From Biochemie-Zentrum Heidelberg, University of
Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany and
¶ Laboratorium für Biochemie, ETH Zürich,
Universtitätstrasse 16, CH-8092 Zürich, Switzerland
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ABSTRACT |
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A site-directed photocross-linking approach was
employed to determine components that act downstream of
ADP-ribosylation factor (ARF). To this end, a photolabile phenylalanine
analog was incorporated at various positions of the putative effector
region of the ARF molecule. Depending on the position of incorporation,
we find specific and GTP-dependent interactions of ARF with
two subunits of the coatomer complex, COPI1-coated vesicles
transport proteins and lipids between intracellular compartments along
the secretory pathway (1, 2). The initial step in the formation of
these vesicles involves the recruitment of two factors from the cytosol
to the Golgi membrane; ADP-ribosylation factor (ARF) binds to Golgi
membranes in a GTP-dependent manner (3-5) with subsequent
recruitment of coatomer, a soluble complex of seven subunits (COPs) (6,
7).
Coatomer has a KKXX-COOH binding site (8), capable of
binding both the dibasic ER retrieval motifs and the
diphenylalanine motifs of members of the p24 family of proteins (9,
10). Conflicting data have been obtained concerning the binding site(s) of these motifs within coatomer. Using an affinity column containing a
KKXX sequence, a coatomer subcomplex of We previously used a site-directed photocross-linking approach to
elucidate components that interact directly with ARF1 during coat
assembly (15). This method is based on the replacement of an endogenous
amino acid with a photolabile analog of phenylalanine, L-4'-(3-trifluoromenthyl-3H-diazirin-3-yl)phenyalanine
((Tmd)Phe) (16, 17). It was found that this analog in position 82 of ARF1 directly and exclusively interacts with the ARF, however, has been implicated in several other biochemical
reactions such as activation of phospholipase D (18, 19) and
interaction with heterotrimeric G proteins (20, 21) and biologically
active phospholipids (22). To determine whether ARF interacts with
additional effector molecules, we inserted the (Tmd)Phe analog at
various positions of the putative effector loop of ARF, identified in
its crystal structure and based on its similarity with Ras (23). We
find that, depending on the site of insertion, the effector loop of ARF
interacts with Synthesis of Site-specific Photolabile ARF Mutants--
For
details, refer to Zhao et al. (15). In short, the codon of
the ARF cDNA corresponding to the amino acid position of interest
was replaced with amber stop codon using the QuickChange site-directed
mutagenesis kit from Stratagene. Amber suppressor tRNA, charged
chemically with the photolabile amino acid (Tmd)Phe, was synthesized as
described (16, 17). In vitro transcription using T7 RNA
polymerase was performed with linearized plasmids according to the
manufacturer's protocol. In vitro translation using
Flexi-lysate (Promega) was performed in the presence of [35S]methionine and 5 µM suppressor tRNA at
30 °C for 2 h. The suppression efficiency was analyzed by
SDS-PAGE (24) and subsequent autoradiography.
