AP-2 adaptors onto the endosomal compartment. This model is based
on one proposed by Traub and Kornfeld for AP-1 recruitment (Traub et al., 1993
According to this model, all of the proteins leading to
coat binding, i.e., ARF, PLD, and the putative docking
protein, could act catalytically and would not necessarily
be found to any great extent in the resulting coated pits or
vesicles. Indeed, there is no evidence for stoichiometric
quantities of any of the known ARFs, or of any other proteins, in purified clathrin-coated vesicles. This model also
suggests that there may not necessarily be a direct interaction between ARF and the docking protein, in contrast to the model proposed by Traub and Kornfeld for AP-1 recruitment (Traub et al., 1993 In some respects, this model is similar to models proposed for proteins such as rabs and ARFs, which use lipid
modifications to associate with membranes. The lipids do
not actually direct such proteins to a particular membrane
since proteins with the same lipid modification may associate with different membranes. Instead, it has been proposed that such proteins initially interact with a specific
docking site (which in the case of ARF may also be its nucleotide exchange factor), which causes a conformational change so that the lipid becomes inserted into the membrane bilayer (Donaldson and Klausner, 1994 How well can this model be extrapolated to account for
other membrane coating events? It seems likely that the
recruitment of AP-2 adaptors onto the plasma membrane
also requires the activation of PLD and the generation of
acidic phospholipids, even though a conventional ARF is
probably not involved. Other coats that require ARF for
membrane binding, such as AP-1 adaptors and coatomer, might be expected to use the same mechanism as that
shown in Fig. 10. Indeed, there is evidence that PLD mediates coatomer binding to Golgi membranes (Ktistakis et
al., 1996 Thus, it seems clear that different binding mechanisms
have evolved for different coats. One common theme may
be that all coats make use of a small GTP-binding protein,
although the role of such a protein in the recruitment of
AP-2 onto the plasma membrane is still open to question.
However, the same GTP-binding protein may regulate different coats in different ways. Thus, although ARF1 may
promote the binding of AP-2 adaptors and coatomer by activating PLD, it must use some other mechanism to promote the binding of AP-1 adaptors. A number of activities
have been attributed to ARF, and a number of interactions with both proteins and phospholipids have been reported (Bowman and Kahn, 1995).
; Palmer et al., 1993
). Similar studies have shown that
ARF1 also allows AP-1 adaptors to be recruited onto the
TGN membrane, after which clathrin binds to the adaptors and the two components coassemble to form a clathrin-coated bud (Stamnes and Rothman, 1993
; Traub et al.,
1993
). Recently, an adaptor-related complex, called AP-3,
has been identified; AP-3 is also recruited onto the TGN,
but it is not associated with clathrin. Although studies
have not yet been carried out using purified components,
AP-3 recruitment is affected by reagents that act on ARF,
indicating that it too requires ARF (Simpson et al., 1996
,
1997
). In contrast, the COPII coat, which is associated with the ER, requires another small GTP-binding protein,
Sar1p, to bind to the membrane (Barlowe et al., 1994
).
; Robinson and Kreis,
1992
; Simpson et al., 1997
). Similarly, in an in vitro system,
BFA prevents the binding of AP-1, AP-3, and coatomer to their target membranes, without affecting the binding of
AP-2 to the plasma membrane (Orci et al., 1991
; Robinson
and Kreis, 1992
; Seaman et al., 1993
; Simpson et al., 1996
).
Intriguingly, however, GTP
S, a poorly hydrolyzable analogue of GTP, which stimulates the recruitment of other
coats onto their target membranes, does not stimulate the
binding of AP-2 adaptors to the plasma membrane but instead causes them to become associated with a late endosomal compartment. This endosomal association can be
prevented by the addition of brefeldin A, indicating that it
is ARF dependent (Seaman et al., 1993
). These observations suggest that docking sites for AP-2 adaptors exist
both on the plasma membrane and on the endosomal compartment, but that normally they are only active at the
plasma membrane, and that somehow GTP
S, presumably acting via ARF, switches on endosomal docking sites
that normally are inactive. Whether recruitment of AP-2
adaptors onto the plasma membrane might also involve an
(unconventional) ARF or ARF-related protein is not known.
; Bobak et al., 1989
; Kahn et al., 1991
; Tsuchiya
et al., 1991
), in addition to the less closely related ARL
(ARF-like) family (Clark et al., 1993
). ARF1 has been localized to the Golgi region in mammalian cells and has
been shown to associate with isolated Golgi membranes
(Stearns et al., 1990
; Serafini et al., 1991
). In other reports,
ARFs 1, 3, and 5 have been detected on crude Golgi/microsomal fractions (Tsai et al., 1992
) and, together with
ARF4, have also been found on an endosome-enriched
fraction (Whitney et al., 1995
), while ARF6 has been localized to the plasma membrane (Cavenagh et al., 1996
) and
endosomes (Peters et al., 1995
). All of the ARFs have
been shown to be BFA sensitive, with the exception of
ARF6, the least related of the ARF family (Tsuchiya et
al., 1991
). ARF6 also differs from the other ARFs in that it appears to be constitutively membrane associated, instead
of cycling back and forth between membranes and cytosol
(Peters et al., 1995
; Cavenagh et al., 1996
).
). A second possibility was proposed in a
study on AP-1 recruitment, in which it was suggested that
ARF might interact with the putative AP-1 docking site,
inducing a conformational change that would allow the
docking site to bind the adaptor complex (Traub et al.,
1993
). A third possibility is that ARF may interact with
neither the coat proteins nor their docking sites, but exert
its effect through some other mechanism. ARF has been
reported to activate phospholipase D (PLD), which catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid (Brown et al., 1993
; Cockcroft et al., 1994
), and a
recent study suggests that PLD may play a key role in
coatomer recruitment (Ktistakis et al., 1996
).
