(Received for publication, July 31, 1996, and in revised form, November 11, 1996)
From the Institut National de la Santé et de la Recherche Médicale, U-338 Biologie de la Communication Cellulaire, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France, and § Biochemie I, Universitaet Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany
The ADP-ribosylation factor (ARF) GTP-binding
proteins are believed to function as regulators of vesicular budding
and fusion along the secretory pathway. To investigate the role of ARF
in regulated exocytosis, we have examined its intracellular
distribution in cultured chromaffin cells by subcellular fractionation
and immunoreplica analysis. We found that ARF6 is specifically
associated with the membrane of purified secretory chromaffin granules.
Chemical cross-linking and immunoprecipitation experiments suggested
that ARF6 may be part of a complex with subunits of trimeric G
proteins. Stimulation of intact chromaffin cells or direct elevation of cytosolic calcium in permeabilized cells triggered the rapid
dissociation of ARF6 from secretory granules. This effect could be
inhibited by AlF4
which
selectively activates trimeric G proteins. Furthermore, a synthetic
myristoylated peptide corresponding to the N-terminal domain of ARF6
strongly inhibited calcium-evoked secretion in streptolysin-O-permeabilized chromaffin cells. The possibility that
ARF6 plays a role in the effector pathway by which trimeric G proteins
control exocytosis in chromaffin cells is discussed.
Studies on diverse secretory cell types have highlighted the importance of GTP-binding proteins in Ca2+-regulated exocytosis. Proteins able to bind and hydrolyze GTP can be subdivided into different families including (i) trimeric G proteins, (ii) ras-related low molecular mass G proteins, and (iii) ADP-ribosylation factor (ARF)1 proteins. Trimeric G proteins have been found associated with the membrane of secretory granules in various secretory cells (1-3), suggesting a role in exocytosis. Indeed, the participation of a plasma membrane-bound Gi3 protein in the late stages of exocytosis in mast cells has been demonstrated (4). Direct control of exocytosis by Gi and Go proteins has also been described in insulin-secreting cells (5) and in chromaffin cells (3, 6). Thus regulated exocytosis appears as a possible effector pathway for trimeric G proteins although the mechanisms by which this class of G proteins relates to the exocytotic machinery remain to be elucidated.
Besides heterotrimeric G proteins, recent studies suggest that a subset of small GTPases of the ras superfamily may also participate in the control of calcium-dependent exocytosis. For example, rab3 isoforms which are specifically localized on the membrane of secretory vesicles (7, 8), have been proposed to serve as a regulator of exocytosis in chromaffin cells (9) and in anterior pituitary cells (10). Other investigations have postulated the participation of ARF in exocytosis (11, 12).
ARF is a monomeric GTP-binding protein that appears to control a wide range of vesicle transport and fusion steps along the secretory pathway. This may include budding, transport, and fusion steps in the Golgi complex (13, 14), in the endoplasmic reticulum (15), and in the endocytotic pathway (16). Initial evidence for the involvement of ARF in the secretory pathway came from genetic studies in yeast Saccharomyces cerevisiae where deletion of the ARF gene resulted in a secretory defect (17). In mammalian cells, ARF proteins are classified into three families according to their size and sequence homology (18). ARF1, ARF2, and ARF3 form class I, ARF4 and ARF5 form class II, and ARF6 forms class III. The best characterized ARF protein is ARF1. It is localized on the Golgi apparatus and is involved in the recruitment of cytosolic coat proteins to membranes during the formation of transport vesicles (19). ARF6 has been recently localized in the endocytotic pathway in transfected mammalian cells, where it appears to control the fusion of endosomes (16).
The aim of the present work is to assess the possible involvement of
ARF in regulated exocytosis. We report that ARF6 is specifically associated to the membrane of secretory chromaffin granules, presumably through an interaction with G subunits. Stimulation of chromaffin cells triggered the dissociation of ARF6 from secretory granules. This
dissociation can be blocked by aluminium fluoride, an activator of
trimeric G proteins. We propose that ARF may be part of the effector
pathway by which trimeric G proteins control the exocytotic pathway in
chromaffin cells.