Irradiation of the Photolabile ARF Mutants--
20 µl of the
in vitro translation samples (performed as described above)
was incubated in 25 mM Hepes/KOH (pH 7.2), 2.5 mM Mg(OAc)2, 20 mM KCl, 1 mg/ml
ovalbumin, 1 mM dithiothreitol, 0.2 M sucrose,
and 50 µM nucleotide (GDP Antibodies and Immunoprecipitation--
In case the proteins
were denatured prior to immunoprecipitation, SDS was added to a final
concentration of 1% and the samples (20 µl) were incubated for 3 min
at 95 °C. For immunoprecipitation, the samples were incubated with
the indicated antibodies in 200 µl of 20 mM Tris-HCl (pH
7.5), 2 mM EDTA, 0.15 M NaCl, and 0.5% Triton
X-100 (immunoprecipitation buffer) for 2 h at 4 °C by
head-over-head rotation. If SDS was present in the sample, the amount
of Triton X-100 was increased to 0.9% to have a 10-fold excess of
Triton X-100 over SDS. Subsequently the samples were incubated with
protein A-Sepharose (Amersham Pharmacia Biotech) for 2 h at
4 °C by head-over-head rotation. The beads were washed five times in
immunoprecipitation buffer and once in phosphate-buffered saline. The
precipitated material was solubilized in sample buffer and analyzed by
Western blotting and autoradiography. For immunoprecipitation of native coatomer complex, anti- In order to analyze proteins or molecules that act downstream of
ARF (effector molecules), we focused on The photolabile ARF molecules were generated by mutating the
corresponding codons of ARF1 cDNA to amber and subsequent in vitro transcription and translation with suppression of this stop codon by addition of suppressor tRNA chemically loaded with the photolabile amino acid analog (see "Experimental Procedures"). Translation in the presence of [35S]methionine allows
visualization of the corresponding photolabile full-length protein by
SDS-PAGE and autoradiography. Each mutated and radiolabeled ARF showed
a GTP-dependent binding to Golgi membranes, excluding the
possibility that replacement of the endogenous amino acid for (Tmd)Phe
had severely affected ARF activity (data not shown).
Incubation of [35S]ARF-(Tmd)Phe-46 with Golgi membranes
in the presence of GTP-COP and
-COP, as well as
an interaction with a cytosolic protein (~185 kDa). In addition, we
observe homodimer formation of ARF molecules at the Golgi membrane.
These data suggest that the binding site of ARF to coatomer is at
the interface of its
- and
-subunits, and this is in close
proximity to the second site of interaction of coatomer with the
Golgi membrane, the binding site within
-COP for cytosolic
dibasic/diphenylalanine motifs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-,
'-, and
-COP could be isolated (8, 11). The diphenylalanine motif was shown to bind to
-,
- and
-COP (9). However, using direct
photolabeling of intact coatomer with various photolabile peptides
containing the dibasic and diphenylalanine motifs, only the
-COP
subunit was labeled, suggesting that coatomer has only one binding site for the various types of these motifs (12, 13). The diphenylalanine sequence is present in the C-terminal tails of the members of p24
family of type I membrane proteins (9, 10, 14), and it has been
proposed that one or more members of this family provide(s) a matrix
for coatomer binding to Golgi membranes and subsequent formation of
COPI-coated vesicles (10).
-COP subunit of
coatomer during the formation of transport vesicles (15). Thus, it was
inferred that a bivalent interaction of coatomer with Golgi membranes
via ARF and via members of the p24 family of proteins is involved in
the budding of a COPI-coated vesicle (15).
-COP,
-COP, and an as yet unidentified protein of
~185 kDa. In addition, evidence is presented that ARF is capable of
forming a homodimer on Golgi membranes, the function of which remains
to be determined.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S or GTP
S) in the presence or absence of isolated CHO Golgi membranes, of purified rabbit liver
coatomer, of recombinant mARF1, and of isolated bovine brain cytosol as
described in the figure legends in a total volume of 500 µl for 15 min at 37 °C. After incubation the Golgi membranes were pelleted in
a microcentrifuge (30 min at 14,000 rpm at 4 °C) and resuspended in
20 µl of 25 mM Hepes/KOH (pH 7.2), 20 mM KCl,
2.5 mM Mg(OAc)2, and 0.2 M sucrose.
Samples were irradiated at 366 nm for 2 min on ice, and thereafter the
membranes were pelleted in a microcentrifuge and analyzed by SDS-PAGE
and sequent autoradiography. When no membranes were present during the
incubation, the total volume of incubation was 50 µl and these
samples were not subjected to centrifugation but rather the complete
incubation subjected to irradiation. Golgi membranes from Chinese
hamster ovary cells and bovine brain cytosol were isolated as described by Beckers et al. (25), coatomer was purified as described
by Waters et al. (26) with modifications according to Pavel
et al. (27), and recombinant mARF1 was produced and purified
as described by Weiss et al. (28) and modified according to
Helms et al. (29).
