(Received for publication, December 29, 1995)
From the
ADP-ribosylation factor (ARF) is a small GTP-binding protein
that has been implicated in intracellular vesicular transport. ARF
regulates the budding of vesicles that mediate endoplasmic reticulum to
Golgi and intra-Golgi transport. It also plays an important role in
maintaining the function and morphology of the Golgi apparatus. Using a
permeabilized cell system derived from GH cells, we provide
evidence that ARF-1 regulates the formation of nascent secretory
vesicles from the trans-Golgi network. Both myristoylated and
non-myristoylated forms of recombinant human ARF-1 enhanced secretory
vesicle budding about 2-fold. A mutant lacking the first 17 N-terminal
residues, as well as one that preferentially binds GDP (T31N) did not
stimulate vesicle formation. In contrast, a mutant defective in GTP
hydrolysis (Q71L) promoted vesicle budding. Strikingly, a peptide
corresponding to the N terminus of human ARF-1 (amino acids 2-17)
also stimulated vesicle budding from the trans-Golgi network,
in marked contrast to its inhibitory effect on vesicular transport from
the endoplasmic reticulum to Golgi. These data demonstrate that in
endocrine cells, ARF-1 and in particular its N terminus play an
essential role in the formation of secretory vesicles.
ADP-ribosylation factor (ARF) ()is a M
20,000 polypeptide that is a member of the Ras superfamily of
small GTP-binding proteins. It was originally discovered as a cofactor
in cholera toxin-mediated ADP-ribosylation of the G
subunit of heterotrimeric G-proteins(1) . In the past
several years, genetic and biochemical studies have shown that ARF also
plays an essential role in intracellular vesicular transport (reviewed
in (2, 3, 4) ). Deletion of one of two ARF genes (ARF1) from yeast Saccharomyces
cerevisiae led to impaired processing and secretion of invertase,
and deletion of both genes was lethal(5) . Subsequent studies
using in vitro systems have demonstrated that ARF is a
necessary component for vesicular transport between the ER and Golgi
apparatus and in the endocytic
pathway(6, 7, 8) . ARF is also required for
the assembly of the coatomer complex (COP I-coated vesicles) and may
therefore function in facilitating intra-Golgi and retrograde
transport(9) . Other in vitro studies have shown that
ARF-1 is required for the binding of
-adaptin to isolated Golgi
membranes(10, 11) , suggesting that it functions in
the assembly of clathrin coats at the TGN. Experiments in which native
and mutant ARF polypeptides were overexpressed in Chinese hamster ovary
and normal rat kidney cells have also established a key role for ARF in
ER to Golgi, intra-Golgi transport, and maintenance of Golgi morphology in vivo(12, 13) . More recently, it was
demonstrated that ARF activates phospholipase D
activity(14, 15) , and this enzyme has now been shown
to be present in Golgi membranes(16) . Furthermore, the
ARF-stimulated phospholipase D activity was shown to affect
-COP
binding to isolated Golgi membranes, raising the exciting possibility
that changes in the membrane lipid composition (via phospholipase D
activity) may influence coatomer recruitment (17) .
Recently, we established a permeabilized cell system that supports
prohormone processing and secretory vesicle formation from rat anterior
pituitary growth hormone (GH) and prolactin (PRL) secreting GH cells(18) . To determine if nascent secretory vesicle
budding from the TGN might also be ARF-1-regulated, we used recombinant
human ARF-1 in our permeabilized cell system. Here we provide evidence
that ARF-1 stimulated vesicle formation from the TGN and that the N
terminus of the polypeptide was essential for this function.
Human ARF-1 was expressed in and the protein purified from E. coli transformed with appropriate expression vectors
encoding either ARF-1 or ARF-1 and yeast N-myristoyl
transferase. In addition three ARF-1 mutants were also constructed,
expressed, and purified; these corresponded to ARF-1 lacking the first
17 N-terminal amino acids (NT), which could not serve as a
myristic acid acceptor; T31N defective in GTP binding; and Q71L
defective in GTP hydrolysis and corresponding to an activated ARF-1.
Permeabilized GH
cells were incubated in the absence and
presence of the ARF-1 polypeptides, and nascent secretory vesicles were
separated by brief centrifugation and analyzed for immunoprecipitable
GH (Fig. 1). Surprisingly, both the myristoylated and
non-myristoylated forms of native ARF-1 enhanced vesicle budding
approximately 2-fold (Fig. 1, A, lanes
5-8, and B). Interestingly, the two mutants,
NT and T31N, did not stimulate vesicle budding above background
levels (lanes 9-12). In contrast, the Q71L mutant
corresponding to GTP-activated ARF-1 enhanced vesicle formation to the
same extent as the native polypeptide (lanes 13 and 14). The concentration needed for 50% stimulation was about 1
µM (data not shown), which is close to that required for
phospholipase D stimulation (14) and promotion of coatomer
binding to Golgi membranes (21) .
