Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
ADP-ribosylation factor (ARF) 6 localizes
to the plasma membrane (PM) in its GTP state and to a
tubulovesicular compartment in its GDP state in HeLa
cells that express wild-type or mutant forms of this
GTPase. Aluminum fluoride (AlF) treatment of ARF6-transfected cells redistributes ARF6 to the PM and stimulates the formation of actin-rich surface protrusions.
Here we show that cytochalasin D (CD) treatment inhibited formation of the AlF-induced protrusions and
shifted the distribution of ARF6 to a tubular membrane
compartment emanating from the juxtanuclear region
of cells, which resembled the compartment where the
GTP-binding defective mutant of ARF6 localized. This
membrane compartment was distinct from transferrin-positive endosomes, could be detected in the absence of
ARF6 overexpression or CD treatment, and was accessible to loading by PM proteins lacking clathrin/AP-2
cytoplasmic targeting sequences, such as the IL-2 receptor subunit Tac. ARF6 and surface Tac moved into this
compartment and back out to the PM in the absence of
pharmacologic treatment. Whereas AlF treatment
blocked internalization, CD treatment blocked the recycling of wild-type ARF6 and Tac back to the PM; these
blocks were mimicked by expression of ARF6 mutants
Q67L and T27N, which were predicted to be in either the
GTP- or GDP-bound state, respectively. Thus, the ARF6
GTP cycle regulates this membrane traffic pathway. The
delivery of ARF6 and membrane to defined sites along
the PM may provide components necessary for remodeling the cell surface and the underlying actin cytoskeleton.
EUKARYOTIC cells internalize material from the external environment through a variety of distinct endocytic pathways (Steinman et al., 1983 Small ras-related GTPases have been implicated in the
regulation of endocytic membrane recycling (Gruenberg
and Maxfield, 1995 The ADP-ribosylation factor (ARF) family of proteins
represent another group of small GTPases that are thought
to function as regulators of membrane traffic (Donaldson
and Klausner, 1994 ARF6 is the most divergent member of the ARF family
and, unlike ARF1, appears to function in the peripheral
plasma membrane/endosomal system (D'Souza-Schorey
et al., 1995 More recently, we have studied the effector function of
ARF6 by identifying pharmacologic reagents that shift the
distribution, and presumably the GTP status, of wild-type
ARF6 after transient expression in HeLa cells (Radhakrishna
et al., 1996 We have now sought to identify reagents that would
shift wild-type ARF6 into the inactive, GDP-bound state.
Although brefeldin A (BFA) inhibits the activation of
ARF1 (Donaldson et al., 1992b Cells, Reagents, and Antibodies
HeLa cells were grown in DME supplemented with 10% FBS, 100 U/ml
penicillin, 100 µg/ml streptomycin, and 500 µg/ml gentamycin sulfate at
37°C with 5% CO2. Rabbit polyclonal antibodies were raised against a
COOH-terminal peptide of ARF6, residues 164-175. The antiserum specifically recognizes ARF6 in transfected cells and on immunoblots; ARF6-specific labeling during immunofluorescence or immunoblotting was
blocked by the immunizing peptide. A mouse mAb (16B12) against the
influenza HA epitope was purchased from BabCo (Berkeley, CA). The
human Tac antigen (IL-2 receptor DNA Manipulations and Transient Transfections
PCR (Perkin-Elmer Cetus Instruments, Norwalk, CT) was used to remove DNA sequences encoding a COOH-terminal HA epitope tag from
the cDNAs encoding human ARF6/T27N and ARF6/Q67L generated previously (Peters et al., 1995 HeLa cells were grown on glass coverslips and transfected using the
calcium phosphate method as described previously (Bonifacino et al.,
1989 Immunofluorescence Microscopy
30-36 h after transfection, transfected cells were treated as indicated,
fixed with 2% formaldehyde in PBS for 10 min at room temperature, and
rinsed with 10% FBS and 0.02% azide in PBS (PBS/serum). Cells were incubated with primary antibodies diluted in PBS/serum containing 0.2%
saponin for 1 h at room temperature and were washed (three times, 5 min
each) with PBS/serum. The cells were then incubated with fluorescently
labeled secondary antibodies diluted in PBS/serum plus 0.2% saponin for
1 h, washed again, and mounted on glass slides.
