From the
A specific role for ADP-ribosylation factors (ARFs) in in
vitro endosome-endosome fusion has been proposed (Lenhard, J. M.,
Kahn, R. A., and Stahl, P. D.(1992) J. Biol. Chem. 267,
13047-13052). However, in vivo studies have failed to
support a function for ARFs in the endocytic pathway, since an
antagonist of ARF activities, brefeldin A, does not interfere with
receptor internalization (Schonhorn, J. E., and Wessling-Resnick,
M.(1994) Mol. Cell. Biochem. 135, 159-164). This
controversy surrounding the exact function of ARF in endocytic vesicle
traffic prompted us to critically re-examine the involvement of ARFs in
cell-free endosome fusion. Cytosol depleted of ARF activity was capable
of supporting in vitro endocytic vesicle fusion but failed to
support inhibition of this reaction in the presence of guanosine
5`-3-O-(thio)triphosphate (GTP
ADP-ribosylation factors (ARFs)
In addition to its proposed function in the secretory pathway, a
specific role for ARF in endosome-endosome fusion was proposed based on
the effects of GTP
These concerns prompted us to re-evaluate the role of ARF
in endocytic vesicle fusion by investigating the mechanics of this
reaction in the absence of the GTP-binding protein. Our results
rigorously demonstrate that cell-free endocytic vesicle fusion does not
require cytosolic ARFs, although in agreement with the initial
observations made by Lenhard et al.(18) , we find that
the presence of ARFs is necessary for GTP
Rat liver Golgi fractions were isolated
according to Tabas and Kornfeld (27). Livers from five male rats were
homogenized in 0.5 M sucrose in homogenization buffer (0.05
M Tris maleate, pH 6.5, 5 mM MgCl
Preparation and characterization of ARF-depleted cytosol and
bovine ARF1 have been described
(2, 11) . Briefly, a
soluble CHO cell extract was centrifuged at 100,00
The complex between avidin
The effects of BFA on cell-free endocytic vesicle fusion were
examined to further investigate the proposed activity of ARFs in this
reaction. As shown by the results of Fig. 1, treatment of cytosol
and membranes with BFA did not inhibit endocytic vesicle fusion. In
fact, the extent of vesicle fusion was the same with or without BFA
treatment. This observation is in agreement with results demonstrating
that BFA interferes with exocytic and not endocytic
processes
(21) . However, it should be noted that in vitro intra-Golgi transport also takes place in the presence of the drug
(26), despite the fact that BFA inhibits secretion in vivo (31).
Our results establish that inhibition of cell-free endocytic
vesicle fusion in the presence of GTP
Nonetheless, one could argue that a
membrane-bound form of ARF that is resistant to BFA mediates
endosome-endosome fusion. Such a proposal would be consistent with our
observation that the effects of GTP
It still remains to be determined if
GTP
S). Addition of purified
ARF1 restored the ability of the ARF-depleted cytosol to inhibit
endosome fusion when incubated with GTP
S. Both endocytic vesicle
fusion and the GTP
S-mediated inhibition of vesicle fusion were
unaffected by brefeldin A. Moreover, the ATP requirement and kinetics
of cell-free fusion are not altered by brefeldin A or depletion of
cytosolic ARFs. These results suggest that cytosolic ARFs are not
necessary for endosomal vesicle fusion in vitro but are
responsible for inhibition of fusion in the presence of GTP
S and
cytosolic factors in a brefeldin A-resistant manner.
(
)
are
members of a family of 20-21-kDa GTP-binding proteins implicated
in protein transport from the endoplasmic reticulum to the cis-Golgi
stacks
(1) , intercisternal Golgi transport
(2) , exocytic
vesicle traffic
(3) , and nuclear vesicle dynamics
(4) .
ARF activity appears to be regulated by guanine nucleotide exchange and
hydrolysis since cytosolic ARF is in a GDP-bound state while GTP-bound
ARF associates with cellular membranes
(5) . Because recruitment
and dissociation of the coat proteins
-COP
(6, 7) and AP-1
(8) are dependent on the membrane binding of
ARF, this factor's guanine nucleotide binding state may play a
critical role in cellular membrane traffic. Based on these and other
observations, it was proposed that ARF functions in coatomer-coated
vesicle formation
(9) , although the GTP-binding protein's
exact activity in the secretory pathway remains unclear (10-12).