S-induced recruitment of AP-2 adaptors onto endosomes. However,
ARF1 does not appear to be required for the recruitment of AP-2 adaptors onto the plasma membrane. To investigate the possibility that ARF may facilitate adaptor recruitment through the activation of PLD, we have tested the effects of exogenous bacterial PLD and of neomycin, which
inhibits endogenous ARF-dependent PLD by binding to
its cofactor phosphatidylcholine 4,5-bisphosphate (PIP2).
We have found that neither reagent has any effect on AP-1 recruitment. However, exogenous PLD stimulates AP-2
recruitment onto the endosomal compartment, while neomycin inhibits this event. Interestingly, neomycin also has
a strong effect on plasma membrane AP-2 binding, indicating that the recruitment of AP-2 adaptors onto both
membranes may require PLD.
Materials and Methods
. Either full-length or
variable regions of the ARFs were then ligated into pGEX-3X using
BamHI and EcoRI or KpnI restriction sites, which were introduced by
PCR using the following primers: ARF 1 forward: 5
CGC GGA TCC
CCA GCA ATG ACA GAG AGC GTG TG 3
, reverse: 5
CCG GAA
TTC AAT CCC GGA GCT CGT CCT CGG C 3
; ARF 4 forward: 5
GCG GGA TCC CCA TGG GCC TCA CTA TCT CCT CC 3
, reverse:
5
CGG GGT ACC AAC TAA TTT TGT TGT AAC AAG CCT 3
;
ARF 5 forward: 5
CGC GGA TCC CCA GTA ATG ACC GGG AGC
GGG TC 3
, reverse: 5
CCG GAA TTC AAT CCC GCA GCT CGT
CCT CCT G 3
; ARF 6 forward: 5
CGC GGA TCC CCT GCG CCG
ACC GCG ACC GCA TC 3
, reverse: 5
CCG GAA TTC AGT CCC
TCA TCT CCC GGT CAT T 3
.
,
and injected into rabbits using 0.5 mg in complete Freund's adjuvant for
the first injection and 0.5 mg in incomplete Freund's adjuvant for two subsequent injections (Page and Robinson, 1995
). Affinity purification of the
resulting antisera was carried out essentially as previously described (Page and Robinson, 1995
). Briefly, 10 ml of each serum was first adsorbed against 1 mg of GST coupled to CNBr-activated Sepharose (Pharmacia LKB Biotech.) and then affinity purified on 1 mg of its own fusion protein
coupled to CNBr-activated Sepharose. Bound antibodies were eluted with
0.2 M glycine, pH 2.3, containing 0.1% gelatine. To select for monospecific antibodies, each of the affinity-purified sera was adsorbed at least
twice against a mixture of the other full-length ARF-GST fusion proteins
(1 mg each) coupled to CNBr-activated Sepharose.
to generate GST-free ARFs. The digests
were subjected to SDS-PAGE on a 15% SDS gel, and Western blots were
probed with the various antisera.
). The transformed cells were induced with 0.3 mM IPTG in the presence of 50 µM myristate and grown at
27°C for 3 h (Franco et al., 1995
). The cells were lysed, and the expressed
ARF1 was purified using DEAE-Sephacel and AcA54 Ultrogel columns
as described by Weiss et al. (1989)
. This particular strain of transformed
bacteria has been shown to myristoylate 10-60% of the protein (Randazzo et al., 1993
).
. 15 g of rat liver was homogenized in 40 ml
of cold 0.25 M sucrose, 10 mM TES, pH 7.4, 1 mM MgCl2 (STM), and a
postmitochondrial supernatant was prepared by centrifuging the homogenate at 1500 g for 10 min. 5-ml samples of this were loaded onto 34-ml linear gradients prepared from 0.25 M sucrose, 10 mM TES pH 7.4, 1 mM
EDTA, and 45% Nycodenz in 10 mM TES pH 7.4, 1 mM EDTA. The gradients were centrifuged at 206,000 g for 60 min in a vertical rotor (model
VTi50; Beckman Instruments, Fullerton, CA), and 1-ml fractions were
collected by upwards displacement.
. The cytosol was
clarified before use by centrifuging at 350,000 g for 15 min in a rotor
(model TL100.2; Beckman Instruments) and was used at a final protein
concentration of ~8 mg/ml.
S, when included, was used at a concentration of 100 µM. After the incubation, the
membranes were diluted with 1 ml of cold STM and collected by centrifugation as before. The pellets were resuspended in 50 µl of SDS-PAGE
sample buffer, and 10-µl aliquots were subjected to SDS-PAGE and
Western blotting. Blots were probed with the brain-specific rabbit anti-
-adaptin antibody, A706-727 (Ball et al., 1995
), or the species-specific mouse anti-
-adaptin monoclonal antibody, mAb100/3 (Sigma Chemical Co., Poole, UK) (Ahle et al., 1988
) and rabbit anti-mouse IgG (Sigma
Chemical Co.), followed by 125I-protein A (Amersham Corp., Indianapolis, IN). Quantification was carried out using a phosphorimager (FujiX
Bas2000 Bio-Imaging Analyzer; Fuji Photo Film Co., Tokyo, Japan). Each
experiment was carried out at least three times to ensure that the results were reproducible.
, bacterial PLD was added to a final concentration of 4 µg/ml, although the specific activity was measured differently (50 U/mg, 1 U being defined as the
amount of enzyme that will hydrolyze 1.0 µmol of phosphatidylcholine per min at 37°C). Neomycin was also obtained from Sigma Chemical Co.
and was added at concentrations ranging from 0.03 to 3.0 mM.