'-COP antibodies were used (30), and for
immunoprecipitation of dissociated coatomer, antibodies against the
respective individual subunits were used (against
-COP (31),
-COP
(32),
'-COP (30), and
-COP (12)). The same antibodies were also
used for analysis by Western blotting. For visualization the enhanced
chemiluminescence system (Amersham Pharmacia Biotech) was used.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet
2 (amino acids
41-46) and loop L4 (amino acids 47-50) (23). These structures constitute the putative effector region in ARF, based on its similarity with the Ras proteins, as revealed by a comparison of the crystal structure of ARF and Ras (23). The following criteria were applied for
selection of amino acids to be replaced with the photolabile (Tmd)Phe:
(i) insertion of the photolabile residue should not change the net
charge of the ARF molecule and (ii) the amino acid to be replaced
should have an optimal orientation (i.e. facing outward from
the ARF molecule) for optimal cross-link efficiencies. Based on these
criteria, we choose Val-43, Ile-46, and Ile-49 to be replaced with
(Tmd)Phe, resulting in ARF mutants designated ARF-(Tmd)Phe-43, ARF-(Tmd)Phe-46, and ARF-(Tmd)Phe-49, respectively.
S and subsequent irradiation of the
membrane-bound ARF resulted in three cross-link products with estimated
molecular masses of between 120 and 140 kDa (Fig.
1A, lane 2). The
molecular masses of the cross-link products are in the range of several coatomer subunits (reviewed in Ref. 33), and therefore we determined whether addition of purified coatomer during the incubation of [35S]ARF-(Tmd)Phe-46 with Golgi membranes would
increase the efficiency of cross-linking. As shown in Fig.
1A (lane 5 versus
lane 2), a much stronger intensity of the
cross-link products is observed under these conditions. In contrast to
several nonspecific cross-reactivities, these three bands are strongly
reduced in the presence of GDP
S (Fig. 1A, lane
3) or in the absence of irradiation (Fig. 1A,
lane 4) or in the presence of an excess of wtARF
(Fig. 1A, lane 6). ARF has to be bound
to Golgi membranes for the cross-linking to occur, as an incubation in
the absence of membranes lead to a complete absence of cross-link
products (Fig. 1A, lane 1).
View larger version (34K):
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Fig. 1.
Photocross-linking of membrane-bound
[35S]ARF-(Tmd)Phe-46. A,
in vitro translated ARF-(Tmd)Phe46 was incubated in the
absence (lane 1) or presence (all
other lanes) of Golgi membranes, with GDP S
(lanes 3 and 7) or GTP
S
(all other lanes), in the presence
(lanes 3-6) or absence (all other
lanes) of purified coatomer, in the presence of 20 µg of
myristoylated ARF1 (lane 6) or in the presence of 2 mg/ml
bovine brain cytosol (lanes 7-9). After incubation, the
samples were centrifuged and the membranes were irradiated (except
lanes 4 and 8), and membrane-bound material was
analyzed by 12% SDS-PAGE (24) and autoradiography. When no membranes
were present during the incubation (lane 1),
1/10th of the complete incubation was irradiated (without prior
centrifugation) and was analyzed as described above. Before addition of
cytosol, Golgi membranes were preincubated for 5 min at 37 °C with
[35S]ARF-(Tmd)Phe-46 to prevent competition of
photolabile ARF with ARF1 from cytosol. B,
immunoprecipitation of membrane-bound coatomer and its individual
subunits. In vitro translated ARF-(Tmd)Phe46 was bound to
CHO Golgi membranes in the presence of GTP
S and coatomer as
described in panel A, lane
5. After irradiation of membrane-bound ARF, the membranes
were solubilized in detergent-buffer and the coatomer complex was
immunoprecipitated using an anti-
'-COP antibody (lane
2), or the individual coatomer subunits were
immunoprecipitated after denaturation of the complex, using
anti-
-COP antibody (lane 3), anti-
-COP
antibody (lane 4), anti-
'-COP antibody
(lane 5), and anti-
-COP antibody
(lane 6). Immunoprecipitation with a preimmune
serum is shown in lane 1. Samples were analyzed
by 7.5% SDS-PAGE and subsequent autoradiography.