Bovine adrenal chromaffin cells were isolated and maintained in primary culture essentially as described previously (6, 20). Cells were loaded with [3H]noradrenaline (14.68 Ci/mmol, Dupont NEN) and washed with Locke's solution (6). To trigger release from intact cells, chromaffin cells were stimulated for an indicated period of time with Locke's solution containing 10 µM nicotine (6). Permeabilization of chromaffin cells was performed with streptolysin-O (SLO; Institut Pasteur, Paris, France) for 2 min in Ca2+-free KG medium (3). Cells were then stimulated for 10 min, in the presence of the compound to be tested, in KG medium containing 20 µM free Ca2+ and 1 mM free Mg2+ (3).
Subcellular Fractionation of Cultured Chromaffin CellsChromaffin granules were purified from cultured chromaffin cells on 1.6 M sucrose gradients as described previously (21). Granule membranes were obtained after three freezing/thawing steps and centrifugation at 50,000 × g. Purified cytosol was obtained from the 20,000 × g supernatant, which was further centrifuged 30 min at 100,000 × g. Golgi membranes were prepared from cultured chromaffin cells essentially as described previously (22).
Alternatively, the crude membrane fraction (20,000 × g pellet) was layered on a continuous sucrose density gradient (sucrose 1-2.2 M, 10 mM Tris, pH 7.4) and centrifuged for 1 h at 100,000 × g. Twelve 1-ml fractions were collected from the top to bottom and analyzed for protein content. The distribution of the chromaffin granule marker chromogranin A in the fractions of the gradient was estimated by the ELISA technique (20). The plasma membrane marker Na+/K+-ATPase was assayed as described previously (3).
AntibodiesThe mouse monoclonal antibody 1D9 against ARF
(23) was kindly provided by Dr. Richard D. Kahn (NIH, Bethesda). The
affinity-purified rabbit polyclonal antibody against ARF1 was
previously characterized (24). The polyclonal anti-ARF6 antibody was a
total IgG fraction prepared against recombinant ARF6, and its
specificity was demonstrated by ELISA (data not shown). Rabbit
polyclonal antisera against sARFII (25), ARF5 (26), and ARF6 (26) were
a generous gift from Dr. J. Moss (NIH, Bethesda). Affinity-purified
antibodies against ARF(23-36) were prepared by injecting
into rabbits the synthetic peptide CVGLDAAGKTWILYK conjugated to
carrier keyhole limpet hemocyanin (27). After ammonium sulfate
precipitation, the ARF IgG was purified by passing the total IgG
fraction through a Affi-gel 10 (Bio-Rad) column coupled to the
ARF(23-36) peptide. The rabbit polyclonal anti-G
subunit antibody was purchased from Upstate Biotechnology Incorporated
(Lake Placid, NY). The rabbit polyclonal anti-dopamine
-hydroxylase
(EC 1.14.17.1) antiserum was prepared in our laboratory, and its
specificity has been demonstrated (28).
Chromaffin granule membranes
(2.5 mg) were immunoprecipitated with 50 µl of anti-dopamine
-hydroxylase antiserum and 250 µl of ImmunoPure immobilized
protein A (Pierce) (7). Proteins linked to the beads were analyzed by
SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting.
The granule-associated ARF protein was immunoprecipitated from purified chromaffin granule membranes (700 µg) solubilized in 2% sodium cholate (29). Solubilized proteins were diluted twice in extraction buffer (140 mM KCl, 20 mM HEPES, pH 7.3, 2 mM EDTA) to reduce the final concentration of sodium cholate to 1%. Immunoprecipitation was performed with 5 µl of affinity-purified antibodies prepared against the synthetic ARF(23-36) peptide followed by the addition of 100 µl of ImmunoPure Immobilized protein A. The beads were collected by centrifugation and bound proteins were analyzed by SDS-PAGE and immunoblotting.