; Seaman et al., 1993
).
- and
-adaptins were labeled for immunofluorescence with A706-727 and
mAb100/3 followed by FITC anti-rabbit IgG or Texas red anti-mouse
IgG (Amersham Corp.), respectively. Each experiment was repeated at
least three times.
.
Briefly, NRK cells grown to ~90% confluency were labeled overnight
with 25 µCi/ml [3H]palmitic acid (Amersham Corp.), and then cells from
two 9-cm-diam dishes were washed with cytosol buffer, frozen in liquid nitrogen and rapidly thawed, scraped from the dishes, and incubated as eight aliquots, each with 50 µl cytosol containing 1% butanol for 20 min.
GTP
S and neomycin were added as indicated. Cellular lipids were then
extracted, and the organic phase was subjected to thin-layer chromatography (TLC) as described by Bi et al. (1997)
. Radioactivity was quantified
using a phosphorimager, and the phosphatidyl butanol produced in each
tube was normalized using other radiolabeled lipids to control for variability in recovery and/or loading. Another two dishes of cells were grown under identical conditions but without the [3H]palmitic acid, permeabilized
as above, and used to assay AP-2 recruitment under the same conditions.
). At the end of the incubation, the cells were washed gently
with cytosol buffer and then fixed for 1 h with 4% paraformaldehyde,
0.1% glutaraldehyde in 0.25 M Hepes, pH 7.2, before scraping, pelleting,
and embedding in 10% gelatine. For examination of rat liver membranes,
recruitment was carried out on gradient fractions as above, and the pellets were fixed and embedded in 10% gelatine. Cryosections were prepared, immunolabeled, and contrasted as described by Simpson et al. (1996)
. Newly recruited AP-2 adaptor complexes were detected using the A706-727 anti-
-adaptin antibody. The anti-lgp110 antibody was a kind gift
from Paul Luzio (University of Cambridge, Cambridge, UK) (Reaves et
al., 1996
).
Results
; Seaman et al.,
1993
). We have adapted this system to allow a more quantitative assessment of adaptor recruitment, using enriched
rat liver fractions as the source of target membranes. To
investigate whether any of the ARFs cofractionate with
membranes that bind adaptors, we have raised antisera
against ARFs 1, 4, 5, and 6 and then cross-adsorbed them
with the other ARF isoforms. Fig. 1 a shows Western blots
probed with the various ARF antisera and demonstrates
that each of the adsorbed antisera recognizes only its appropriate ARF, while an antiserum raised against ARF1 and not adsorbed recognizes ARFs 1, 4, 5, and also 6 to a
lesser extent.
Fig. 1.
Recruitment of cytosolic proteins onto rat liver membranes. (a) To determine the specificity of the anti-ARF antibodies raised in this study, samples of ARF-GST fusion proteins were digested with Factor X and the digests subjected to SDS-PAGE on 15% gels and Western blotted. Identical panels were probed with affinity-purified and cross-adsorbed anti-ARF antisera as indicated. Non-cross-adsorbed anti-ARF1 labels all four ARF isoforms, while after cross-adsorption each antibody is specific for its own isoform. ARF3,
which is 97% identical to ARF1, was not included in this study. (b) Membranes from a Nycodenz gradient were incubated with pig brain
cytosol in the presence or absence of GTPS. The incubation mixture in this experiment and in all subsequent experiments also contained ATP and an ATP-regenerating system. After the incubation, membranes were pelleted and assayed by Western blotting for the
various ARF isoforms, or for newly recruited AP-2 or AP-1 adaptor complexes using brain-specific anti-
- or species-specific anti-
-adaptin antibodies, respectively. Significant amounts of both
-adaptin and ARF6 are associated with membranes even in the absence of GTP
S, although they show only partial overlap. In the presence of GTP
S, the binding of AP-2 is increased and the fractionation profile shifted to less dense membranes. The other ARF isoforms and AP-1 are also recruited onto membranes and show similar fractionation profiles to each other but not to AP-2. (c) Frozen thin sections were prepared from samples equivalent to 8-19 in b. The sections were labeled with brain-specific anti-
-adaptin, followed by 10-nm protein A gold. In the absence of GTP
S, AP-2 is recruited onto small vesicles, presumably derived from the plasma membrane, while in the presence of GTP
S, AP-2 is recruited onto larger structures that often contain internal membranes, characteristic of endosomes. Bar, 200 nm.
[View Larger Version of this Image (75K GIF file)]
S.
The various ARF isoforms were detected by probing
Western blots with the antisera shown in Fig. 1 a, while
newly recruited adaptor complexes were detected with a
brain-specific anti-
-adaptin antibody (A706-727) (Ball et
al., 1995
) for AP-2 adaptors and a species-specific anti-
-adaptin antibody (mAb100/3) (Ahle et al., 1988
) for AP-1
adaptors (Fig. 1 b).
