(Received for publication, August 25, 1995; and in revised form, November 14, 1995)
From the
Clathrin coat assembly in the trans-Golgi network, leading to
the sequestration of the mannose 6-phosphate receptors (MPRs) into
nascent vesicles, requires the ARF-1-dependent translocation of the
cytosolic AP-1 Golgi assembly proteins onto the membranes of this
organelle. The mechanistic role of the MPRs, i.e. the cargo
molecules, in coat assembly is at present unclear. Using a
GTP-dependent, brefeldin A-sensitive in vitro AP-1 binding
assay, we have determined here the parameters of the AP-1 binding
reaction. We demonstrate that, in addition of ARF-1, the MPRs
contribute to create high affinity AP-1 binding sites (K
25 nM), since their
number correlates the number of MPR molecules expressed in MPR-negative
cells. The quantitative electron microscopy shows that these high
affinity binding sites are present on trans-Golgi network membranes, as
expected, and to some extent on early endosomes. The high affinity
binding sites are lost when the MPRs or ARF-1 become rate-limiting
components. Conversely, GTP
S (guanosine
5`-O-(3-thiotriphosphate)), which increases the amount of
membrane-bound ARF-1, mostly uncovers low affinity AP-1 binding sites (K
150 nM) on trans-Golgi
network membranes, normally not detected in its absence. Collectively,
these results argue that MPR sorting is highly coupled to the first
step of coat assembly and that the MPRs, ARF-1, and possibly other
proteins cooperate for high affinity interactions of AP-1.
In eukaryotic cells, vesicular transport from the trans-Golgi
network (TGN) ()and from the plasma membrane to endosomes
involves the formation of clathrin-coated vesicles as transport
intermediates (Kornfeld and Mellman, 1989; Pearse and Robinson, 1990).
Although the plasma membrane- and the Golgi-derived vesicles share
clathrin as one of the major coat components, their coat can be
distinguished by the nature of the underlying assembly proteins (APs).
Immunofluorescence localization studies indicate that AP-1 is
restricted to Golgi-derived vesicles, whereas AP-2 is associated with
plasma membrane-derived vesicles (Robinson, 1987; Ahle et al.,
1988). Although it is still not understood how the APs specifically
interact with their target membranes (Robinson, 1992, 1994), these coat
proteins are now well characterized biochemically (Morris et al. 1989; Pearse and Robinson, 1990), and the cDNAs encoding their
different subunits have been isolated (Robinson, 1989; Kirchhausen,
1989; Robinson, 1990; Ponnanbalam, 1990). AP-1 is a heterotetrameric
complex composed of two
100-kDa subunits (the
and
`
adaptins) associated with a 47-kDa and a 19-kDa polypeptide. AP-2 has a
similar protein composition also made of two large subunits of
100
kDa (the
and
adaptins) associated with a 50-kDa and a
17-kDa polypeptide.
Due to their topological position, APs are
likely to play a major role in coat assembly by interacting with both
clathrin triskelions and the appropriate membrane. Their
heterotetrameric structure is probably a reflection of these multiple
functions. In vitro reconstitution studies indicate that
purified APs can bind to reconstituted clathrin cages (Pearse and
Robinson, 1984). It has been proposed that the and
`
adaptins, two closely related polypeptides (Ahle et al.,
1988), mediate this interaction, because these purified adaptins (Ahle et al., 1988; Ahle and Ungewickell, 1989) or the recombinant
and
` adaptins (Gallusser and Kirchhausen, 1993) also bind
to reconstituted clathrin cages via their NH
-terminal,
trunk domains. In vitro assays also indicate that, besides
interacting with clathrin, APs can bind to cytoplasmic domains of
receptors, thereby mediating their clustering into plasma membrane- or
Golgi-derived clathrin-coated vesicles (Pearse and Robinson, 1990).
Accordingly, purified AP-2 binds to immobilized cytoplasmic domains of
receptors recycling via the plasma membrane, including the low density
lipoprotein receptor, the mannose 6-phosphate/IGF II receptor (Pearse,
1988), the asialoglycoprotein receptor (Beltzer and Spiess, 1991) and
the lysosomal acid phosphatase (Sosa et al., 1993). In all
cases, this binding is dependent on specific endocytosis motifs in the
cytoplasmic tail of these proteins. In contrast, purified AP-1 has,
until now, only been found to bind to the immobilized cytoplasmic tail
of mannose 6-phosphate/IGF II receptor (Glickman et al., 1989)
which sorts lysosomal enzymes in the TGN (Kornfeld, 1992). The
and
adaptins, the two most divergent polypeptides of APs
(Robinson, 1990), are suspected to play a role in these interactions
(Pearse and Robinson, 1990).