Figure 1:
Recombinant human
ARF-1 protein promotes secretory vesicle formation. A,
permeabilized cells were incubated in the absence of added ARF-1
protein (lanes 1-4) or with 100 µg/ml purified human
recombinant native ARF-1 (lanes 5 and 6);
myristoylated ARF-1 (Myr, lanes 7 and 8); a mutant
ARF-1, NT lacking residues 1-17 (lanes 9 and 10); a mutant defective in GDP-GTP exchange T31N (lanes 11 and 12); or a mutant defective in GTP hydrolysis Q71L (lanes 13 and 14). Following incubation, the nascent
secretory vesicles were separated from permeabilized cells by
centrifugation, and both fractions were analyzed for immunoreactive GH. B, quantitation of vesicle formation by native and mutant
human ARF-1 protein. Data are from three experiments. Vesicle budding
efficiency was calculated as GH or PRL-immunoreactive material in the
supernatant (S)
the total GH or PRL material (pellet (P) + supernatant)
100. ERS,
energy-regenerating system.
Earlier work has shown
that the N terminus of ARF-1 is required for its activity in vesicular
transport and that a peptide corresponding to residues 2-17
prevented ER to Golgi transport in vitro(7) . The
NT mutant also failed to activate cholera toxin-catalyzed
ADP-ribosylation of G
or to rescue the yeast arf1
arf2
lethal
mutant(7) . To determine if secretory vesicle budding from the
TGN might be similarly inhibited, we used the same peptide in our
permeabilized cells. In marked contrast to its inhibitory effects in
other vesicular transport systems(6, 7, 8) ,
the ARF-1 peptide stimulated formation of GH-containing vesicles from
the TGN about 2-fold (Fig. 2A, compare lanes 3 and 4 with 7 and 8); identical results
were obtained for prolactin-containing vesicles (Fig. 2B). Since our permeabilized cell system is not
cytosol-dependent unless pretreated with high ionic strength
buffers(18) , ARF-1 stimulation did not require addition of
cytosol (lanes 7-10). The specificity of the ARF-1
effect was demonstrated by incubating permeabilized cells with a
peptide having the identical composition as the ARF-1 peptide but
possessing a random sequence. Even at a concentration of 200 µM (4-8-fold higher than that used for the native peptide)
there was no stimulation of GH or PRL vesicle budding (Fig. 2, C and D). ATP and GTP were required for vesicle
formation (lanes 1 and 2), and in their absence the
ARF-1 peptide did not promote vesicle budding from the TGN (lanes
11 and 12).
Figure 2: The ARF-1 N-terminal peptide stimulates budding of nascent secretory vesicles from the TGN. A, the release of nascent secretory vesicles (S) containing GH was used to measure formation of immature secretory granules from permeabilized cells (P). Similar results were obtained for prolactin not shown. B, quantitation of growth hormone and prolactin vesicle budding from the TGN by densitometry. C, formation of growth hormone-containing vesicles in the presence of 50 or 200 µM random ARF-1 peptide (R, M, lanes 5-8) or 50 µM native peptide (lanes 9 and 10). D, densitometric analysis of vesicle formation shown in C. ARFp, human ARF-1 peptide; ERS, energy-regenerating system. Budding efficiency was calculated as in Fig. 1.
Since vesicle budding in our system was
ATP- and GTP-dependent ( Fig. 1and Fig. 2)(18) ,
it is unlikely that the ARF-1 peptide had detergent-like properties
that caused membrane lysis resulting in leakage of GH and PRL into the
supernatant(22) . However, to exclude this possibility,
protease protection studies were performed (Fig. 3A).
Proteinase K digestion of the released vesicle fraction (supernatant)
and residual permeabilized cells (pellet) showed that in control and
ARF-1-treated permeabilized cells, PRL was largely protease-resistant (lanes 1-4); in the presence of Triton X-100, it was
degraded quantitatively (lanes 5 and 6); identical
results were obtained for GH-containing vesicles. The ARF-1 peptide
stimulation of vesicle formation was further confirmed by high speed
centrifugation (Fig. 3B). Significantly more PRL was
recovered in the high speed pellet (nascent secretory vesicles)
isolated from ARF-1 peptide-treated permeabilized cells than from
control incubations (lanes 2 and 5). The residual
immunoreactive PRL present in the 150,000 g supernatant corresponded to the small fraction of hormone that had
leaked from disrupted Golgi membranes and was not therefore
sedimentable (lanes 3 and 6). Together these results
indicate that the released hormones were present in membrane-bound
vesicles.
Figure 3:
The ARF-1 peptide stimulates budding of
intact secretory vesicles. A, resistance of PRL-containing
vesicles to proteolysis. Following the budding assay performed without
or with the ARF-1 peptide (ARFp), samples were incubated with
25 µg of proteinase K/ml at 4 °C for 30 min in the absence (lanes 1-4) or presence of 1% Triton X-100 (lanes 5 and 6). The pellet (P) and supernatant (S) fractions were separated by brief centrifugation and
analyzed by SDS-PAGE following immunoprecipitation with anti-PRL or
anti-GH (not shown) antibodies. B, sedimentation of nascent
secretory vesicles containing PRL. Following incubation at 37 °C,
permeabilized cells (lanes 1 and 4, CP) were
separated by centrifugation (11,000 g for 20 s) from
the vesicle-containing supernatant. This was further centrifuged in a
Beckman Airfuge (150,000
g, 10 min) to separate
nascent vesicles (lanes 2 and 5, SP) from
supernatant material (lanes 3 and 6, SS).