Cross-linking of Chromaffin Granule MembranesChromaffin granule membranes were cross-linked with 5 mM disuccinimyl suberate exactly as described previously (29). Membranes were collected by centrifugation at 100,000 × g for 15 min, and proteins were analyzed by SDS-PAGE and immunoblotting.
Electrophoresis and ImmunoblottingOne-dimensional SDS-PAGE was performed on 15% acrylamide gels in Tris-glycine buffer (30). Proteins were transferred to nitrocellulose sheets (30) at a constant current of 120 mA for 1 h. Blots were developed with secondary antibodies coupled to horseradish peroxidase (Amersham, Les Ulis, France), and immunoreactive bands were detected with the ECL system (Amersham). The conditions for separation of proteins by electrophoresis on two-dimensional gels were as described previously (30).
PeptidesThe synthetic ARF(23-36) peptide CVGLDAAGKTWILYK was obtained from Neosystem (Strasbourg, France). Myristoylated (myrARF6(2-13)) N-terminal ARF6 peptide (GKVLSKIFGNKE), nonmyristoylated (ARF6(2-13)) N-terminal ARF6 peptide, and myristoylated (myrARF1(2-17)) N-terminal ARF1 peptide (GNIFANLFKGLFGKKE) were synthesized in our laboratory (432A Peptide Synthesizer SYNERGY, Applied Biosystems, Warrington, UK). Purity was checked on high performance liquid chromatography. Sequence analysis was performed by Edman degradation on an automated gas phase protein sequencer (Applied Biosystems, Warrington, UK), and mass spectrometry was used to assess the structure of the final product.
The
intracellular distribution of ARF proteins in chromaffin cells was
estimated by immunoreplica analysis using a monoclonal anti-ARF
antibody (1D9). As illustrated in Fig. 1A,
ARF was detected in the cytosol and in the Golgi apparatus, displaying
an apparent molecular mass of 20 kDa. ARF was also found associated to
chromaffin granule membranes but with an apparent molecular mass of 18 kDa. The subtype of ARF proteins was further defined with several
antibodies able to distinguish between the different classes of ARF. We
found that the 20-kDa ARF protein detected in the cytosolic and Golgi fractions was specifically labeled with the affinity-purified anti-ARF1
antibody (Fig. 1A) and with the anti-sARFII antiserum (data
not shown) but it was not recognized by the anti-ARF6 antibody (Fig.
1A) or by antisera against ARF5 or ARF6 (data not shown). In
contrast, the 18-kDa ARF protein found associated to chromaffin granule
membranes was selectively labeled with the anti-ARF6 antibody (Fig.
1A) and with anti-ARF6 antiserum (data not shown).
Accordingly, ARF6 migrates slightly faster than ARF1 on
SDS-polyacrylamide gel electrophoresis (31).
The specific association of ARF6 with chromaffin granule membranes was further substantiated by immunoblotting analysis of fractions collected from a sucrose density gradient layered with a crude membrane preparation (Fig. 1B). The highest immunosignal for ARF1 was detected in fractions 2 and 3 containing the plasma membranes as estimated by the Na+/K+-ATPase activity. Fractions 11 and 12, enriched in chromaffin granules revealed by the peak of chromogranin A, were strongly labeled with the anti-ARF6 antibody but showed no immunoreaction with anti-ARF1 antibody (Fig. 1B).
We also used an immunoadsorption procedure to exclude the association
of ARF6 with a contaminating organelle that may comigrate with
chromaffin granules in the sucrose density gradients. Dopamine -hydroxylase is a specific marker for chromaffin granules. Fig. 1C shows that preincubation of purified granule membranes
with an anti-dopamine
-hydroxylase antibody followed by protein
A-Sepharose addition resulted in the coadsorption of ARF6 and dopamine
-hydroxylase. Thus ARF6 is an associated component of the chromaffin
granule membrane.