Recent studies have shown that the
small GTPase ADP-ribosylation factor (ARF) not only regulates coatomer
assembly in the early secretory pathway (Donaldson et al.,
1992a; Palmer et al., 1993), but also acts as a potent
regulator of clathrin coat assembly in the TGN. The evidence comes from in vivo studies showing that the fungal metabolite brefeldin A
inhibits binding of AP-1 on Golgi membranes (Robinson and Kreis, 1992;
Wong and Brodsky, 1992) as was previously observed for -COP (Orci et al., 1991), a subunit of the coatomer required for vesicle
budding in the early secretory pathway (Rothman and Orci, 1992). It is
now known that this drug prevents the exchange of GDP for GTP on the
small GTPase ARF which is required for its membrane insertion via its
myristoylated moiety (Donaldson et al., 1992b: Helms and
Rothman, 1992). Several members of the ARF family have now been
identified in mammalian cells (Tsuchiya et al., 1991; Kahn et al., 1991), but their function remains elusive. It has been
shown that binding of AP-1 to membranes present in Golgi-enriched
fractions is stimulated upon addition of recombinant, myristoylated
ARF-1 (Stamnes and Rothman, 1993; Traub et al., 1993). ARF-1
has been involved in several transport reactions occurring along the
secretory (Balch et al., 1992; Taylor et al., 1992)
and the endocytic pathways (Lenhard et al., 1992) leading to
the proposal that ARF-1 may act as a common molecular switch for coat
assembly. Recent data suggest that ARF-6 regulates the process of
endocytosis of transferrin receptor (D'Souza-Schorey et
al., 1995). ARF-6 localizes to the plasma membrane and endosomes
and overexpression of GTP hydrolysis mutants results in extensive
plasma membrane invaginations as well as a depletion of endosomes
(Peters et al., 1995).
It is currently believed that cargo proteins are segregated into transport vesicles when the first steps of coat assembly have occurred. According to this view, the mannose 6-phosphate receptors (MPRs), two trans-membrane proteins sorted in the TGN (Kornfeld, 1992; von Figura, 1991) are clustered into nascent vesicles when AP-1 has already been recruited onto TGN membranes (Pearse and Robinson, 1990; Robinson, 1994; Traub et al., 1995). Our ealier studies have suggested that the MPRs may play a role in the recruitment of AP-1, because AP-1 binding is drastically reduced in MPR-deficient cells and the addition of the soluble cytoplasmic domain of the mannose 6-phosphate/IGF II receptor inhibits AP-1 binding almost completely (Le Borgne et al., 1993). In order to get more insight on the role of the MPRs in AP-1 recruitment, we have determined the parameters of the AP-1 binding reaction under conditions in which the concentration of the MPRs or ARF would vary in membranes. We report that AP-1 binds with high affinity to membranes in a MPR-dependent manner when ARF-1 is also present, strongly suggesting that protein sorting in the TGN is highly coupled to the first step of clathrin coat assembly. These results suggest that the first step of clathrin coat assembly on TGN membranes, namely the recruitment of AP-1, requires some cooperation between MPRs, i.e. cargo proteins and ARF-1.
The bovine brain cytosol (16 mg of
protein) was separated on a preparative Superose 6 column (1.6
50 cm) equilibrated in KOAc buffer, pH 7. The column was eluted at 0.4
ml/min with the same buffer, and 2-ml fractions were collected. AP-1
present in the different fractions were detected by Western blotting
using the mAb 100/3 anti-
-adaptin monoclonal antibody. The elution
profiles of ARF proteins was determined by GTP overlay and Western
blotting after SDS-gel electrophoresis using the 1D9 anti-ARF
monoclonal antibody. AP-1 containing fractions were pooled, dialyzed
overnight against 50 mM Tris/HCl, pH 7.5, 0.1 M NaCl,
1 mM phenylmethylsulfonyl fluoride, and applied onto a 1-ml
DEAE-cellulose column equilibrated in the same buffer. AP-1 was eluted
with 0.2 M Tris-HCl, pH 7.5, 0.1 M NaCl, 1 mM phenylmethylsulfonyl fluoride and dialyzed overnight against KOAc
buffer, pH 7. AP-1 and ARF containing fractions were concentrated in
order to reach the same concentration as that found in cytosol.