S, newly recruited AP-2 adaptors were found to bind to membranes that were broadly
distributed throughout the upper half of the gradient, with
a slight peak in fractions 15-18. ARF6 could also be detected in the absence of GTP
S and was found to peak in
fractions 15-19. The other ARF isoforms, and newly recruited AP-1 adaptors, were unable to be detected under
these conditions (data not shown). In the presence of
GTP
S, the binding of AP-2 adaptors was found to be enhanced and was shifted somewhat to less dense membranes. This shift is consistent with GTP
S inducing the
recruitment of AP-2 onto a different (i.e., endosomal)
compartment (Seaman et al., 1993
). GTP
S also facilitated the binding of the other ARF isoforms and AP-1 adaptors
to membranes. The membranes that bound AP-1 and the
membranes that bound the various ARFs had similar fractionation profiles, peaking between fractions 14 and 18 or
19. The nonadsorbed ARF1 antibody, which cross-reacts
with all of the known ARFs and possibly also with other as
yet unidentified ARFs, produced a strong signal that also
peaked between fractions 14 and 19 but which extended
into the lighter portion of the gradient. In contrast, the
membranes that bound AP-2 in the presence of GTP
S
had a different fractionation profile, with a broad peak between fractions 9 and 17. These results suggest either that
low levels of a conventional ARF may facilitate the
GTP
S-dependent recruitment of AP-2 onto these membranes, or alternatively that a novel ARF could be involved.
-adaptin antibody followed by 10-nm protein A
gold. Without GTP
S, low levels of newly recruited AP-2
were observed associated with small vesicles. In the presence of GTP
S, enhanced labeling was seen that tended to
be associated with larger membranous structures. These
structures often contained internal membranes and are
likely to correspond to the endosomal compartment to
which AP-2 is targeted in permeabilized cells (Seaman et
al., 1993
), while the smaller vesicles labeled in the absence of GTP
S are likely to be derived from the plasma membrane.
S. The addition of GTP
S increased the
binding of AP-2 adaptors to the membranes and also
caused trace amounts of ARF1 to bind. In contrast, membranes incubated with ARF-depleted cytosol alone showed only a basal level of AP-2 recruitment, which was
not increased with GTP
S (Fig. 2, fourth and fifth lanes).
To determine whether ARF1 could substitute for the ARF-containing fractions, recombinant myristoylated ARF1
was included with ARF-depleted cytosol. The last four
lanes of Fig. 2 show that the recombinant ARF1 protein fully restored the GTP
S-enhanced binding of AP-2 adaptors and was itself recruited onto the membrane as well, although at lower levels.
Fig. 2.
Role of ARF1 in AP-2 recruitment. Aliquots of peak
fractions from a Nycodenz gradient were incubated with either
reconstituted cytosol or with high-molecular weight, ARF-
depleted fractions of cytosol that had been gel filtered. GTPS
and recombinant myristoylated ARF1 were included as indicated. Newly recruited AP-2 adaptors (
) and membrane-associated ARF1 were detected by Western blotting. When reconstituted cytosol was added, AP-2 recruitment onto the membranes
was enhanced by the addition of GTP
S. No such enhancement was seen with the ARF-depleted cytosol, but it was restored by the addition of recombinant myristoylated ARF1.
[View Larger Version of this Image (35K GIF file)]
S is likely to have multiple effects, we
also tested whether the Q71L mutant of ARF1, which hydrolyzes GTP poorly (Tanigawa et al., 1993
), could mimic
the effect of GTP
S on AP-2 recruitment. For these experiments, unfractionated cytosol was used since the Q71L
mutant is expected to have a dominant effect. Fig. 3 a
shows that 50 µg/ml of the mutant ARF1 strongly stimulated the binding of AP-2 to membranes, giving levels of
recruitment approaching that seen with GTP
S. Higher
concentrations (100 and 200 µg/ml) of Q71L ARF1 increased AP-2 binding slightly further. Equivalent amounts
of the dominant negative ARF1 mutant, T31N (Dascher
and Balch, 1994
), did not enhance AP-2 binding over levels seen without GTP
S. Q71L ARF1 also promoted the
membrane recruitment of
-adaptin (Fig. 3 b), as expected
since ARF1 has been demonstrated to have a role in AP-1
adaptor recruitment to the TGN (Stamnes and Rothman,
1993
; Traub et al., 1993
). However, the effect of the mutant ARF1 on AP-1 was more moderate than on AP-2
adaptors in that even 200 µg/ml Q71L ARF1 did not
stimulate AP-1 recruitment to quite the levels seen with
GTP
S.
Fig. 3.
Effects of ARF1 mutants on adaptor recruitment. Aliquots of pooled peak fractions from a Nycodenz gradient were used as
acceptor membranes for the recruitment of (a) AP-2 () or (b) AP-1 (
). These membranes were incubated with cytosol containing either GTP
S, Q71L ARF1, or T31N ARF1 and then subjected to SDS-PAGE and Western blotting. Q71L ARF1, but not T31N ARF1,
stimulates the recruitment of both types of adaptors even in the absence of GTP
S. (c-h) Permeabilized NRK cells were incubated with
either cytosol alone (c) or cytosol containing 100 µg/ml T31N ARF1 (d), GTP
S (e and f), or 100 µg/ml Q71L ARF1 (g and h), and
newly recruited AP-2 or AP-1 adaptors were detected using anti-
(c, d, e, and g) or anti-
(f and h) antibodies. Q71L ARF1 has a
GTP
S-like effect on both types of adaptors, indicating that in both cases the GTP
S is acting via ARF. Bar, 10 µm.
[View Larger Versions of these Images (14 + 116K GIF file)]
-adaptin antibody. After
incubating the cells with cytosol alone, a punctate plasma
membrane pattern was seen (Fig. 3 c). This same type of labeling was seen when T31N ARF1 was included (Fig. 3
d). Only faint AP-1 recruitment was detected under either
of these conditions (not shown). When GTP
S was included in the incubation, intense perinuclear labeling of
-adaptin, characteristic of endosomal targeting, was observed (Fig. 3 e). A very similar pattern was seen when
Q71L ARF1 was included instead of GTP
S (Fig. 3 g). With either GTP
S or Q71L ARF1, perinuclear labeling
of newly recruited
-adaptin was also detected (Fig. 3, f
and h), consistent with its TGN localization (Robinson
and Kreis, 1992
). However, this pattern was distinct from
the perinuclear labeling seen for AP-2 adaptors (see Seaman et al., 1993
).