Two-dimensional gel electrophoresis of purified chromaffin granule membranes revealed the presence of two spots labeled with the anti-ARF6 antibody. These two spots displayed only a different isoelectric point value (respectively, 7.7 and 7.9), but no difference in the apparent molecular mass (Fig. 1D). Occasionally, the ARF-labeled band present in chromaffin granule membranes could also be resolved into two components on monodimensional gels (Fig. 1A). This observation may indicate some post-translational modification, such as phosphorylation or sulfation of the granule-associated ARF protein, although such modifications have not yet been reported for ARF proteins.
ARF6 Interacts with theThe presence of trimeric G proteins
on the chromaffin granule membrane (1, 3), together with the recent
in vitro data indicating an interaction between ARF and
G subunits (32, 33), encouraged us to examine whether ARF6 may
bind to G
in chromaffin granule membranes. For this purpose, sodium
cholate extracts of granule membranes were incubated with protein
A-Sepharose beads coated with affinity-purified antibodies raised
against a synthetic peptide corresponding to the consensus
ARF(23-36) sequence. As illustrated in Fig.
2A, immunoprecipitation with the polyclonal
anti-ARF(23-36) antibody which recognized the chromaffin
granule-associated ARF protein, resulted in the coprecipitation of an
additional 34-kDa protein labeled with the anti-G
antibodies.
We performed protein cross-linking experiments to confirm the possible
ARF6-G interaction. Fig. 2B shows that cross-linking of
chromaffin granule-associated proteins with disuccinimyl suberate resulted in the appearance of a 50-kDa adduct that reacted with antibodies against both ARF and G
. With the exception of bands corresponding approximately to the mass of ARF dimers, no additional adducts were observed (Fig. 2B). Therefore, it is likely
that this additional 50-kDa band is due to the cross-linking of ARF6 (18 kDa) to G
(34 kDa), which is in agreement with the idea that ARF6 and G
may be bound to each other in the granule membrane.
In order to probe the role of ARF6 in the exocytotic
pathway, we investigated, in SLO-permeabilized chromaffin cells, the effects of the myristoylated (myrARF6(2-13)) and the
nonmyristoylated (ARF6(2-13)) synthetic peptides
corresponding in sequence to residues 2-13 of ARF6.
MyrARF6(2-13) produced a dose-dependent inhibition in Ca2+-dependent catecholamine
secretion with a mean inhibitory dose, ID50, of
approximately 40 µM (Fig. 3). The
secretory response was almost completely abolished in the presence of
100 µM peptide. In contrast, ARF6(2-13) did
not modify Ca2+-evoked secretion (Fig. 3). Neither
myrARF6(2-13) nor ARF6(2-13) affected the
basal catecholamine release measured in the absence of calcium,
indicating that in the range of the tested concentrations, the peptides
had no membrane-perturbing effects due to their amphipathic properties.
We also examined the effect of a myristoylated synthetic peptide
corresponding to the N-terminal region of ARF1
(myrARF1(2-17)). However, myrARF1(2-17)
reduced only slightly the Ca2+-evoked secretory response in
SLO-permeabilized cells, indicating that the strong inhibition of
secretion observed in the presence of myrARF6(2-13) was
not due to some unspecific effect of the myristoyl group. Thus, ARF6
may represent an important component of the regulated exocytotic
machinery in chromaffin cells.
ARF6 Dissociates from Chromaffin Granule Membranes during Exocytosis
In order to compare the intracellular distribution of
ARF6 in resting and stimulated chromaffin cells, secretory granules were purified by sucrose density gradients from a crude membrane fraction obtained either from intact cells stimulated with 10 µM nicotine or from SLO-permeabilized cells challenged
with 20 µM free Ca2+. The amount of ARF6
associated to granule membranes was analyzed by immunodetection with
the 1D9 anti-ARF antibody (Fig. 4). Stimulation of
chromaffin cells strongly reduced the quantity of ARF6 associated with
chromaffin granule membranes. In intact cells, nicotine reduced by 70%
the amount of ARF6 bound to granules (Fig. 4). In permeabilized cells,
calcium decreased the quantity of ARF6 recovered in the chromaffin
granule membrane fraction by approximately 60% (Fig. 4). Parallel
analysis with the anti-ARF6 antibody revealed a similar decrease in the
amount of granule-associated ARF6 in stimulated cells (data not shown).