The specific cross-link products are also observed in the presence of
high concentrations of bovine brain cytosol (a high speed supernatant
from bovine brain homogenate), demonstrating the highly specific
interaction between ARF and these proteins (Fig. 1, lanes
7-9). Under these conditions, Golgi membranes were preincubated for 5 min at 37 °C with
[35S]ARF-(Tmd)Phe-46 before addition of cytosol in order
to prevent competition of photolabile ARF1 with ARF1 from cytosol.
Surprisingly, another cross-link product of ~205 kDa appears in the
presence of cytosol (Fig. 1, lane 9). Subtracting
the molecular mass of ARF, this protein has an apparent molecular mass
in the range of about 180-185 kDa. We considered the possibility that
under these circumstances, ARF might also interact with clathrin heavy chain (CHC), a protein with similar molecular mass, or with AP180, a
clathrin assembly-promoting phosphoprotein, or with subunits of the
adaptor protein (AP) complex, all of which are recruited to the
membrane to form a clathrin coated vesicle (34-37).
Immunoprecipitation with an -CHC antibody did immunoprecipitate CHC
from cytosol but failed to immunoprecipitate this radioactive band
(data not shown). Likewise, antibodies against AP180,
-adaptin (a
subunit of the AP-1 complex), and
3B (a subunit of the AP-3 complex) did immunoprecipitate their respective antigen but failed to
immunoprecipitate the cross-linked product. Although we cannot exclude
the possibility that the cross-link occurs at the epitope of the
antibody (making it inaccessible for immunoprecipitation), these data
make it unlikely that the cross-link-product is due to an interaction
of ARF involved in the formation of clathrin-coated vesicles.
Immunoprecipitation studies in the absence of cytosol confirmed the
presence of the cross-link products (~120-140 kDa) in the coatomer
complex (Fig. 1B). An anti-coatomer antibody that immunoprecipitates native coatomer complex did immunoprecipitate all
three cross-link products (Fig. 1B, lane
2). Coatomer has four subunits (-,
-,
'-, and
-COP) with a molecular weight in the range in the cross-link
product. Antibodies directed against each of these subunits were used
to determine which subunit(s) is bound to ARF-(Tmd)Phe-46 (Fig.
1B, lanes 3-6). To this end, the
samples were treated with SDS in order to dissociate coatomer before
immunoprecipitation (for details see "Experimental Procedures"). Antibodies against
-COP (lane 3) and
'-COP
(lane 5) did not immunoprecipitate the cross-link
products, whereas antibodies against
-COP (lane 4) and
-COP (lane 6) efficiently immunoprecipitated two different cross-link products. The third band might be derived from
-COP, as it is faintly seen with the
-COP antibody (Fig. 1B, lane 4). One possibility is that
more than one ARF binds to
-COP under these conditions.
Alternatively, it could be that a small fraction of ARF cross-links at
a different position within
-COP, affecting its migration.
Incubation of [35S]ARF-(Tmd)Phe-49 with isolated CHO
Golgi membranes in the presence of GTPS and subsequent irradiation
of the membrane-bound [35S]ARF-(Tmd)Phe-49 gave a
relatively complex cross-link pattern (Fig.