S in several cell-free assay
systems
(13, 14, 15) . Stahl and co-workers
(16) have demonstrated that GTP
S has a dual effect on
endosomal fusion, stimulating fusion at low cytosolic concentrations
(<0.5 mg/ml) and inhibiting fusion at concentrations of cytosol that
support maximal fusion activity (0.5-2.0 mg/ml). Using a
different cell-free assay, Wessling-Resnick and Braell
(14) described the inhibition of endocytic vesicle fusion in
response to the preincubation of cytosol with GTP
S. Treatment of
cytosol with GTP
S causes the recruitment of cytosolic factor(s) to
vesicle membranes in both of these systems
(14, 17) .
Further work by the Stahl laboratory
(18) demonstrated that an
N-terminal ARF peptide prevents the effects of GTP
S on endocytic
vesicle fusion at low or high cytosol concentrations and that
recombinant, myristoylated ARF1 inhibits this reaction in the presence
of GTP
S. Based on this evidence, Lenhard et al.(18) proposed that ARF activity is required for
endosome-endosome fusion although stringent functional criteria to
support this idea are lacking. For example, the N-terminal
peptide's actions may be rather indirect since it alone does not
affect in vitro fusion
(18) . In fact, nonspecific
effects associated with this peptide have been reported
(19) ,
and a thorough investigation of the peptide's activity has
revealed that this cationic amphipathic helix can cause irreversible
membrane damage
(20) . Moreover, recent in vivo experiments fail to support a direct role for ARF in the endocytic
pathway. Brefeldin A (BFA), an agent that blocks the catalyzed exchange
of guanine nucleotides necessary to activate ARF, was found to
interfere with transferrin receptor membrane traffic by slowing the
rate of exocytosis and not endocytosis(21) .
In addition, BFA does not prevent endocytosis of the low density
lipoprotein receptor but causes its missorting from endosomes
and the trans-Golgi network
(22) . The failure of BFA to
interfere with early events of the endocytic pathway suggests that
endosome fusion continues under conditions that would inactivate ARF
function.
S-mediated inhibition.
Moreover, endocytic vesicle fusion and GTP
S inhibition of vesicle
fusion are not responsive to BFA under conditions where BFA inhibits
the binding of ARF to Golgi membranes
(23, 24, 25) and can antagonize GTP
S-mediated inhibition of
intra-Golgi transport
(26) .
Preparation of Cell Extracts
K562 cells and
Chinese hamster ovary (CHO) cells were maintained in -minimum
Eagle's medium containing 7.5% fetal calf serum as
described
(14) . Cells (K562 or CHO) were collected by
centrifugation and washed three times in phosphate-buffered saline
(PBS) on ice. Cell pellets were resuspended in 3 volumes of uptake
buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 1 mg/ml
glucose, 1 mg/ml bovine serum albumin) and incubated with either 0.5
mg/ml avidin
-galactosidase or 100 nM biotin-transferrin
for 60 min at 20 °C. Endocytic uptake was quenched by diluting the
cell suspension with 5 volumes of ice-cold PBS, followed by five washes
in the same buffer at 4 °C. Cells were mechanically disrupted with
a stainless steel ball homogenizer with 2-7
10
cells/ml in ice-cold breaking buffer (20 mM HEPES, pH
7.4, 0.1 M KCl, 85 mM sucrose, 20 mM EGTA)
until 80-90% of the cells were broken. Postnuclear supernatants
(PNS) were prepared by centrifugation at 800
g for 5
min at 4 °C. Vesicle fractions were separated from cytosolic
proteins over a step gradient
(14) . Briefly, PNS was layered on
0.25 M sucrose, 10 mM acetic acid, 10 mM
triethanolamine, 1 mM EDTA, pH 7.4, and membranes were
pelleted on a cushion of isotonic Nycodenz (Accurate Chemical Co.) upon
centrifugation at 380,000
g for 5 min at 4 °C.