Furthermore, the quantity of ARF6 detected with the 1D9 antibody in the
crude membrane fraction remained unchanged in resting and
nicotine-stimulated cells (data not shown), indicating that the
reduction of ARF6 associated to the granule membrane fraction was not
due to a possible degradation or a covalent modification of the protein
in stimulated cells. These results suggest that the exocytotic process
may be accompanied by the translocation of ARF6 from the secretory
granule to some unknown subcellular compartment. Since ARF6 was not
detected among the cytosolic proteins that escape through the pores
created in the plasma membrane of permeabilized cells (data not shown),
we assume that ARF6 translocates from the granule membrane to another
membrane-bound compartment in stimulated cells.
We examined the time course of ARF6 translocation in
nicotine-stimulated cells. As illustrated in Fig. 5, the
dissociation of ARF6 from the chromaffin granules was rapid and almost
complete within 1 min of stimulation. By comparison, maximal
[3H]noradrenaline release required 2-3 min of
stimulation (Fig. 5), suggesting that the dissociation of ARF6 may
represent an early event in the pathway of regulated exocytosis. Note
that G remained attached to the granule membrane during the entire stimulation period (Fig. 5).
The presence of Go on the chromaffin granule membrane
(3), which displays a high affinity for G
and may thereby compete with ARF for the binding to G
(32, 33), led us to investigate whether AlF4
may interfere with the
translocation of ARF6 in stimulated cells. AlF4
is an activator of trimeric G
proteins which prevents the association of the
subunit with
subunits (34). In the presence of
AlF4
, the amount of ARF6 detected on
chromaffin granule membranes was comparable in resting and
calcium-stimulated cells (Fig. 6), indicating that
AlF4
was able to inhibit the
translocation of ARF6 induced by a rise of cytosolic calcium.
Studies with purified proteins indicate that ARF binds to G in
its GDP-liganded form (32, 33). We examined the effect of the
nonhydrolyzable GTP analogue, GTP
S, on the attachment of ARF6 to
chromaffin granule membranes. As shown in Fig. 6, the presence of
GTP
S in the incubation medium did not significantly affect the
calcium-induced dissociation of ARF6 from secretory granules in
permeabilized chromaffin cells.
The current general model of vesicular fusion suggests that the so-called SNARE complex functions in many fusion events throughout the secretory pathway, including transport at the endoplasmic reticulum and the Golgi apparatus, and exocytotic and endocytotic fusions (35). Cumulative evidence also implicates ARF as a molecular switch in the regulation of vesicular transport and fusion through early compartments of the secretory pathway (reviewed in Refs. 19, 36, and 37). These apparent similarities of the mechanisms involved in intracellular vesicular trafficking led us to investigate whether ARF may also be a component of the regulated exocytotic machinery. We report here the presence of an immunologically related ARF6 protein on the membrane of purified secretory chromaffin granules. Stimulation of chromaffin cells by a cholinergic agonist or a direct elevation of cytosolic calcium triggers a rapid decrease of the amount of ARF6 associated to granule membranes, suggesting that exocytotic fusion is accompanied by the release of ARF6 from secretory granules in chromaffin cells. It is noteworthy that a similar sequence of events has been described in the Golgi where the release of ARF from vesicles seems to be a prerequisite for fusion with the acceptor membrane (35).
ARF was originally discovered as a cofactor required for the
ADP-ribosylation by cholera toxin of the heterotrimeric G protein Gs (39). Further studies confirmed the close relationships
between trimeric G proteins and ARF. For example trimeric G proteins
seem to regulate the association of ARF and coat components onto the Golgi membrane (23, 40, 41). A collaboration among trimeric G proteins
and ARF has also been suggested in the control of endosomal fusion
(32). Finally direct interactions between ARF and the G subunits
have been described (32, 33). Indeed, ARF proteins have more in common
with G protein
subunits than with other small GTPases when
comparing GTP-binding concensus sequences and cotranslational
modifications such as myristoylation of the N terminus (37). By
cross-linking and coimmunoprecipitation experiments, we observed here
that ARF6 interacts with G
in chromaffin granule membranes. Since
G
exists only in close association with G
(38), we propose that
the G
complex may represent the membrane receptor that could
stabilize the interaction of ARF6 with secretory granules.