We also determined the
cytosolic concentration of AP-2. For the quantitative Western blotting,
several antibodies against AP-2 were used: the mAb AP-6 (a generous
gift from Dr. F. Brodsky) against the -subunit and the mAb 100/2
(Sigma ImmunoChemicals) against the
-subunit and two
immunopurified polyclonal antibodies raised against two synthetic
peptides corresponding to the A and C forms of the bovine
-adaptin
(these latter recognized by Western blot a single protein with the
expected molecular weight). Similar values were obtained with three
antibodies (the two polyclonal antipeptide antibodies and the mAb
100/1), the mAb AP-6 which recognizes an epitope on the
carboxyl-terminal region giving lower values. The cytosolic AP-2 was
estimated to be 450 nM.
Immunodetection of -adaptins and ARF-1 was performed as
followed. Immediately after transfer, the nitrocellulose was incubated
for 2 h in blocking buffer (1
PBS, 0.05% Tween 20, 0.1%
gelatin, 5% (w/w) defatted milk). The filter was then incubated
overnight at 4 °C with the appropriate antibody (mAb 100/3
anti-
-adaptin monoclonal antibody and 1D9 anti ARF-1 monoclonal
antibody) diluted in blocking buffer. Filters were washed 10 min in 1
PBS, two times for 10 min with 1
PBS, 0.05% Nonidet
P-40, and 10 min in 1
PBS. Filters were then incubated for 1 h
with a rabbit anti-mouse IgG coupled to horseradish peroxidase in
blocking buffer. After washing, the immunocomplexes were detected using
the ECL detection kit.
To deplete acceptor membranes of endogenous ARF proteins, the cells were incubated for 10 min at 37 °C with 100 µg/ml BFA prior to permeabilization.
The number of moles of AP-1 bound to membranes was determined by ELISA as described above using as a standard curve several concentrations of bovine brain cytosol immobilized on plastic. This determination was also performed by Western blotting after incubation of the permeabilized cells with cytosol or semi purified AP-1 and solubilization of membranes with 1% Triton X-100 using as a standard curve bovine brain cytosol or purified AP-1.
In order to understand the contribution of the MPRs in the
recruitment of AP-1 on membranes, we have re-expressed the MPRs in
MPR-negative fibroblasts and calculated the parameters of the AP-1
binding reaction (K and number of binding sites)
using the in vitro assay that we have previously described (Le
Borgne et al., 1993). This assay relies on the incubation of
SLO-permeabilized rodent cells (NRK or mouse embryonic fibroblasts)
with a bovine brain cytosol followed by the detection of the newly
bound bovine AP-1 using a species-specific monoclonal antibody against
its
-subunit. These determinations were performed in the presence
of GTP
S, as this slowly hydrolyzable analogue of GTP prevents the
uncoating of COP-coated vesicles thereby blocking vesicular transport
in the secretory pathway (Rothman and Orci, 1992; Tanigawa et
al., 1993). GTP
S also blocks the late stages of clathrin coat
assembly at the plasma membrane (Carter et al., 1992).
Figure 1: AP-1 binding and ARF-1. NRK cells were permeabilized and incubated for 10 min at 37 °C with cytosol or with partially purified AP-1 in the absence (AP-1) or the presence of 2 µM recombinant myristoylated ARF-1 (AP-1+mARF-1) or with an excess of cytosolic ARF-enriched fractions (AP-1+ARF FRACTION). NRK cells were also pretreated for 10 min with 100 µg/ml BFA, permeabilized in the absence (BFA I) or in the presence (BFA II) of 100 µg/ml BFA, and finally incubated for 10 min at 37 °C with cytosol in the presence of 100 µg/ml BFA. The amount of bound AP-1 was then quantitated by ELISA.
AP-1 recruitment becomes temperature-independent when the acceptor
membranes have been primed at 37 °C with ARF-enriched fractions. To
show this, SLO-permeabilized NRK cells, treated with BFA to remove
endogenous ARFs (Donaldson et al., 1992b; Helms and Rothman,
1992), were first incubated with ARF-enriched fractions and GTPS
at 37 °C to allow efficient membrane insertion of these GTPases.