Internalization of Transferrin
Transfected cells were rinsed briefly three times with 0.5% BSA in DME,
and then incubated in the same medium for 30 min at 37°C in the absence
or presence of the indicated drugs. Cells were then incubated with 30 µg/
ml of iron-saturated human transferrin in the continued presence of drugs
for an additional 30 min at 37°C, rinsed quickly three times with DME
containing 10% FBS, fixed, and processed for immunofluorescence as
above.
Internalization and Recycling of Anti-Tac Antibodies
For Tac antibody internalization, cells cotransfected with Tac and ARF6
constructs were chilled to 4°C and incubated with mouse anti-Tac antibodies
for 30 min in an ice/water bath. The cells were rinsed briefly with ice-cold
DME containing 10% FBS (complete medium), and they were incubated
with prewarmed (37°C) complete medium in the absence or presence of
the indicated drugs for 30 min at 37°C. Cells were then fixed either immediately or after washing with low pH buffer (Klausner et al., 1983 To detect ARF6 and the internalized Tac antibody, fixed cells were labeled with rabbit anti-ARF6 antiserum, washed, and then incubated with
fluorescently labeled donkey anti-rabbit IgG to detect ARF6 staining,
and incubated with the appropriate fluorescently labeled donkey anti-
mouse IgG to detect the internalized Tac antibody. To detect Tac antibody remaining at the cell surface, some cells were incubated with fluorescently labeled donkey anti-mouse IgG in the absence of saponin; the cells
were then washed and incubated in the presence of saponin with anti-ARF6 antibodies, followed by the appropriate fluorescently labeled donkey anti-rabbit IgG.
To monitor the recycling of Tac antibody, cells cotransfected with Tac
and ARF6 constructs were labeled with mouse anti-Tac antibodies at 4°C
as described above, and were then warmed to 37°C for 30 min either in the
absence or presence of 1 µM CD. The cells were chilled to 4°C, quickly
washed with low pH buffer, as described above, to remove Tac antibody
remaining at the cell surface, and were warmed again to 37°C for 30 min in
the absence or presence of CD before fixation. The surface reappearance
of Tac antibody was detected by incubating the fixed cells with fluorescently labeled donkey anti-mouse IgG in the absence of saponin; ARF6 was then labeled with anti-ARF6 antiserum and the appropriate secondary antibodies in the presence of saponin.
CD Redistributes ARF6 from the PM to a Novel,
Tubular Compartment That Resembles the
Compartment Where the GTP Binding-defective
Mutant of ARF6, T27N, Resides
We previously observed that inhibition of actin polymerization, by treatment of cells with CD, inhibited the AlF-induced protrusions in HeLa cells overexpressing ARF6,
and additionally, we noticed that CD treatment shifted the
distribution of ARF6 to an internal membrane compartment (Radhakrishna et al., 1996
A striking enhancement of ARF6 labeling of the tubular
structures was observed in cells treated with 1.0 µM CD
(Fig. 1, CD). Morphologically, these tubular structures
were similar to those observed in the absence of CD, but
they were more extensive, suggesting that CD treatment
was not creating but merely shifting more ARF6 to these
structures. Other inhibitors of actin polymerization, cytochalasin B and latrunculin B (Spector et al., 1989 These tubular elements were internal structures, and not
invaginations of the PM, as recently described (van Deurs
et al., 1996 Immunoelectron microscopy had previously shown that
wild-type ARF6 localized to both the PM and internal tubulovesicular membranes (Peters et al., 1995 The ability of CD to enhance the tubular morphology of
the ARF6 compartment was reminiscent of the effects of
BFA on increasing the tubular morphology of the transferrin receptor endosomal compartment (Hunziker et al.,
1991
PM Markers Colocalize with ARF6 in the
Tubular Compartment
Although ARF6 redistributed from the PM to the tubular
membrane compartment during CD treatment, the mechanism of transfer between these two compartments was
not clear. Since ARF1 cycles between the cytosol and
Golgi membranes (Donaldson and Klausner, 1994 We used Tac, the IL-2 receptor
The movement of Tac and ARF6 into the tubular endosomal compartment in transfected cells prompted us to examine whether an endogenous PM protein would redistribute to such a tubular compartment with or without
ARF6 overexpression. MHC class I proteins (MHC-I) are
found on the PM and apparently do not contain cytoplasmic tails conferring clathrin/AP-2 localization (Neefjes et
al., 1990 Surface Tac and ARF6 Move from the PM to the
Tubular Compartment
Although ARF6 and Tac colocalized in the tubular compartment with CD or at surface protrusions with AlF, it
was difficult to discern the ARF6- and Tac-localized structures in the juxtanuclear region. However, it is reasonable
to assume that some of this labeling may represent Tac in
the Golgi complex. Indeed, a tight juxtanuclear structure
labeled with Tac antibody is observed in some cells. This
raised the issue of whether the Tac in the tubular compartment was newly synthesized Tac en route to the PM or
surface Tac redistributing from the PM.