; Cockcroft et al., 1994
). PLD hydrolyzes phosphatidylcholine to phosphatidic acid (PA), which
along with PIP2 has been proposed to contribute to
coatomer binding to membranes (Ktistakis et al., 1996
). If
ARF1 acts in AP-2 recruitment via activation of PLD,
then the addition of exogenous active PLD should bypass
the requirement for activated ARF to give enhanced recruitment.
). Under
these conditions, the membrane association of AP-2 adaptors in both the absence and the presence of GTP
S is reduced, although a GTP
S enhancement of recruitment is
still observed (Fig. 4 a, first five lanes). When the effect of
adding exogenous PLD was tested, we found that bacterial
PLD can effectively substitute for GTP
S in promoting
AP-2 recruitment. At low concentrations of PLD (e.g., 0.1 µg/ml), the addition of 1 mM EGTA was found to inhibit
PLD-induced recruitment by ~70%, but at concentrations
of 2.5 µg/ml or above, 1 mM EGTA only inhibited by
~40% (data not shown). In addition, because EGTA itself
promotes AP-2 recruitment, at high concentrations of
PLD similar amounts of AP-2 were found to be recruited
onto the membrane in the absence and in the presence of
EGTA (Fig. 4 a, last lane).
Fig. 4.
Effect of exogenous PLD on adaptor recruitment. (a and b) Membrane pools (see Fig. 3) were incubated with cytosol containing bacterial PLD, EGTA (100 µM), and GTPS in combinations as indicated, and either
(a) or
(b) recruitment was detected by
Western blotting. PLD mimics the GTP
S effect on AP-2 recruitment, but not on AP-1 recruitment. (c-h) Recruitment onto permeabilized NRK cells was carried out with cytosol alone (c and d), with GTP
S (e and f) or with 12.5 µg/ml PLD (g and h). The same cells
were double labeled for immunofluorescence using either the anti-
(c, e, and g) or anti-
(d, f, and h) antibodies. PLD only has a
GTP
S-like effect on the AP-2 adaptors. Bar, 10 µm.
[View Larger Versions of these Images (13 + 122K GIF file)]
S and Q71L ARF1 stimulate both AP-1 and AP-2
recruitment, the finding that only AP-2 recruitment is
stimulated by PLD suggests that the mechanisms of
ARF1-dependent AP-1 and AP-2 recruitment may differ.
). As previously shown, in the absence of GTP
S or PLD, AP-2 adaptors
were recruited onto the plasma membrane (Fig. 4 c) and
AP-1 adaptors showed a faint perinuclear localization
(Fig. 4 d), while in the presence of GTP
S, AP-1 recruitment was increased (Fig. 4 f) and AP-2 adaptors bound to
a perinuclear compartment (Fig. 4 e). When bacterial PLD
was included in the incubation mixture, the enhanced AP-2 recruitment detected biochemically on rat liver membranes (Fig. 4 a) was seen to correlate with perinuclear
staining (Fig. 4 g), morphologically similar to that induced
by GTP
S (Fig. 4 e). Some punctate plasma membrane
staining was also observed, but this did not seem to be increased in the presence of PLD. Double labeling of the
same cells for
-adaptin confirmed the lack of effect of PLD on AP-1 adaptor recruitment (Fig. 4 h).
S exerts its effect through the
cell's endogenous PLD. If this is the case, then inhibitors
of the endogenous PLD activity would be expected to prevent or reduce the GTP
S-stimulated AP-2 recruitment.
Although multiple PLD activities have been described, it
has been reported that the mammalian ARF-dependent
PLD requires PIP2 as a cofactor for optimum activity
(Hammond et al., 1995
). Thus, we investigated the effects
of including the drug neomycin, a high-affinity ligand of
PIP2 that inhibits membrane-bound PLD activity (Liscovitch et al., 1994
), in the incubation.
S (Fig. 5 a). 0.1 mM
neomycin gave a lesser but still detectable inhibition. We
also assessed the effect of neomycin on AP-2 recruitment
induced by the dominant active Q71L ARF1 since it is formally possible that ARF and PLD may independently lead to the binding of AP-2 adaptors to endosomes. However,
neomycin had the same effect on Q71L-induced recruitment as on GTP
S-induced recruitment, indicating that
the activities of ARF1 and PLD are in fact coupled (data
not shown). In contrast, the recruitment of AP-1 adaptors
was unaffected by neomycin, even at 1 mM (Fig. 5 b).
Fig. 5.
Effect of neomycin on the recruitment of adaptors onto
rat liver membranes. Rat liver membrane pools (see Fig. 3) were
incubated with cytosol in the absence or presence of GTPS and
varying concentrations of neomycin. The effect of neomycin on
AP-1 and AP-2 recruitment was assessed by Western blotting of
the membrane samples using anti-
(a) or anti-
(b) antibodies.
Neomycin, which indirectly inhibits PLD activity, decreases the
amount of AP-2 recruitment both in the absence and in the presence of GTP
S but has no appreciable effect on AP-1 recruitment.
[View Larger Version of this Image (30K GIF file)]
S
was much reduced by the presence of 1 mM neomycin in
the incubation (Fig. 6, compare a and c). In the same cells,
neomycin did not appear to affect the basal levels of AP-1
(Fig. 6, b and d). Similarly, the GTP
S-induced perinuclear targeting of AP-2 adaptors was also strongly inhibited (Fig. 6, compare e and g), while AP-1 recruitment in
the same cells was apparently unaffected by treatment
with neomycin (Fig. 6, f and h). Thus, these experiments
confirm a role for PLD activity in the binding of AP-2 to
endosomal membranes in vitro, and in addition they implicate PLD in the normal recruitment of AP-2 adaptors
onto the plasma membrane.