Cytosol was prepared by centrifugation of PNS at 380,000
g for 15 min at 4 °C. The supernatant was removed and stored at
-85 °C until use.
,
containing 5 mM phenylmethanesulfonyl fluoride, 1 mg/ml
soybean and lima bean trypsin inhibitors, 1 mg/ml leupeptin, and 1
mg/ml aprotinin). The homogenate was centrifuged at 600
g for 10 min to remove nuclei and intact cells. The PNS was layered
on top of 1.3 M sucrose in homogenization buffer and
centrifuged in an SW 28 rotor at 63,000
g for 2 h at 4
°C. The crude smooth membrane fraction, collected above the 1.3
M sucrose layer, was adjusted to 1.1 M sucrose and
layered on a sucrose step gradient as described
(27) . Golgi
membranes were collected at the interface between 0.5 and 1.0
M sucrose and pelleted by centrifugation in a Ti-70 rotor at
165,000 rpm for 30 min at 4 °C. The Golgi membrane pellet was
resuspended in breaking buffer and frozen at -85 °C until
use.
g for 1 h at 4 °C, and the resulting supernatant was
fractionated over a Superdex-75 size-exclusion column. Column fractions
were analyzed for the presence of 20-21-kDa GTPases using
[
P]GTP-ligand blot analysis. An ARF(-)
pool consisted of fractions of apparent molecular mass greater than
35 kDa and smaller than
15 kDa, which were concentrated to
the original volume by ultrafiltration.
Assay Conditions for in Vitro Endocytic Vesicle
Fusion
Vesicle fusion was performed by incubating 2.5-µl
samples of avidin -galactosidase and biotin-transferrin vesicle
fractions together for 30 min at 37 °C in 25 µl of breaking
buffer supplemented with 1 mM MgATP, 50 mg/ml creatine kinase,
8 mM phosphocreatine, 10 mg/ml biotin-cytochrome c,
and 1 mM dithiothreitol
(14) . The fusion reaction was
terminated by addition of 5 µl of lysis buffer (10% Triton X-100,
1% sodium dodecyl sulfate, 50 mg/ml biotin-cytochrome c) and
200 µl of dilution buffer (0.05% Triton X-100, 50 mM NaCl,
10 mM Tris, pH 7.4, 1 mg/ml heparin); lysates were clarified
by centrifugation.
-galactosidase
and biotin-transferrin resulting from vesicle fusion was measured by a
modified enzyme-linked immunosorbent assay
(23) . Microstrip
wells (Labsystems) were precoated with rabbit anti-human transferrin
antibodies (1:500 in 50 mM Na
CO
, pH
9.6). Antibody-coated wells were incubated with clarified lysates for 3
h at 37 °C (or overnight at 4 °C) and then rinsed free of
unbound material by three washes with PBS, followed by four washes with
10 mM Tris, pH 7.5, 1% Triton X-100, 0.1% SDS, 10 mM
NaCl, 1 mM EDTA. The wells were incubated in the final wash at
37 °C for 30 min and were rinsed once with PBS. Avidin
-galactosidase activity was then measured by incubating the wells
for 3 h at 37 °C with 250 µl of substrate solution (0.3
mM 4-methylumbelliferyl
-galactoside in 0.1 M
NaCl, 25 mM Tris, pH 7.4, 1 mM MgCl
, 12
mM
-mercaptoethanol). Samples were diluted into 15
volumes of 133 mM glycine, 83 mM
Na
C0
, pH 10.7, and the fluorescence of the
hydrolysis product (365 nm excitation, 450 nm emission) was measured
with an Hitachi F-2000 spectrophotometer. The resulting signal, in
fluorescence units, is directly proportional to the extent of vesicle
fusion in vitro(14, 28) .