In chromaffin cells, trimeric G proteins control the
ATP-dependent priming step and the late calcium-regulated
fusion event of exocytosis (3, 6). However, their downstream effectors in the exocytotic machinery remain unknown. We found that
AlF4 can prevent the calcium-induced
uncoupling of ARF6-G
on the secretory granule membrane in
stimulated cells. Chromaffin granule membranes contain both
G
o and G
subunits (3). Thus, upon the activation
by AlF4
, G
o is likely
to dissociate from G
which may thereby provide a site of
interaction for ARF6 and prevent its translocation in response to
calcium stimulation. Conversely, calcium may trigger the dissociation
of ARF6 from the granule membrane by allowing the reassociation of
G
o with G
which becomes then unable to retain
ARF6. AlF4
, as well as mastoparan,
inhibit calcium-evoked catecholamine secretion in permeabilized
chromaffin cells by a mechanism which can be selectively reversed by
anti-G
o antibodies (6). This suggests that
G
o blocks the exocytotic machinery when activated and
dissociated from G
. Thus, our data support a model in which exocytosis requires the inactivation of the granule-associated Go. In stimulated cells, the activation of
G
o may be impaired by a calcium-dependent
process and G
o reassociates with G
. This triggers
the release of ARF6 from G
, an event which may be necessary for
the subsequent exocytotic steps to occur.
We investigated the possible function of ARF6 in the exocytotic machinery by using synthetic peptides corresponding in sequence to the N-terminal residues of ARF1 and ARF6. The N-terminal region of ARF was chosen because it has been implicated as the putative effector domain. Indeed, the N terminus of ARF seems to be critical for its function since deletion of this region results in a global reduction of ARF activities (42). Furthermore, N-terminal ARF peptides have proved to be potent inhibitors of ARF functions, including endoplasmic reticulum to Golgi transport, intra-Golgi transport and endocytotic vesicle fusion (15, 40, 42, 43). The strong inhibition of calcium-evoked catecholamine secretion by the myristoylated ARF6(2-13) peptide implicates ARF6 as a key component of the exocytotic pathway in chromaffin cells. Recent studies in HL60 cells have shown that ARF contributes to the restoration of secretion in permeabilized cytosol-depleted cells (44). Hence, the data presented here are convergent with the idea that ARF proteins may play a general role in the exocytotic pathway in endocrine and neuroendocrine cells.
In conclusion, based on subcellular fractionation techniques and
immunological detection, the present results strongly suggest that ARF6
dissociates from secretory granules in stimulated chromaffin cells.
Thus, an important future undertaking will be to confirm this
observation by immunofluorescence and confocal microscopy since we
cannot completely rule out some artefactual redistribution due to cell
lysis. Nevertheless, the finding that ARF6 interacts with G in the
secretory granule membrane offers an attractive speculation: the
dissociation of ARF6 from secretory granules may allow the interaction
of ARF6 with its nucleotide exchange factor resulting in activation in
the target membrane. In view of the current proposed functions for ARF
proteins in vesicular transport, ARF6 is a good candidate to be
involved in the assembly of a multisubunit complex required for fusion
or to contribute to the generation of fusogenic lipids at the plasma
membrane.
We thank Pascal Garcia for skillful technical assistance and Gerard Nullans for the synthesis of peptides. We gratefully acknowledge Dr. Joel Moss for his generous gift of anti-sARFII, anti-ARF5, and anti-ARF6 antisera and Dr. Richard A. Kahn for kindly providing us with anti-ARF 1D9 antibody. We also thank Dr. Susan Lyons for revising the manuscript.