Fig. 6.
Effect of neomycin on adaptor recruitment in permeabilized cells. Permeabilized NRK cells were allowed to recruit adaptors from cytosol in the absence (a-d) or presence (e-h) of GTPS. Neomycin (1 mM) was included in c, d, g, and h. For each of the conditions, the cells were double labeled for immunofluorescence using anti-
(a, c, e, and g) or anti-
(b, d, f, and h) antibodies. Photographs
in the presence and in the absence of neomycin were taken and printed under identical conditions. Neomycin inhibits the recruitment of
AP-2 adaptors both onto the plasma membrane in the absence of GTP
S and onto the endosomal compartment in the presence of
GTP
S, without showing any effect on AP-1 recruitment. Bar, 10 µm.
[View Larger Version of this Image (79K GIF file)]
Fig. 7.
Comparison of the effects of neomycin on AP-2 recruitment and on PLD activity. (a) Permeabilized NRK cells were allowed to recruit adaptors from cytosol either without GTPS,
with GTP
S at 4°C, or with GTP
S plus increasing concentrations of neomycin and then assayed by Western blotting and
phosphorimager quantification for AP-2 recruitment. The relatively weak effect of GTP
S is due to the use of whole cells rather
than membrane fractions. Neomycin inhibits AP-2 recruitment in
a dose-dependent manner, with essentially complete inhibition at
3 mM. (b) NRK cells were labeled overnight with [3H]palmitic
acid, permeabilized, and then incubated with cytosol containing
1% butanol either without GTP
S or with GTP
S plus increasing
concentrations of neomycin. Lipids were extracted and subjected
to TLC, and PLD activity was assayed by quantifying the production of phosphatidyl butanol. In one sample, the butanol was
omitted as a control. PLD activity is stimulated ~30% by the addition of GTP
S and inhibited by neomycin, although even at 3 mM it is only inhibited ~40%. This probably reflects the existence of multiple types of PLD in the cell, not all of which can be inhibited by neomycin, and may also indicate that neomycin has a
direct effect on AP-2 recruitment, as well as an indirect effect by
inhibiting PLD.
[View Larger Version of this Image (26K GIF file)]
S on
AP-2 recruitment was less pronounced than on membrane
fractions, although recruitment could be inhibited by incubating the cells at 4°C (Fig. 7 a). When neomycin was
added to the cells, there was a steady decline in recruitment with increasing concentrations of the drug, and at 3 mM neomycin, AP-2 binding was essentially undetectable. Increasing concentrations of neomycin also inhibited PLD
activity, but the effect was less pronounced, and even at 3 mM the production of phosphatidyl butanol in GTP
S-treated cells was only diminished by ~40% (Fig. 7 b).
However, it is worth noting that the addition of GTP
S
only stimulated PLD activity by ~30% over the level
found in the absence of GTP
S. This result suggests that
much of the PLD activity is ARF independent and might
not be susceptible to neomycin. In addition, it is possible
that PIP2 may play a direct role in AP-2 recruitment, as
well as an indirect role via PLD (see Discussion).
S, Q71L ARF1, and PLD all cause AP-2
adaptors to bind to a perinuclear compartment. However,
there are many organelles concentrated in the perinuclear
region of the cell. To confirm that all three treatments
cause the adaptors to bind to the same compartment, we
investigated the localization of the newly recruited AP-2
adaptors by subcellular fractionation and by immunogold
electron microscopy.
S,
Q71L ARF1, or PLD, and the recruitment of adaptor
complexes onto each pool was assessed by Western blotting and quantified using a phosphorimager. Membranes binding AP-1 and AP-2 adaptors in the presence of
GTP
S had distinct fractionation profiles, as shown previously (Fig. 8 a; see also Fig. 1 b). AP-2 adaptors recruited
in the presence of Q71L ARF1 gave a very similar profile
to that seen with GTP
S (Fig. 8 a). In a separate experiment, fractions were incubated with cytosol containing either GTP
S or exogenous PLD and then assayed for newly recruited AP-2, and again the profiles were found to
be similar (Fig. 8 b).
Fig. 8.
Fractionation of
membranes binding adaptors
under various conditions.
Pools of pairs of membrane
fractions from Nycodenz gradients were incubated with
cytosol in the presence of
GTPS or 100 µg/ml Q71L
ARF (a) or in a separate experiment in the presence of
GTP
S or 12.5 µg/ml PLD
(b). Newly recruited AP-2 or
AP-1 adaptors were detected
on Western blots using anti-
-
or anti-
-adaptin antibodies, respectively. The signal in
each sample was quantified
using a phosphorimager. As
also shown in Fig. 1 b,
- and
-adaptin-binding membranes
have distinct fractionation
profiles. However, the profile of the
-adaptin-binding
membranes is similar
whether GTP
S, Q71L ARF1,
or PLD is added.
[View Larger Version of this Image (20K GIF file)]
S, Q71L ARF1, or PLD
and then prepared frozen thin sections of the cells for immunogold electron microscopy. When the recruitment was
carried out in the presence of GTP
S, the AP-2 adaptors
(labeled with large gold particles) were detected on endosome-like structures, often with internal membranes, and
on smaller tubulovesicular elements, often in close proximity to the Golgi stack (Fig. 9, a and b). A proportion of
these structures were also positive for lgp110, a marker for
late endosomes and lysosomes (small gold particles) (Reaves
et al., 1996
). These data are consistent with the previous
identification of this compartment as a late endosome (Seaman et al., 1993
). When either Q71L ARF1 or PLD
were included in the cytosol, AP-2 adaptors were recruited
to structures of a similar appearance to those observed
with GTP
S, including multivesicular bodies that sometimes colabeled for lgp110 and smaller tubulovesicular profiles (Fig. 9, c-f). We could detect no significant morphological difference in the membrane structures onto
which AP-2 adaptors were recruited under these three
conditions, indicating that GTP
S, Q71L ARF1, and PLD
all activate the same pathway.