Measurement of GTP
Membranes were isolated from PNS by centrifugation at
380,000 S-dependent Membrane Association
of ARF
g for 15 min, washed once, and then
resuspended in breaking buffer and stored at -85 °C. ARF
binding reactions were carried out with PNS membranes (1.25-2
mg/ml) or rat liver Golgi (2 mg/ml) and 2-3 mg/ml K562 cell
cytosol, with or without 100 µM GTP
S; these
components were incubated for 30 min at 37 °C in 100 µl of
breaking buffer supplemented with 1 mM MgATP, 50 mg/ml
creatine kinase, 8 mM phosphocreatine, 10 mg/ml
biotin-cytochrome c, and 1 mM dithiothreitol.
MgCl
was added to 3 mM, and the binding reaction
was terminated by centrifugation at 14,000 rpm for 30 min at 4 °C.
Membrane pellets were washed once in breaking buffer plus 3 mM
MgCl
and then repelleted at 14,000 rpm for 5 min at 4
°C. The ability of membrane-associated ARF to stimulate the
[
C]NAD:agmatine ADP-ribosyltransferase activity
of cholera toxin A subunit (CTA) was then
assayed
(29, 30) . Briefly, membranes (100 µg of
protein) were incubated in the presence of 50 mM potassium
phosphate, pH 7.5, 0.3 mg/ml ovalbumin, 20 mM dithiothreitol,
4 mM MgCl
, 1 mM GTP, 2 mg/ml cardiolipin,
60 mM Cibachrome blue (Fluka), 20 mM agmatine,
[
C]NAD (3
10
cpm/reaction)
with or without 0.5 mg CTA (List Laboratories) in a total volume of 100
µl. After a 60-min incubation at 30 °C, reactions were
terminated by the addition of 1 ml of 1% sodium dodecyl sulfate, and
the entire sample was transferred to a column (0.5
2 cm) of
Dowex AG 1-X2. Eluates were collected by washing columns with 5 ml of
distilled, deionized H
0, and
[
C]ADP-ribosylagmatine was measured using a
Beckman LS 5000-TD liquid scintillation counter. GTP
S-dependent
ARF binding was defined as the difference in
[
C]ADP-ribosylagmatine produced in assays with
membranes that had been incubated with cytosol in the presence and
absence of GTP
S. Activation of CTA ADP-ribosyltransferase activity
was the same in reactions with membranes incubated with or without
GTP
S but in the absence of cytosol, i.e. the effect was
dependent on cytosolic ARFs.
Figure 1:
Brefeldin A does not prevent cell-free
endocytic vesicle fusion or GTPS inhibition of fusion. K562 cell
membrane vesicles and cytosol were separately incubated in the presence
(openbars) or absence (filledbars) of 200 µM BFA for 10 min at 37 °C.
Cytosol was further incubated for 30 min with or without 100
µM GTP
S as indicated. The membrane and cytosol
fractions were then combined at final concentrations of 1 mg/ml
(leftpanel) or 3 mg/ml (rightpanel) cytosol in order to assay for endocytic vesicle
fusion. Reaction conditions for the fusion assay were as described
under ``Experimental Procedures.'' The results shown are the
average of duplicate samples (±S.E.); data are representative of
three independent experiments.
Although BFA does not affect cell-free intra-Golgi
transport, it does block the GTPS-mediated inhibition of this
reaction
(26) . To investigate BFA effects on inhibition of
endosome fusion, cytosol was also incubated with GTP
S before
addition to fusion reactions (Fig. 1). At a final concentration
in the assay of 1 mg/ml, GTP
S-treated cytosol inhibited fusion by
40% while at 3 mg/ml,
80% of this activity was blocked; these
observations are compatible with the idea that a cytosolic factor promotes inhibition of endocytic vesicle
fusion
(14) . However, the presence of BFA did not block
GTP
S-mediated inhibition at either cytosol concentration of
cytosol. Finally, although the concentration of BFA used in these
experiments (200 µM) was previously documented to disrupt
ARF membrane association in vitro(23, 24) , we
also examined the effects of BFA over a wide concentration range
(Fig. 2). The dose-response curve reveals that treatment of
membranes and cytosol with up to 500 µM BFA did not
diminish that extent of endocytic vesicle fusion or prevent the
inhibition of vesicle fusion by GTP
S. The results of
Fig. 1
and Fig. 2indicate that if ARFs play a role in
endosome fusion or the GTP
S-mediated inhibition of fusion, as has
been previously suggested (18), they most likely function by a
BFA-resistant mechanism.