2A, lane
4). However, most of these cross-links are unspecific, as
they also appear in the presence of GDP
S or in the absence of
irradiation (Fig. 2A, lanes 2 and
3, respectively). Two bands at ~40 kDa are specific
according to these criteria and only appear in the complete incubation
(Fig. 2A, lane 4 versus
lanes 2 and 3). These bands are
dependent on the orientation of ARF in the membrane as they do not
appear without membranes (Fig. 2A, lane 1). Several potential candidates could be involved in the
formation of the cross-link product of ~40 kDa, including an
interaction of ARF with the p24 family of proteins (10, 14). However, antibodies against p23 and p24 did not immunoprecipitate the cross-link products, although they did immunoprecipitate their respective antigens
(data not shown). Alternatively, the cross-link products at ~40 kDa
could be due to dimerization of ARF itself. This possibility cannot, of
course, be tested by immunoprecipitation with
-ARF antibodies, as
they would also immunoprecipitate ARF-(Tmd)Phe-49 together with its
cross-link product. Therefore, we added increasing amounts of wtARF to
the incubation of ARF-(Tmd)Phe-49 with Golgi membranes and GTP
S
(Fig. 2B). If a cross-link exists with a protein other than
ARF, this would be expected to reduce the efficiency of cross-linking
as the specific activity of [35S]ARF-(Tmd)Phe-49 is
reduced (see Fig. 1 and Ref. 15). However, the efficiency of the
cross-linking was increased with increasing amounts of wtARF (Fig.
2B). This strongly suggests that ARF-(Tmd)Phe49 forms a
dimer with ARF present on the membranes, because according to the law
of mass effect, only in this case an increase of the cross-link signal
is expected.
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When ARF-(Tmd)Phe-49 is incubated under standard conditions but in the
presence of coatomer, an additional cross-link product appears at
~120 kDa (Fig. 2A, lane 7). This
band is not observed in the presence of GDPS or in the absence of
irradiation (Fig. 2A, lane 7 versus
lanes 5 and 6). Addition of cytosol to
the complete incubation (under conditions as described for
ARF(Tmd)Phe-46) also yielded the specific cross-link products at ~40
kDa and at ~120 kDa (Fig. 2A, lane
10 versus lanes 8 and
9). The dependence on the presence of coatomer or cytosol
for the ~120-kDa products to appear suggested a cross-linking of
ARF-(Tmd)Phe-49 to a coatomer subunit. Indeed, immunoprecipitation of
coatomer with an anti-coatomer antibody immunoprecipitated the 120-kDa
cross-link product (Fig. 2C, lane 2).
After dissociation with SDS, antibodies against
-COP (lane 3) and
'-COP (lane
5) did not immunoprecipitate of the cross-link product,
whereas antibodies against
-COP (lane 4), and,
to a much lesser extent, also
-COP, immunoprecipitated the cross-link product. Interestingly, both with ARF-(Tmd)Phe-46 and with
ARF-(Tmd)Phe-49, in fact two bands (migrating at ~120 and 140 kDa)
are immunoprecipitated with the
-COP antibody (Fig. 1B,
lane 6; Fig. 2C, lane
6). Whereas the upper band predominates with ARF-(Tmd)Phe-46
(Fig. 1B), the lower band predominates with ARF-(Tmd)Phe-49
(Fig. 2C). As the immunoprecipitation of coatomer-subunits after dissociation of the coatomer complex is subunit-specific and no
other subunits co-immunoprecipitate (15), it is likely that ARF can
cross-link to two different positions of
-COP, affecting its
migration by SDS-PAGE. Depending on the location of the photolabile analogue in ARF, one cross-link of
-COP prevails over the other.
ARF-(Tmd)Phe-43 was bound to Golgi membranes under the same conditions
as described above for ARF-(Tmd)Phe-46 and ARF-(Tmd)Phe-49, but did not
result in any detectable cross-linking to an interacting protein. This
does not exclude the possibility that this site is important for
interaction with other proteins, as the distance of the interaction
partner with the photolabile residue is critical for cross-link
efficiency (17).
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DISCUSSION |
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Using a site-directed photocross-linking approach, we previously
identified a direct interaction between ARF and -COP (15). With the
photolabile group at a different positions within the ARF molecule, we
now find that ARF also interacts with the
-subunit of coatomer.