Fig. 9.
Immunogold EM labeling of newly recruited AP-2 adaptors. Frozen thin sections were prepared of permeabilized NRK cells
that had been allowed to recruit proteins from cytosol in the presence of either GTPS (a and b), Q71L ARF1 (100 µg/ml) (c and d), or PLD (12.5 µg/ml) (e and f). The sections were labeled with antibodies against newly recruited
-adaptin (15-nm gold) and lgp110 (8-nm gold). The arrows show membranes that are positive for both antigens, the large arrowheads show membranes that are positive for
-adaptin only, and the small arrowheads show membranes that are positive for lgp110 only. The same types of membranes are labeled
under all three conditions. Bar, 200 nm.
[View Larger Version of this Image (109K GIF file)]
Discussion
S. In
the case of AP-1 adaptors, GTP
S stimulates their recruitment onto the TGN, the membrane with which they are
normally associated, and this event has been shown to be
mediated by ARF1 (Robinson and Kreis, 1992
; Stamnes
and Rothman, 1993
; Traub et al., 1993
; Seaman et al.,
1996
). In contrast, GTP
S causes AP-2 adaptors to be recruited onto an endosomal compartment, even though
they are normally associated with the plasma membrane
(Seaman et al., 1993
). Excess Ca2+ was found to have a
similar effect (Seaman et al., 1993
), and a recent study reported that AP-2 adaptors can also bind to lysosomes in
vitro, although in this case the recruitment was carried out under essentially physiological conditions, so it is not clear why the adaptors bound to this compartment (Traub et al.,
1996
). In addition, in living cells certain cationic amphiphilic drugs such as chlorpromazine cause AP-2 adaptors to bind to late endosomes and/or lysosomes (Wang et
al., 1993
). Taken together, these studies indicate that AP-2
docking sites are present on membranes of the endolysosomal system as well as the plasma membrane and that a number of stimuli can switch them on. Whether the sites
on these membranes are normally completely inactive,
perhaps acting as a storage pool that can be mobilized to
the plasma membrane, or whether there is a very low level
of recruitment onto endosomes and lysosomes to facilitate
a particular trafficking pathway, is still not known.
S on AP-2 adaptors. We found that when ARFs and
other low molecular weight GTP-binding proteins were
removed from cytosol by gel filtration, the GTP
S effect
was lost but could be restored by the addition of recombinant ARF1, indicating that ARF1 is the target of the GTP
S. Further evidence for a role for ARF1 came from
our finding that the constitutively active ARF1 mutant,
Q71L ARF1, can substitute for GTP
S. Whether other
ARFs are equally effective at driving AP-2 onto endosomes is still not known. The endosomal membranes that
recruit AP-2 adaptors have relatively little ARF1 or any of
the other ARFs associated with them, suggesting that
ARF is acting catalytically rather than stoichiometrically.
Studies from other labs have shown that ARF activates
PLD (Brown et al., 1993
; Cockcroft et al., 1994
), and PLD
has been implicated in the recruitment of coatomer onto
Golgi membranes (Ktistakis et al., 1996
). Similarly, we
found that exogenous PLD causes AP-2 adaptors to bind
to endosomes, indicating that ARF1 promotes recruitment
onto endosomes by activating endogenous PLD. Interestingly, two other treatments that cause AP-2 to bind to endosomes, elevated Ca2+ levels and cationic amphiphilic
drugs, have also been reported to activate PLD (Brown et
al., 1995
; Liscovitch et al., 1994
; Siddiqi et al., 1995
). In
contrast, exogenous PLD has no apparent effect on AP-1
adaptors, even though both GTP
S and Q71L ARF1 greatly stimulate the recruitment of AP-1 adaptors onto
the TGN.
). In our in vitro system, it inhibits not only the
recruitment of AP-2 onto endosomes but also the recruitment of AP-2 onto the plasma membrane, without affecting the recruitment of AP-1 onto the TGN. These observations suggest that under physiological conditions, endogenous
PLD associated with the plasma membrane may play a
role in the recruitment of AP-2 adaptors. However, it is
difficult to dissociate the effects of PIP2 and PLD because
once the PLD is activated, it generates PA from phosphatidylcholine, and the PA can in turn activate phosphatidylinositol 4-P 5 kinase to generate more PIP2 (Jenkins et al., 1994
). Thus, either one or both of these lipids
may be involved in AP-2 recruitment.
; Gaidarov et al., 1996
). Second, AP-2 adaptors require ATP in order to be recruited onto membranes (Seaman et al., 1993
; Traub et al., 1996
), and one possible role for the ATP may be in the production of PIP2. Third, neomycin has a stronger inhibitory effect on AP-2 recruitment
than on PLD activity, and although this result may be due
at least in part to interference from other types of PLD
that do not require PIP2, it also suggests that PIP2 may
play a more direct role in AP-2 recruitment. Fourth, we
have found that neomycin inhibits the recruitment of AP-2
adaptors onto endosomes not only in cells that have been
treated with GTP
S or Q71L ARF1, both of which presumably work by stimulating the cell's endogenous PLD,
but also in cells that have been treated with exogenous PLD
from bacteria (data not shown). Since it is unlikely that
bacterial PLD requires PIP2 as a cofactor, this result also
indicates that PIP2 plays a direct role in AP-2 recruitment.