Therefore, we assessed the movement of surface Tac
from the PM into the tubular compartment in cells coexpressing ARF6 and Tac by monitoring Tac antibody internalization. Surface Tac was labeled by incubating cells at
4°C with anti-Tac antibody. The cells were then washed
and incubated at 37°C in the presence or absence of CD to
allow internalization of the bound antibody. After fixation, the distribution of the antibody-bound Tac was revealed by secondary antibody staining. Localization of the
Tac antibody after binding at 4°C showed only staining of
the cell surface, where it colocalized with PM-associated
ARF6, but ARF6 was also observed in the internal perinuclear and tubular structures (Fig. 5, 4°C Binding). Subsequent incubation of cells at 37°C in the absence of CD
revealed that Tac antibody was internalized into the perinuclear and tubular compartment in many cells, where, it
then colocalized with ARF6; some of the Tac antibody labeling also colocalized with ARF6 staining at peripheral
PM sites (Fig. 5, 37 °C No drug). This observation indicated that Tac normally moved from the PM to the tubular
compartment in the absence of any perturbants. Warming
the cells in the presence of CD resulted in a more dramatic
redistribution of the Tac antibody and ARF6 to the tubular compartment, with little Tac antibody or ARF6 localization at the peripheral edges of the cells (Fig. 5, 37 °C CD).
To assess Tac antibody remaining on the surface after
CD treatment, the fixed cells were incubated with the secondary antibodies in the absence of detergent permeabilization. After CD treatment, the Tac antibody remaining at
the PM was evenly distributed along the cell surface (Fig.
5, CD, Surface only). No staining of the tubular compartment was observed, indicating that the Tac antibody taken
up into the tubular compartment was inaccessible to secondary antibodies and thus not continuous with the PM.
The internalization of surface Tac was not induced by antibody cross-linking during the course of the experiment,
since the localization of Tac in the tubular compartment
was also observed by immunofluorescence after fixation
(see Fig. 3). In contrast to the internalization of PM proteins, this compartment could not be loaded with fluid phase markers (not shown). Unlike the movement of surface Tac into the tubular compartment in either the presence or absence of CD, when cells were incubated with
AlF at 37°C, the Tac antibody localized to the surface protrusions along with ARF6 and did not redistribute into the
tubular compartment (Fig. 5, AlF).
CD Blocks Recycling of Tac and ARF6 from the
Tubular Compartment Back Out to the PM
Since surface Tac appears to move from the PM to the tubular compartment, and CD treatment causes Tac to accumulate in this compartment (see above), we wanted to determine whether internalized Tac can recycle from the
tubular compartment back out to the PM. To study the fate
of internalized Tac antibody, we had to remove the anti-Tac
antibody that remained at the cell surface after internalization at 37°C. Rinsing cells with low pH buffers containing high salt has been shown to remove transferrin bound to
its receptor at the cell surface (Klausner et al., 1983 To determine the efficiency of removing surface-bound
antibody with low pH buffer, cells expressing ARF6 and
Tac were incubated with Tac antibody at 4°C to label surface Tac. Some cells were warmed to 37°C to allow internalization in the presence of CD. The cells were then either fixed immediately (Fig. 6, Total) or rinsed briefly with
low pH buffer (0.5% acetic acid, 0.5 M NaCl, pH 3.0; Fig.6,
Internal) before fixation and immunolabeling for ARF6
and Tac antibody. The Tac antibody labeled only the cell surface at 4°C, whereas ARF6 localized both to the PM
and to internal structures (Fig. 6, 4°C Total). Rinsing with
low pH buffer completely removed the surface-bound Tac
antibody, but did not alter the distribution of ARF6 (Fig.