Because single amino acids at position 46 as well as position 49 can
interact both with
-COP and with
-COP (Figs. 1 and 2), this
implies that these two subunits are close neighbors within the coatomer
complex. Several coatomer subcomplexes have previously been described
(8, 11, 27, 31), including a
/
/
subcomplex (9). It is
important to note, however, that the cross-linking approach used here
is qualitative and does not allow a quantitative determination of,
e.g., the stoichiometry between ARF and coatomer. The main
reason is that, in an aqueous environment, the generated carbene will
react predominantly with water molecules, making cross-link yields
1% quite typical (38). This high reactivity, however, renders this
photoprobe so specific, because a very close proximity of a protein is
needed to compete with the ubiquitously abundant water molecules. Under
these conditions, it can be calculated (based on the specific activity
of [35S]methionine incorporated into in vitro
translated ARF) that low picogram amounts of coatomer subunits are
covalently bound to suppressed ARF, which does not permit quantitation
of the amount of coatomer, bound to suppressed ARF (by bandshift on
SDS-PAGE).
A schematic drawing of how the coatomer complex is bound to Golgi
membranes is shown in Fig. 3. This model
is based on the following experimental data. (i) The cytoplasmic tail
of the p24 family of proteins specifically binds to coatomer (9, 10, 39). This tail contains a diphenylalanine motif and in some instances,
like p23, an additional dilysine motif. The diphenylalanine motif
sets these proteins apart from ER-resident proteins that contain only
the dilysine ER retrieval signal (10, 40). A single binding site exists
within coatomer for both dilysine retrieval motifs and the cytoplasmic
domain of p23, and this site resides in -COP (13). (ii) By use of
site-directed photocross-linking, we have shown a direct and
GTP-dependent interaction of ARF with
-COP, which
persists in COPI-coated vesicles and thus is not restricted to the
budding process (15). These interactions indicate a bivalent
interaction of coatomer with the Golgi membrane during vesicle
formation, i.e. an anchoring of coatomer to Golgi membranes by ARF as well as by the C-terminal tails of the p24 family of proteins. (iii) The results described here demonstrate that ARF interacts with coatomer at the
/
-COP interface. Thus,
-COP interacts both with ARF and the p24 family of proteins, indicating that
the bivalent interaction occurs in close proximity of one to another.
In the absence of ARF, coatomer cannot bind to Golgi membranes (6, 7),
and therefore it is tempting to speculate that the initial recruitment
of coatomer to Golgi membranes by ARF affects the
-subunit so that
it subsequently allows the C-terminal tail of p24 protein(s) to bind.
This binding then would lead to a conformational change of coatomer,
leading to polymerization of the complex and subsequent shaping of the
membrane to form a bud (41). The uncoating of COPI-coated vesicles
would simply be the reversal of this process: hydrolysis of ARF-bound
GTP by a GTPase-activating protein results in uncoating of COPI-coated vesicles (42). Thus, hydrolysis of ARF-bound GTP reduces the affinity
of coatomer for ARF and ARF dissociates from COPI-coated vesicles. This
in turn could reverse the conformational change in
-COP to reduce
the affinity for the p24 family of proteins, resulting in dissociation
from transport vesicles of the coatomer complex.
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Together with our previous study that shows an interaction of Phe-82 in
ARF with -COP, we have now mapped the interaction site of ARF and
coatomer to a surface area of about 400 Å2, typical for
protein-protein interactions. Interestingly, Ile-46, Ile-49, and Phe-82
are located on the switch 1 and 2 regions of ARF, respectively, and
exactly these regions have been predicted to interact with the Sec7
domain of Arno (43), a guanine nucleotide exchange factor for ARF (44).
It thus appears that one interface on ARF protein is used both for the
interaction with the Sec7 domain (likely in the ARF GDP-bound form) and
with the coatomer complex (in the GTP-bound form). It has been shown
that N-terminally deleted ARF in its GDP-bound form can functionally
interact with the Sec7 domain (45). It will be interesting to determine
whether corresponding N-terminally deleted photolabile ARF proteins,
mutated at the positions described in this study, can also cross-link to the Sec7 domain under these conditions.