They were then washed and subsequently incubated at 4 or 37 °C with
semi-purified AP-1. Fig. 2shows that AP-1 binds to membranes
whether this subsequent incubation is performed at 4 or 37 °C and
with the same efficiency as when ARF proteins and AP-1 are
simultaneously incubated with membranes at 37 °C. Therefore, AP-1
behaves as the coatomer which is also recruited in a
temperature-independent manner, provided that the membranes have been
primed at 37 °C in order to incorporate ARF (Palmer et
al., 1993).
Figure 2:
Effect of the temperature on AP-1 binding.
NRK cells were permeabilized with SLO, preincubated at 37 °C as
indicated. They were re-incubated at 4 or 37 °C in the presence or
the absence of GTPS with cytosol or with ARF-enriched fractions (ARF) and semi-purified AP-1 (AP-1) as indicated. The
amount of membrane-bound AP-1 was then determined. The values are the
means ± S.E. of two independent experiments performed in
duplicates.
Figure 3:
Effect of GTPS on AP-1 binding. A, NRK cells were pretreated for 10 min at 37 °C with
Brefeldin A (100 µg/ml), permeabilized with SLO in the absence of
Brefeldin A, and incubated in the presence of GTP
S with increasing
amounts of partially purified AP-1 in the absence (
) or the
presence of an excess of ARF-enriched fractions (
) or 2
µM recombinant myristoylated ARF-1 (
). For
comparison, BFA-treated NRK cells were incubated with increasing
amounts of cytosol in the presence (
) or the absence of
GTP
S (
). The amount of bound AP-1 was then quantitated, and
the concentration curves were linearized according to the Scatchard
method (B and C). The indicated values are means
± S.E. of three independent experiments performed in
duplicate.
Figure 4:
Effect of GTPS on AP-1 distribution
in HeLa cells. HeLa cells expressing a VSV-G-tagged sialyltransferase
were first incubated with fluorescein isothiocyanate-transferrin for 90
min at 37 °C. Then, they were either permeabilized with saponin in
the cold and fixed (A, B, E, F) or permeabilized with SLO,
incubated with bovine brain cytosol in the presence of GTP
S (C, D, G, H), and then fixed. The different samples were
processed for double immunofluorescence confocal microscopy. A, B,
C, and D are focal planes of the Golgi region, and E,
F, G, and H are focal planes of the bottom of the cells
close to the substratum. A, C, E, and G, AP-1
staining; B and D, sialyltransferase staining; F and H, internalized transferrin. The processing of the
images does not take into account the differences in the intensity of
fluorescence associated with the
-adaptin as observed between the
nonpermeabilized and the permeabilized cells incubated with cytosol and
GTP
S. The same AP-1 distribution was obtained in permeabilized
cells incubated with cytosol alone (not
shown).
The electron microscopy confirmed and extended these
results. Both in vivo and in permeabilized cells systems, the
anti--adaptin antibody decorated two distinct kinds of structures (Fig. 5). First, it labeled membrane structures in the vicinity
of the Golgi stack that also contained the sialyltransferase. Second,
it labeled some early endosomal structures identified by the presence
of endocytosed BSA-gold. Both in vivo and in vitro,
the late endocytic structures as well as other intracellular organelles
remained unlabeled, thereby confirming the specificity of the in
vitro interactions. Table 2shows the quantitation of these
experiments. While the permeabilization allows an apparent better
detection of the membrane-bound AP-1, it does not appear to modify its
distribution on membranes when compared with nonpermeabilized HeLa
cells. In permeabilized cell systems, GTP
S induced a strong,
3-4-fold increase in the density of AP-1 labeling on the
membranes of the sialyltransferase-rich compartment and a more modest
2-fold increase on membranes of early endosomes. This quantitation is
consistent with the biochemical experiments showing that GTP
S
induces an overall 2-3-fold increase of AP-1 binding. Thus, the
results show that the low affinity AP-1 binding sites generated by the
addition of GTP
S are localized to the same compartments as the
high affinity binding sites.