Figure 2:
Dose-response curve for brefeldin A
effects on cell-free endocytic vesicle fusion and GTPS inhibition
of fusion. Endocytic vesicle fusion measurements were performed exactly
as detailed under Fig. l, except that membrane and cytosol fractions
were incubated for 10 min at 37 °C in the presence of BFA at the
concentrations indicated. In vitro fusion was supported by 3
mg/ml cytosol incubated with (opensymbols) or
without (closedsymbols) 100 µM
GTP
S. Data presented are the average of duplicate reactions
(±S.E.) and are representative of three independent
experiments.
The lack of effect on cell-free endocytic
vesicle fusion might suggest that in the presence of BFA, membrane
fusion proceeds via an alternate pathway independent of ARF function.
For example, a mechanism of ``uncoupled'' fusion was proposed
to account for the significant level of in vitro Golgi
transport observed in the absence of ARF
(10) . To test this
possibility, the kinetic parameters of the in vitro reaction
were examined, and it was found that BFA treatment did not
significantly alter the rate or extent of endocytic vesicle fusion
(Fig. 3). Rates of fusion of BFA-treated samples, 27.0 ±
7.1 units/min (n = 4), were comparable with those
measured in control reactions, 24.1 ± 4.4 units/min (n = 4). The fact that the time course of endosome fusion is
unaffected by BFA argues that the mechanism of endocytic vesicle fusion
is not substantially altered by the presence of the drug.
Figure 3:
Time course of endocytic vesicle fusion in
the absence or presence of BFA. K562 cell cytosol and PNS fractions
were separately incubated with (opencircles) or
without (closedcircles) 200 µM BFA for
30 min at 20 °C. The samples were then combined (final
concentration, 3 mg/ml protein), and fusion assays were carried out as
described for Fig. 1. At the times of incubation indicated, samples
were placed on ice, and fusion was rapidly quenched with lysis buffer.
Data presented are the average of duplicate samples (±S.E.) and
are representative of results obtained in four independent
experiments.
As a
positive control for BFA effects related to ARF activity, we examined
the membrane association of the GTP-binding protein in our in vitro system based on the well characterized activity of ARF as a
stimulator of cholera toxin ADP-ribosyltransferase activity (29). As
shown by the results of Fig. 4, BFA reduced GTPS-dependent
ARF binding to rat liver Golgi membranes by approximately 60%, similar
to previous reports
(23, 24, 25, 30) . In
contrast, GTP
S-mediated ARF binding to the K562 cell membranes
present in our assay was relatively unaffected by BFA. Since the PNS
membrane fraction includes Golgi, endosomes, endoplasmic reticulum,
plasma membrane, and other intracellular membrane components, it is
unclear from these experiments whether ARFs specifically associated
with endocytic vesicle membranes persist in binding in the presence of
BFA. The combined results of Fig. 1-3 do suggest, however,
that BFA-insensitive membrane binding of cytosolic ARFs could
account for the GTP
S-mediated inhibition of endocytic vesicle
fusion seen under our assay conditions.
Figure 4:
Brefeldin A reduces GTPS-dependent
membrane association of ARF. K562 cell PNS membranes or rat liver Golgi
and cytosol were incubated in the presence (filled bars) or
absence (open bars) of GTP
S and BFA exactly as described
in Fig. 1. This binding reaction was terminated by centrifugation;
membranes were then washed and assayed for the ability to activate CTA
ADP-ribosyltransferase activity as detailed under ``Experimental
Procedures.'' Data are calculated as the percent
GTP
S-dependent binding activity (±S.D.) from results
obtained in four independent experiments.