With the photolabile amino acid residue at positions 46 and 49 of the
ARF molecule, we have also found interactions with proteins other than
coatomer. In the presence of cytosol, ARF-(Tmd)Phe-46 cross-links to a
protein with an apparent molecular mass of ~185 kDa. Due to steric
hindrance, it seems unlikely that, in addition to coatomer, a third
protein binds to ARF at the same molecular interface. We consider it
more likely that, under these conditions, part of the membrane-bound
ARF is bound to coatomer and the other part of ARF is associated with
p185. As ARF is also involved in the budding of clathrin-coated
vesicles from the trans-Golgi network (34, 35), we considered the
possibility that CHC, a protein with a molecular mass of ~180 kDa,
was bound to ARF under these conditions. However, immunoprecipitation
studies with antibodies against CHC failed to immunoprecipitate the
cross-link product. Similarly, antibodies against AP180 and various
subunits of the adaptor-protein complex that are recruited to the Golgi
complex via ARF did not immunoprecipitate the cross-link product of
~205 kDa. As both coatomer and the AP-1 complex are recruited by ARF to the Golgi complex and the trans-Golgi network, respectively (in the
presence of GTPS), it is remarkable that so far we have not
cross-linked one of the subunits of the adaptor protein complex. With
respect to ARF involved, there are notable differences between COPI-coated vesicles and clathrin coated vesicles that bud from the
Golgi. In the presence of GTP
S, only COPI-coated vesicles are
observed, the reason for which is not clear. Possibly this guanine
analogue simultaneously inhibits dynamin, a putative pinchase for
clathrin-coated vesicles, which is a GTP-binding protein itself (46,
47). In addition, it has recently been reported that ARF1 only
transiently activates high affinity adaptor protein complex AP-1
binding sites and that hydrolysis of ARF bound GTP can occur even
before AP-1 binds (48). Finally, although ARF is involved in the
recruitment of coat proteins for the formation of clathrin-coated
vesicles, in contrast to COPI-coated vesicles, ARF1 is not present on
coathrin-coated vesicles (48). Thus, whereas ARF is a stoichiometric
component in COPI-coated vesicles, ARF seems only transiently involved
in the formation of clathrin-coated vesicles and this might be the
reason for the fact that we have only detected interactions with
components of the COPI coat.
We are currently trying to elucidate the identity of the 205-kDa cross-linked protein by fractionation of cytosol and subsequent cross-link studies. One interesting alternative possibility is a spectrin homologue (~220 kDa), recently shown to be recruited to the Golgi membranes by ARF1 (49, 50).
Surprisingly, we have found evidence for the formation of ARF dimers on the Golgi membrane. ARF also crystallizes as a dimer (23), but it remains to be determined whether ARF dimers on Golgi membranes have the same orientation to one another as in the crystal structure. The N termini of both ARF molecules, thought to be involved in membrane anchorage of the ARF molecule, are on one interface of the crystallized dimer, theoretically allowing both N termini to interact with the membrane in this orientation. Another argument for the crystallized dimer orientation also occurring on the Golgi membranes is that according to the crystal structure, amino acid 49 in ARF is in close proximity to the other ARF molecule, i.e. in an optimal orientation for cross-linking. In this case, (Tmd)Phe-49 would result in a cross-link between ARF-(Tmd)Phe-49 (in L4) with amino acid residues 159-164 (in L12) in the second ARF molecule. If the cross-link represents an ARF homodimer, then the presence of a double band (cf. Fig. 2B) is not as easily explained, unless the two ARF molecules are different in, e.g., their myristoylation, known to affect the migration of ARF on SDS-PAGE (51). The purified recombinant myristoylated ARF added in excess is in fact a mixture of non-myristoylated and myristoylated ARF (29). Alternatively, intramolecular cross-linking may affect the migration of ARF on SDS-PAGE and might explain this phenomenon (see below). Finally, the two cross-link products at ~40 kDa might represent dimerization of different ARF isoforms. At least three different isoforms of ARF have been shown to bind to Golgi membranes (52).