6, Internal). Incubation of cells at 37°C for 30 min in the
presence of CD resulted in the internalization of Tac antibody into the tubular compartment (Fig. 6, 37 °C +CD Total). Washing with low pH buffer before fixation removed
the surface Tac antibody, but did not remove the internalized Tac antibody that colocalized with ARF6 in the tubular compartment (Fig. 6, 37 °C +CD Internal). The inability of the low pH wash to remove Tac antibody lends
additional support to the argument that this compartment
is internal and not continuous with the PM. The tubular
compartment had a beaded appearance possibly because of the low pH wash. Nevertheless, ARF6 and Tac remained colocalized in this compartment.
Having demonstrated that the low pH wash could efficiently remove surface-bound Tac antibody, we examined
whether internalized Tac recycled from the tubular compartment back to the PM. Cells were labeled with Tac antibody at 4°C and then warmed to 37°C in the presence of
CD to accumulate Tac antibody in the tubular compartment. Antibodies remaining at the cell surface were then removed with a low pH wash at 4°C (as in Fig. 6), leaving
only the internalized Tac antibody (Fig. 7, Load).
To determine whether the internalized Tac could recycle back to the PM, the loaded cells were subsequently incubated at 37°C in the presence or absence of CD for 30 min. Tac antibody that reappeared on the surface was detected by labeling fixed cells with fluorescent secondary
antibody without detergent permeabilization; ARF6 was
subsequently localized in these cells after permeabilization. The internalized Tac antibody reappeared on the surface at protrusive sites along the edges of cells, together
with ARF6, in the absence of CD (Fig. 7, These results demonstrate that surface Tac moves into
and back out of this tubular endosomal compartment
along with ARF6. In the presence of AlF, internalization into
this compartment is blocked (Fig. 5), whereas in the presence of CD, recycling of Tac back out to the PM is inhibited.
ARF6 Regulates the Cycling of Membrane between the
PM and the Tubular Compartment
The distribution of wild-type ARF6 in cells treated with
AlF and CD mimics the localization of mutant ARF6
in cells expressing the active ARF6/Q67L and inactive
ARF6/T27N mutants, respectively (Radhakrishna et al.,
1996 We first examined the uptake of Tac antibody into cells
cotransfected with plasmids encoding Tac and ARF6/
Q67L, following the protocol used in Fig. 6. After the
binding at 4°C, Tac antibody was uniformly localized along
the PM (Fig. 8, Binding). Upon warming to 37°C in the
presence of CD, no change in the distribution of Tac antibody was observed (Fig. 8, CD Total). Washing these cells with low pH buffer before fixation removed all of the Tac
antibody (Fig. 8, CD Internal), indicating that the Tac antibody remained at the cell surface during the 37°C incubation. The same results were observed when these cells
were warmed in the absence of CD (not shown). This indicates that expression of the active ARF6/Q67L alone blocks
the internalization of surface Tac, suggesting that GTP hydrolysis by ARF6 is required for Tac internalization.
We next examined both the internalization and recycling of Tac antibody in cells cotransfected with plasmids
encoding Tac and ARF6/T27N. After binding at 4°C, Tac
antibody localized to the surface of cells and did not colocalize with ARF6/T27N (Fig. 9, 4°C Binding). Cells were
then incubated at 37°C in the absence of CD, followed by
low pH removal of remaining surface antibody to assess
the internalized Tac antibody. The Tac antibody accumulated in the tubular compartment, where it colocalized in
part with ARF6/T27N (Fig. 9, Uptake). After the low pH
wash, cells that had taken up the Tac antibody were then
further incubated at 37°C to allow recycling, and surface
reappearance of Tac antibody was determined as described above (Fig. 7). The internalized Tac antibody did
not appear on the PM in these cells (Fig. 7, Surface Reappearance). This indicates that expression of the inactive
ARF6/T27N mutant (ARF6-GDP) alone, in the absence
of cytochalasin treatment, blocks the recycling of internalized Tac from the tubular compartment back to the PM,
suggesting that activation of ARF6 is required for exit
from this compartment.
In this study, we describe a novel PM-endosomal recycling
pathway that is regulated by the ARF6 GTP-binding protein. We previously showed that the distribution of the
wild-type protein could be acutely shifted to the active PM
location by treatment of the cells with AlF; dynamic, actin-rich protrusions were induced in these cells as a consequence of this treatment (Radhakrishna et al., 1996 A recent study reported that endogenous ARF6 is associated with a PM fraction isolated from CHO cells and is
not associated with early endosomal membranes, identified by loading with the fluid phase marker HRP (Cavenagh et al., 1996 We propose a tentative working model for how ARF6
regulates this membrane recycling pathway (Fig. 10).