To critically examine
whether cytosolic ARFs are responsible for GTPS-mediated
inhibition of cell-free endocytic vesicle fusion, we measured the
ability of ARF-depleted CHO cytosol
(11) to support in vitro endocytic vesicle fusion. As shown in Fig. 5, ARF(-)
cytosol stimulated endocytic vesicle fusion to the same extent as
wild-type CHO cytosol. However, while GTP
S-treated CHO cytosol
inhibited the fusion reaction by
40%, ARF(-) cytosol
incubated with GTP
S failed to block vesicle fusion. If the
ARF(-) cytosol was first supplemented with 135 ng of purified
bovine ARF1 and then incubated with GTP
S, inhibition of endocytic
vesicle fusion was recovered to levels comparable with the
GTP
S-mediated inhibition observed for wild-type CHO cytosol. Under
these assay conditions, the amount of ARF1 added is roughly equivalent
to the native concentration of ARF in wild-type cytosol (
40
ng/µl); we estimate that
200 ng of ARF is present in the
wild-type CHO cytosol supporting fusion reactions in this experiment.
Control assays were also performed in the presence of ARF1 but in the
absence of GTP
S. Despite the fact that ARF1 alone causes slight
inhibition of in vitro fusion activity, this effect is clearly
potentiated by preincubation with the nucleotide. Thus, factors
contained in ARF-depleted cytosol are both necessary and sufficient to
support cell-free endocytic vesicle fusion, although an ARF or
ARF-dependent function must be responsible for the inhibition of
cell-free endocytic vesicle fusion in the presence of GTP
S. These
results are consistent with data obtained from investigations of
cell-free intra-Golgi transport in which ARF(-) cytosol was shown
to support Golgi transport yet failed to inhibit transport in the
presence of GTP
S
(11) .
Figure 5:
ARF-depleted cytosol supports fusion but
not GTPS inhibition of fusion. CHO or ARF(-) cytosol was
incubated with (filledbars) or without (openbars) 100 µM GTP
S and then assayed for
the ability to support endocytic vesicle fusion (final concentration,
1.5 mg/ml); purified bovine ARF1 was also added to some samples (135 ng
final in assay). Fusion activity is expressed relative to the maximal
fusion signal (ARF(-) cytosol + GTP
S). Data shown are
representative of results obtained in four independent experiments.
Conditions were: A, CHO cytosol alone; B,
CHO cytosol with ARF1 added; C, ARF-depleted cytosol; and
D, ARF-depleted cytosol with ARF1
added.
The reaction parameters in the
absence of ARFs were also evaluated. As shown in the time course
experiments of Fig. 6, the rates of fusion reactions performed in
the presence of CHO cytosol or ARF(-) cytosol were found to be
identical. Moreover, the extent of vesicle fusion in the presence and
absence of ARFs is the same, confirming the results shown in
Fig. 5
. Finally, not only is our cell-free endocytic vesicle
fusion reaction dependent on cytosolic factors, but this activity also
requires ATP and is inhibited by ATPS
(14, 32) .
Therefore, to verify that fusion in the absence of cytosolic ARFs is an
ATP-dependent process, CHO and ARF(-) cytosols were added to
fusion assays with or without the nucleotide. The results of
Fig. 7
demonstrate that in vitro fusion activity
supported by either CHO or ARF(-) cytosols requires ATP. This is
an important observation because other cell-free assay systems display
an alternate pathway supporting endosome fusion that can be detected in
the absence of ATP and cytosol
(33) . Based on the identical
kinetics and ATP dependence of vesicle fusion supported by either
wild-type CHO or ARF(-) cytosol, we conclude that
endosome-endosome fusion in the presence or absence of cytosolic ARFs
is mediated by the exact same mechanism.
Figure 6:
Depletion of cytosolic ARFs does not
change the time course of endocytic vesicle fusion. CHO (opensquares) or ARF(-) (closedcircles) cytosol was added to vesicle fractions at a
final concentration of 1.5 mg/ml, and fusion activity was measured
exactly as described for Fig. 3. Data shown are representative of
results obtained on three separate
occasions.