It may also be significant that AP-2 adaptors bind unusually tightly to hydroxylapatite (i.e., calcium phosphate)
(Pearse and Robinson, 1984
), suggesting that AP-2 adaptors may have an affinity for phosphate groups in general.
), and some of its aspects can be
adapted to describe other coating events as well. ARFs are
recruited from the cytosol through interaction with a
BFA-sensitive nucleotide exchange factor (Donaldson et al., 1992
; Helms and Rothman, 1992
; Randazzo et al., 1993
;
Peyroche et al., 1996
), which catalyzes the exchange of
bound GDP for GTP. This leads to the exposure of the
myristoyl group on ARF, facilitating its association with
the membrane. The active ARF1, in conjunction with PIP2,
stimulates an endogenous PLD resulting in a local increase in PA. PA then stimulates phosphatidylinositol 4-P
5 kinase to produce PIP2, which in turn further stimulates
PLD to generate more PA and in turn more PIP2. Such
positive feedback could rapidly lead to a local increase in
these acidic phospholipids. It seems unlikely that AP-2
binding is mediated by acidic phospholipids alone since
AP-2 is only recruited onto a specific subset of membranes. Thus, we favor the hypothesis that there is an
AP-2-specific docking protein or protein complex, localized both on the plasma membrane and on endosomes and
lysosomes, which is responsible for the initial binding of
AP-2 to the membrane. This docking site may act only transiently, after which AP-2 may become more stably associated with the membrane through interactions with
acidic phospholipids, and possibly also through interactions with the cytoplasmic domains of membrane proteins
that carry internalization signals.
Fig. 10.
Schematic model for the recruitment of AP-2 adaptors onto endosomal membranes. This model is based on the model of
Traub and Kornfeld for AP-1 adaptor recruitment (Traub et al., 1993), but with several important modifications. We propose that
ARF1 does not bind directly to the putative docking protein, but instead activates PLD, either directly or indirectly. Activated PLD, in the presence of PIP2, may lead to rapid local changes in the concentrations of negatively charged phospholipids such as PA and PIP2.
These negatively charged phospholipids, together with the putative docking protein, would constitute an AP-2 binding site.
[View Larger Version of this Image (17K GIF file)]
).
; Pfeffer,
1994
). Similarly, AP-2 adaptors may interact with certain
lipid head groups after their initial binding to a specific
docking site. If the lipid head groups are rapidly turning
over, as is the case for both PA and PIP2, this would suggest a means for releasing the adaptors once the coated
vesicle had formed (De Camilli et al., 1996
). Possibly such
lipids are normally generated on the plasma membrane
rather than on endosomes, but addition of reagents such as
GTP
S, constitutively activated ARF1, Ca2+, cationic amphiphyllic drugs, or exogenous PLD may cause unusually high levels of these lipids to be generated on endosomes
and/or lysosomes. Whether the production of such lipids at
the plasma membrane depends upon an ARF or another
small GTP-binding protein is still not known, although
ARF6 is a potential candidate. ARF6, like the other
ARFs, has been shown to stimulate PLD (Brown et al.,
1995
), but it differs from the other ARFs in that its
membrane association is unaffected by GTP
S or BFA.
Overexpressed ARF6 has been localized to the plasma
membrane and endosomes, and constitutively active or
constitutively inactive forms of ARF6 cause cells to accumulate plasma membrane or endosomal membranes, respectively, consistent with a role in endocytosis (DsouzaSchorey et al., 1995
; Peters et al., 1995
; Cavenagh et al.,
1996
).
), and coatomer has also been shown to interact
with inositol phosphates (Fleischer et al., 1994
). In addition, membrane recruitment of the AP-3 adaptor-related complex, which is BFA sensitive and therefore likely to require an ARF (Simpson et al., 1996
), was significantly enhanced in the presence of exogenous PLD (data not
shown). Like AP-2, AP-3 requires ATP for its recruitment
in vitro (Simpson et al., 1996
) and binds tightly to hydroxylapatite (Simpson, F., and M.S. Robinson, unpublished observations). However, from the data presented here for
AP-1 adaptors, it is clear that the model may not always be
entirely applicable and that there may be variations on the
general mechanism. For example, although there is much
evidence to indicate that ARF1 mediates AP-1 binding to
membranes (Seaman et al., 1996
; Stamnes and Rothman,
1993
; Traub et al., 1993
), we found that neither exogenous
PLD nor neomycin had any effect on AP-1. In addition,
AP-1 recruitment does not require ATP (Simpson et al., 1996
), indicating that PIP2 is not involved, it does not bind particularly well to hydroxylapatite (Pearse and Robinson,
1984
), and there is no evidence that it binds inositol phosphates or inositol phospholipids.
; Kanoh et al., 1997
). As
coat proteins and small GTP-binding proteins coevolved,
it seems likely that different coats became specialized to
make use of different regulatory mechanisms, while still
retaining a general requirement for ARF or an ARF-related protein.
Received for publication 10 April 1997 and in revised form 27 June 1997.
Address all correspondence to Margaret S. Robinson, University of Cambridge, Department of Biochemistry, Cambridge CB2 2QR, U.K. Tel.: 44 1223 330163. Fax: 44 1223 330598.This work was supported by grants from the Wellcome Trust and the Medical Research Council.
AFR, ADP-ribosylation factor; BFA, brefeldin A; NRK, normal rat kidney; PA, phosphatidic acid; PIP2, phosphatidylcholine 4,5-bisphosphate; PLD, phospholipase D.
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