Through its GTP cycle, ARF6 moves between the PM and
this recycling compartment. Nucleotide exchange onto
ARF6-GDP is required for ARF6 and membrane to exit from the recycling compartment. The reappearance of
ARF6-GTP and membrane at peripheral exit sites correlates with the formation of actin-based protrusions. Hydrolysis of the GTP bound to ARF6 is required to allow
internalization of PM and ARF6-GDP back into this internal compartment. The specific mechanism whereby the membrane is internalized, i.e., whether by nonclathrin pinocytosis or tubular membrane invaginations that become
discontinuous with the PM, is yet to be identified. The internal endosomal compartment includes ill-defined structures in the juxtanuclear region and associated tubular elements that extend out to the periphery. We speculate that
treatment of cells with CD blocks ARF6-GTP exchange and membrane recycling, and that treatment with AlF
blocks ARF6-GTP hydrolysis and membrane internalization. We are currently developing methods to investigate
the nucleotides bound to ARF6 under these different conditions, and to further characterize the membrane intermediates involved in the movement into and out of this recycling compartment.
In some respects, the requirement in our model for
ARF6 activation (i.e., GTP exchange) for membrane recycling to occur is similar to the observations made by
D'Souza-Schorey et al. (1995) In HeLa cells, the ARF6-regulated recycling compartment has a distinctive tubular morphology that emanates
out of the juxtanuclear area near the microtubule organizing center. Tooze and Hollinshead (1991) Although the tubular morphology is a hallmark of the
ARF6-regulated membrane compartment in HeLa cells,
this morphology may not be exhibited by ARF6-regulated
compartments in other cells. For example, it was recently
reported that ARF6 cofractionates with isolated chromaffin granules and may be involved in the regulated exocytosis of these granules (Galas et al., 1997 Having demonstrated the existence of a membrane recycling pathway regulated by ARF6, one might ask what
function such a pathway serves in the cell. We showed that
bulk PM was capable of being internalized and recycled
out of such a compartment, and that this apparently occurs
on a slower time scale (loading and unloading of this compartment on the order of ~30-60 min) than the transferrin receptor internalization and recycling pathway (loading
and unloading on the order of 5-10 min; Mellman, 1996 It is intriguing that the trafficking and possibly GTP status of ARF6 are influenced by actin polymerization. The
observation that recycling of ARF6 and membrane back
to the PM is blocked by CD treatment or expression of the
T27N mutant suggests that membrane movement out of
the tubular compartment requires actin polymerization
and ARF6-GTP. At the present time, we cannot determine whether the requirement for actin polymerization is
needed for nucleotide exchange, and thereby conversion
to ARF6-GTP, or if it is required for ARF6-GTP and
membrane to recycle back to the PM, for example,
through an actin-myosin based transport step (Titus,
1997). These
pathways include clathrin-dependent endocytosis (Mellman, 1996
) and a variety of clathrin-independent endocytic processes including pinocytosis (Sandvig and van
Deurs, 1994
; Lamaze and Schmid, 1995
), macropinocytosis
(Swanson and Watts, 1995
), and phagocytosis (Swanson
and Baer, 1995
). A common feature shared by these pathways is that once cargo is delivered to its cellular destination, much of the internalized membrane is recycled back
to the plasma membrane (PM).1 Studies of endocytosis using fluorescent lipid analogues and human transferrin
(Koval and Pagano, 1989
; Mayor et al., 1993
) have shown
that most of the membrane taken up by cells is returned to
the cell surface. Although much of our knowledge about
endocytic membrane recycling has come from studies of
the clathrin-mediated transferrin receptor cycle (Gruenberg and Maxfield, 1995
), it is not clear whether all recycling membrane returns to the cell surface along the same
pathway as the transferrin receptor.
; Mellman, 1996
). In particular, the rab
family GTPases, rab4 and rab11, have been implicated in
the recycling of transferrin receptors. After the release of
iron, transferrin bound to transferrin receptor recycles
back to the PM either rapidly from "sorting" endosomes
or more slowly from a perinuclear compartment termed the "recycling" endosome (Hopkins and Trowbridge,
1983
; Yamashiro et al., 1984
; Hopkins et al., 1994
). Rab4 is
thought to regulate rapid recycling from sorting endosomes (van der Sluijs et al., 1992
), and rab11 has been implicated in traffic between the sorting and recycling endosomes (Ullrich et al., 1996
). It is not known whether rab
proteins are also involved in the recycling of membrane internalized by other endocytic pathways or whether other
regulators are involved.