Figure 7:
Endocytic vesicle fusion in the absence or
presence of cytosolic ARFs requires ATP. K562 cell membrane fractions
were combined with CHO or ARF(-) cytosol and incubated in the
presence of ATP and an ATP-regeneration system (1 mM ATP, 100
µg/ml creatine kinase, 8 mM phosphocreatine) or in the
absence of ATP plus an ATP-depleting system (10 units/ml hexokinase, 5
mM 2-deoxyglucose). Fusion reactions were carried out as
described for Fig. 1. The data presented are the average of duplicate
points (±S.E.) and are representative of results obtained in
three independent experiments.
S is absolutely dependent on
cytosolic ARFs, in agreement with previous observations
(18) .
However, the specific role originally proposed for ARFs in endosome
fusion fails to be supported by our demonstration that neither the rate
nor the extent of fusion are altered in the absence of cytosolic ARFs.
Instead, our data suggest a rather promiscuous membrane association of
cytosolic ARFs in the presence of GTP
S, which disrupts
vesicle-vesicle interactions and which may or may not reflect a true
physiologic function. For example, ARF is known to associate with both
PC12 cell membranes and liposomes in a manner that is entirely
dependent on guanine nucleotides rather than specific membrane
factors
(5) . Furthermore, Helms et al.(34) also
demonstrated the existence of a significant pool of ARFs that are
loosely associated with Golgi membranes and readily extracted with
liposomes. Such nonspecific interactions would not only account for our
in vitro results with GTP
S but would also readily explain
the pleiotropic effects observed in vivo with a
GTPase-defective mutant of ARF1, which disrupts membrane traffic,
including endocytosis (35, 36).
S on endosome fusion are not
reversed by BFA, unlike the results obtained for intra-Golgi transport
assays
(26) , and that less than 10% of GTP
S-dependent ARF
binding is blocked by BFA in the crude PNS fractions that provide the
endocytic vesicles under study. In fact, recent studies on the
overexpression of ARF6 demonstrate a plasma membrane/endosomal
distribution of this isoform
(37, 38) . However, the fact
that GTP
S does not inhibit the endosome fusion assay in the
absence of cytosolic ARFs clearly argues against this idea. A model in
which membrane-associated BFA-insensitive ARFs play a role in endosome
fusion would require that GTP hydrolysis is not essential for ARF
function or that ARFs remain stably associated with membranes in the
GTP-bound state and unable to bind GTP
S. Neither of these caveats
is compatible with known properties of ARFs, since GDP-bound ARF does
not bind membranes
(5) and GTP hydrolysis is rapidly catalyzed
upon ARF association with the Golgi
(23, 24) . Curiously,
a defective GTP-binding mutant of ARF6 does appear to remain associated
with endosomal structures (presumably in the GDP-bound state); however,
this mutant disrupts recycling of transferrin receptors
(37) and
is localized on endosomal domains that appear to be coated
(38) .
Thus, it has been postulated ARF6 may promote budding of transport
vesicles from the endosome
(37) , making a role for this isoform
in endosome fusion unlikely.
S-mediated inhibition of endosome fusion by ARF involves other
factors. Since specific binding of ARFs to Golgi membranes and their
function in coatomer complex recruitment have been well
documented
(6, 7, 8, 9, 10) , it
is possible that these events contribute to the pattern of inhibition
observed in our assay system. One can envision that during coatomer
assembly, universal factors necessary for membrane traffic may be
sequestered. This recruitment by ARF of important elements of the
fusion machinery onto the surface of other membranes may consequently
result in the inhibition of our assay. If this possibility is correct,
then inhibition of endosome-endosome fusion by ARF in the presence of
GTP
S must involve other cytosolic factors, a prediction that we
are currently evaluating. These ongoing experiments may elucidate novel
features of ARF's function in the regulation of intracellular
membrane traffic.
S, guanosine
5`-3-O-(thio)triphosphate; BFA, brefeldin A; CHO, Chinese
hamster ovary; PBS, phosphate-buffered saline; PNS, postnuclear
supernatants; CTA, cholera toxin A subunit.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.