Figure 5:
Distribution of AP-1 on cryosections of
HeLa cells. Hela cells expressing a VSV-G tagged sialyltransferase were
first incubated with BSA-gold under conditions to label early endosomes (EE, 5 nm gold) (C) or late endocytic structures (LE, 16 nm gold) (B and C). The cells were
permeabilized, incubated with cytosol in the presence of GTPS, and
then processed for electron microscopy. In A and B,
the sections were double-labeled with an anti-VSV-G tag polyclonal
antibody (5 nm gold, small arrows) which identifies the TGN (T) on one side of the Golgi stacks (G) and the 100/3
anti-
-adaptin monoclonal antibody (10 nm gold, arrowheads). In C, single labeling with the 100/3
anti-
-adaptin monoclonal antibody was also performed for
localization on endosomes. A and B, examples of AP-1
labeling over the Golgi region. Most of the AP-1 labeling is
distributed in a nonclustered fashion on the TGN membrane (small
arrowheads). Some coated buds (large arrowheads) are also
labeled. C shows an example of
-adaptin-labeled buds on
early endosome elements (arrowheads) identified by 5 nm
BSA-gold (small arrows). Late endosomes identified by 16 nm
BSA-gold do not label for
-adaptin (B, C). Bars:
100 nm. The same distribution was observed in nonpermeabilized cells or
in permeabilized cells incubated with cytosol. The quantitation of
these experiments is shown in Table 2.
Figure 6:
AP-1 binding in MPR-positive and -negative
cells. A, wild type fibroblasts, MPR-negative fibroblasts
(vector alone), or MPR-negative fibroblasts stably re-expressing either
the cation-independent mannose 6-phosphate/IGF II receptor (CI-MPR) or the cation-dependent (CD-MPR) mannose
6-phosphate receptor were permeabilized with streptolysin O, incubated
with bovine brain cytosol in the presence of GTPS, and the
membrane-bound AP-1 was then quantitated as described under
``Experimental Procedures.'' The values represent the means
± S.D. of two experiments performed on independent clones
re-expressing physiological levels of cation-dependent MPR or
cation-independent MPR (
1.5- and
1.9-fold the endogenous level,
respectively). B, MPR-negative fibroblasts (
) or the
corresponding wild type fibroblasts (
) were permeabilized with
streptolysin O, incubated with increasing amounts of cytosol in the
presence of GTP
S, and the membrane-bound AP-1 was then
quantitated. The indicated values are means ± S.E. of three
independent experiments performed in duplicate. The concentration
curves were linearized according to the Scatchard method (C).
Scatchard analyses were performed on these MPR-positive
and -negative fibroblasts in the presence of GTPS (Fig. 6, B and C). Linearization of the concentration curves
shows that permeabilized MPR-positive fibroblasts also exhibit two
types of binding sites for AP-1 in the presence of GTP
S (Table 1): high affinity binding sites with an apparent K
22 nM and low affinity binding
sites with an apparent K
150 nM.
These two types of binding sites could still be detected in
MPR-negative embryonic fibroblasts. However, the number of high
affinity binding sites was drastically reduced to 25% of the control
values and that of the low affinity binding sites to 35% (Fig. 6C and Table 1). We believe that these
residual high affinity binding sites are due to additional proteins
that are similarly sorted via this clathrin-dependent pathway.
Scatchard analyses were also performed on MPR-negative fibroblasts
expressing different amounts of the cation-dependent mannose
6-phosphate receptor and the parameters of the AP-1 binding reaction
determined. Fig. 7shows that the number of high affinity
binding sites for AP-1 that are detected depends on the concentration
of MPR in membranes. These results clearly demonstrate that the MPRs, i.e. cargo proteins are key components for the interactions of
AP-1 with membranes and that they are essential to create the high
affinity binding sites for AP-1.
Figure 7:
MPR expression and high affinity AP-1
binding sites. The mouse cation-dependent MPR was re-expressed in
MPR-negative fibroblasts and clones expressing different levels of the
cation-dependent MPR were selected. The level of expression refers to
the endogenous cation-dependent MPR expressed in control fibroblasts
expressing the two MPRs. The cells were permeabilized with SLO,
incubated with increasing amounts of cytosol in the presence of
GTPS. The concentration curves were linearized according to the
Scatchard method and the number of high affinity binding sites
calculated. The indicated values are means ± S.E. of two or
three independent experiments performed in
duplicate.