In the course of these site-directed photocross-link studies, we have
tried several additional positions for cross-link products. An overview
of all the point mutations that we have analyzed is given in Table
I. Positions 5 and 13 are present in two
structural features that sets ARF apart from other GTP-binding proteins
and are unique to ARF: the N-terminal -helix (amino acid 2-11) and the connecting loop L1 (amino acid 10-17). These two elements are not
present in small GTP-binding proteins, but are present in the G
subunit of heterotrimeric G proteins(23). Mutations in both these
elements (Phe-5 and Phe-13) did not cross-link a protein but caused a
small shift (
1 kDa) of the apparent molecular mass of ARF-(Tmd)Phe
after irradiation. Compared with the input (membrane-bound,
myristoylated ARF), it was observed that after cross-linking, the
cross-link product migrated like non-myristoylated ARF, which can be
separated from myristoylated ARF by high resolution SDS-PAGE. This
shift is not likely due to a cross-link with membrane lipids, as
treatment with several lipases did not affect the migration of the
cross-link product (data not shown). It is possible that cross-linking
at these positions reflects an intramolecular cross-linking with
e.g. the myristoyl moiety (which lies in close proximity), causing myristoylated ARF to migrate like non-myristoylated
ARF.
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Likewise, introduction of (Tmd)Phe at positions Lys-142 in -helix E
and Tyr-154 in
-sheet 7 of the ARF molecule did not yield
protein-cross-links.
In summary, depending on the position of (Tmd)Phe in the ARF molecule,
strikingly different cross-link products are obtained. The strict
discrimination underlines the high specificity of the interactions
observed with site-directed photocross-linking. We have identified the
interface of ARF that interacts with coatomer as it is covered by
Ile-46, Ile-49, and Phe-82, all on one site of the ARF molecule. With
the many additional functions attributed to ARF, generation of
additional photolabile ARF proteins with photocross-link residues at
positions distal from the ones described here will allow one to
pinpoint other partners than coatomer of this remarkable small GTP
binding protein.
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ACKNOWLEDGEMENTS |
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We thank Dr. M. (Scotty) Robinson for
the antibodies directed against clathrin heavy chain, -adaptin of
the AP-1 complex, and the
3B subunit of the AP-3 complex; Dr. E. Ungewickell for antibodies against AP180; Dr. S. Tooze for antibodies
directed against
-adaptin (hinge region); and Daniel Gommel from the
Wieland laboratory for help with the immunoprecipitation of the p24
family of proteins. Fig. 3 was originally designed and created by Drs. K. Sohn and W. Nickel.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft grants (to F. T. W and J. B. H.) and SFB 352 (to F. T. W.), by the Swiss National Science Foundation (to J. B.), by a grant from the Human Frontiers Science Organization Program (to F. T. W.), and by a grant of the German-Israeli Foundation of Scientific Research (to X. E., F. T. W., and J. B. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 49-6221-546758 or -544688; Fax: 49-6221-544366; E-mail: helms{at}urz.uni-heidelberg.de.
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ABBREVIATIONS |
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The abbreviations used are:
COPI, coat protein
complex type I;
COP, coat protein;
(Tmd)Phe, L-4'-(3-trifluoromethyl-3H-diazirin-3-yl)-phenylalanine;
CHO, Chinese hamster ovary;
ARF, ADP-ribosylation factor;
GDPS, guanosine 5'-(
-thio)diphosphate;
GTP
S, guanosine
5'-(
-thio)triphosphate;
CHC, clathrin heavy chain;
ER, endoplasmic
reticulum;
wt, wild type;
AP, adaptor protein.
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REFERENCES |
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