All the samples were treated with anti-PRL antibodies and analyzed by
SDS-PAGE.
Kinetic analysis (Fig. 4, inset) showed
that the ARF-1 peptide enhanced both the rate and level of vesicle
budding. We therefore determined at which stage in the budding reaction
the ARF-1 peptide might act (Fig. 4). Permeabilized cells were
incubated in the absence and presence of cytosol, and the ARF-1 peptide
was added at various times after initiating the reaction. In the
absence of cytosol, the ARF-1 peptide stimulated vesicle budding only
early during incubation (within the first 30 min); thereafter its
addition had little effect. Conversely, in the presence of cytosol, the
peptide continued to stimulate vesicle budding up to about 50 min. We
suggest that ARF-1 acts early in the budding reaction and may interact
with a factor(s) that in the absence of cytosol is rapidly consumed and
is therefore no longer available for vesicle formation. In addition,
the time dependence of the ARF-1 reaction and our observation (not
shown) that prohormone cleavage occurred in the presence of the ARF
peptide, a reaction that requires generation of an acidic pH via a
vacuolar H pump in the TGN and nascent
vesicles(23) , argue against membrane leakiness having induced
hormone release. Instead, the data of Fig. 2, Fig. 3, and Fig. 4strongly suggest that ARF-1 stimulated nascent secretory
vesicle release from the TGN.
Figure 4:
The ARF-1 peptide stimulates vesicle
formation early in the budding reaction. Permeabilized cells were
incubated at 37 °C in the absence (open circles) or
presence (filled circles) of 1 mg/ml GH cell
cytosol. At the indicated times after initiating the budding reaction,
30 µM ARF-1 peptide was added, and the incubation
continued for a total of 90 min after which the level of immunoreactive
GH in the nascent vesicle fraction was determined by densitometric
analysis of an SDS gel. Inset, kinetics of nascent GH and PRL
vesicle budding in the absence and presence of the ARF-1 peptide.
Permeabilized cells were incubated in the absence of added cytosol with
and without 30 µM ARF-1 peptide. At the indicated times
aliquots were removed and centrifuged to generate immature secretory
vesicles and residual permeabilized cells, which were treated with
appropriate antisera.
Previous studies have shown that ARF-1
peptides and full-length myristoylated ARF-1 potently inhibit transfer
of vesicular stomatitis virus G- protein from the ER to cis/medial
Golgi and through the Golgi stacks in vitro and in
vivo(6, 7, 12) . In marked contrast, our
data demonstrate that the ARF-1 peptide as well as myristoylated ARF-1
stimulated nascent vesicle release from the TGN, suggesting competition
with components that regulate vesicle formation and/or budding. The
exogenously added ARF-1 is unlikely to act directly on its effector
because, in contrast to other observations, demonstrating a requirement
of the myristoylation for ARF-1 activity (6, 12, 14, 24) as well as for ARF
binding to Golgi membrane(25, 26) , we observed no
such requirement for the budding of post-TGN vesicles. Two hypotheses
could explain our data. First, that ARF-1 functions in concert with a
negative regulator of its activity to promote nascent secretory vesicle
formation and budding from the TGN. Excess ARF-1 peptide or the
recombinant polypeptide would compete with endogenous ARF for the
putative ``ARF-1 suppressor,'' thus alleviating its
inhibitory effect. Alternatively, ARF-1 may negatively regulate a
downstream effector to prevent premature vesicle release from the TGN.
In this model exogenous ARF-1 would block the effector interaction
resulting in enhanced vesicle budding. Our data underscore the
importance of the N terminus in mediating ARF-1
function(7, 27) . A mutant lacking this domain,
NT, did not enhance vesicle budding (Fig. 1) presumably
because it was unable to compete with either the putative ARF-1
regulator or the downstream effector domain in ARF-1. Similarly, the
T31N mutant, which preferentially binds GDP, also did not stimulate
vesicle budding (Fig. 1). A likely explanation is that this
mutant cannot undergo a GTP-induced conformation change, which normally
exposes the ARF-1 N terminus affecting its membrane
localization(27) . Consequently the N terminus of the T31N
mutant would be inaccessible to and could not compete with those
molecules regulating or binding to the ARF-1 effector domain. The
identification of the putative ARF-1 interacting proteins will help
distinguish between these models and enable us to dissect further the
mechanism of secretory granule formation in endocrine cells; these
studies are currently in progress. We speculate that these proteins in
association with endogenous myristoylated ARF-1 enhance secretory
vesicle budding via stimulation of phospholipase D activity (14, 15, 28) or recruitment of clathrin (10, 11) to the TGN or both.