The results described above
indicate that ARF proteins and the MPRs are simultaneously required in
order to provide the high affinity binding sites for AP-1. We have
previously observed that the addition of the soluble, phosphorylated
mannose 6-phosphate/IGF II receptor cytoplasmic domain fused to the
glutathione S-transferase could completely abolish AP-1
binding onto membranes (Le Borgne et al., 1993). Thus, one
could anticipate that this soluble, phosphorylated cytoplasmic domain
could become a better competitor of the AP-1 binding reaction when low
affinity interactions of AP-1 with membranes are measured in
ARF-limiting conditions. As shown before, the addition of the
phosphorylated tail fused to the glutathione S-transferase
completely abolished AP-1 binding in the ARF-complemented system with a
50% inhibition observed at 1 µM, while the glutathione S-transferase alone or a 50 molar excess of phosphorylated or
nonphosphorylated synthetic peptides corresponding to the last 13
highly charged amino acids of the MPR tail remained without any effect (Fig. 8). However, the fusion protein becomes a stronger
inhibitor of AP-1 binding in the ARF-limiting system, and 50%
inhibition was obtained with a much lower concentration of
300
nM.
Figure 8:
Competition of AP-1 binding in the absence
or the presence of ARF. A fusion protein corresponding to the
full-length soluble cytoplasmic domain of the mannose 6-phosphate/IGF
II receptor fused to the glutathione S-transferase was first
phosphorylated in vitro as described under ``Experimental
Procedures.'' NRK cells pretreated with BFA before
permeabilization were then incubated with partially purified AP-1 and
increasing concentrations of the phosphorylated fusion protein in the
absence () or the presence (
) of an excess of ARF-enriched
fractions. The bound AP-1 was then quantitated. As controls, 5
µM glutathione S-transferase (
) and 50
µM peptide corresponding to the highly charged COOH
terminus of the Man-6-P/IGF II receptor (AAATPISTFHDDSDEDLLHV)
phosphorylated (
) or not phosphorylated (
) on the more
distal serine (Méresse et al., 1990)
were used. The indicated values represent the means ± S.E. of
two independent experiments performed in
duplicate.
The formation of clathrin-coated vesicles in the TGN requires the recruitment of the cytosolic Golgi-specific assembly protein AP-1 and clathrin on the membrane of this organelle. The interaction of AP-1 with its target membrane is regulated by the small GTPase ARF-1 (Stamnes and Rothman, 1993; Traub et al., 1993). During this process, the MPRs and their bound lysosomal enzymes are clustered into the nascent vesicles (Geuze et al., 1992). We show here that the high affinity binding of AP-1 to membranes also requires the presence MPRs, i.e. the cargo transmembrane proteins, strongly suggesting that MPR sorting in the TGN is highly coupled to the first step of clathrin coat assembly. These high affinity interactions are lost when either the MPRs or ARF proteins become rate-limiting. This suggests that these components cannot promote by themselves efficient recruitment of AP-1 and that they cooperate to trigger efficient coat assembly on TGN membranes.
Both in vivo and in vitro, AP-1 recognizes
specific features of the binding sites provided by the TGN (or
endosome?) membranes. ARF-1 probably acts as a general factor for coat
assembly, since it regulates both AP-1 and coatomer binding onto
enriched Golgi membranes (Stamnes and Rothman, 1993; Traub et
al., 1993; Palmer et al., 1993), coatomer-mediated
transport in the early secretory pathway (Balch et al., 1992;
Taylor et al., 1992; Dasher and Balch, 1994; Zhang et
al., 1994a) as well as in vitro endosome fusion (Lenhard et al., 1992). The MPRs could potentially provide some of the
features required for a specific interaction of AP-1 with its target
membrane. Although the MPRs are present in several compartments that do
not recruit AP-1, the different pools of receptors are not totally
equivalent owing to the phosphorylation of their cytoplasmic domains
(Méresse et al., 1990;
Méresse and Hoflack, 1993; Hemer et al.,
1993). Indeed, our subsequent studies show that these phosphorylation
sites are important for the high affinity interaction of AP-1 with
membranes (Mauxion et al., 1996). It is likely that the high
affinity binding sites for AP-1 also contain additional unknown
proteins that may also contribute to the specificity of interaction.
Some support to this view comes from our observation showing that the
addition of GTPS, which largely uncovers low affinity AP-1 binding
sites, does not affect the specificity of interaction of AP-1 with its
target membranes. The putative ARF receptor (Helms et al.,
1993; Traub et al., 1995) or other proteins similar to those
described by Anderson and colleagues for the interaction of AP-2 with
membranes (Mahafrey et al., 1990; Chang et al., 1993;
Peeler et al., 1993; Zhang et al., 1994b) could
potentially fulfill this function.