; Moss and Vaughan, 1995
). ARF proteins, originally identified as cofactors in the cholera toxin-
catalyzed ADP ribosylation of Gs
(Kahn and Gilman,
1986
), have been identified in all eukaryotes tested so far
(Kahn et al., 1991
) and are widely expressed in most mammalian tissues (Tsuchiya et al., 1991
). ARFs also stimulate phospholipase D activity in vitro (Brown et al., 1993
;
Cockroft et al., 1994
; Massenburg et al., 1994
; Hammond
et al., 1995
), and a recent study suggests that this interaction may be important for ARF1 function at the Golgi
complex (Ktistakis et al., 1996
). Among the five known
human ARF proteins, ARF1 is the most thoroughly studied and plays a critical role in the secretory pathway. Both in vivo and in vitro studies have demonstrated that ARF1
cycles between the cytosol (GDP form) and the Golgi
complex (GTP form), where it mediates the binding of soluble coat complexes to Golgi membranes (Donaldson et
al., 1992a
; Robinson and Kreis, 1992
; Palmer et al., 1993
;
Traub et al., 1993
). The regulated cycle of coat assembly/
disassembly is necessary to maintain both the structural integrity of the Golgi complex and transport along the secretory pathway (Melançon et al., 1987
; Donaldson et al., 1991
; Tanigawa et al., 1993
; Dascher and Balch, 1994
; Teal
et al., 1994
; Zhang et al., 1994
). In contrast to ARF1, less is
known about the functions of the other ARF proteins.
; Peters et al., 1995
; Radhakrishna et al., 1996
).
We have been studying the function of ARF6 by examining its localization and the cellular phenotypes conferred
by transient expression of either the wild-type or mutant
forms in mammalian cells (Peters et al., 1995
; Radhakrishna
et al., 1996
). Peters et al. (1995)
showed that wild-type
ARF6 containing an HA epitope tag localizes along the
cytoplasmic face of the plasma membrane and to an internal, tubulovesicular compartment. The morphology of
cells expressing the HA-tagged, wild-type ARF6 appears
normal. A mutant of ARF6 (Q67L) predicted to be defective
in GTP hydrolysis, and thus mainly in the GTP-bound, active form, localizes exclusively to the PM and results in the
formation of elaborate and protrusive PM extensions. Another mutant of ARF6 (T27N), predicted to be defective
in GTP binding, and thus predominantly in the GDP-bound, inactive state, is mostly localized to the internal, tubulovesicular compartment. By immunoelectron microscopy, these
tubulovesicular structures appear to be abundant within
the cells (Peters et al., 1995
).
). We reasoned that whereas the mutant forms
of ARF6 would be locked into either the GTP or GDP
states, the wild-type protein should be capable of cycling
between these two nucleotide states. We found that the G
protein activator, aluminum fluoride (AlF) (Sternweis and Gilman, 1982
), shifts hemagglutinin (HA)-tagged, wild-type ARF6 to the plasma membrane in transiently transfected HeLa cells and stimulates the formation of actin-rich surface protrusions (Radhakrishna et al., 1996
) that
are distinct from the actin rearrangements stimulated by
the rho-related GTPases rac1 or rhoA. Interestingly, whereas there is an apparent increase in macropinocytosis
at these protrusive sites, no effect on transferrin endocytosis is observed (Radhakrishna et al., 1996
). The AlF-stimulated protrusions seen in cells expressing wild-type ARF6
resemble, in part, those observed in cells expressing
ARF6/Q67L alone. This suggests that AlF treatment may
result in the accumulation of ARF6-GTP at the PM, consistent with the observation that AlF can protect ARF1-GTP from GTP hydrolysis (Finazzi et al., 1994
).
; Helms and Rothman,
1992
; Randazzo et al., 1993
), it has no effect on ARF6 (Peters et al., 1995
; Cavenagh et al., 1996
; Radhakrishna et al.,
1996
). An inhibitor of ARF6 activation would be expected to block AlF-induced protrusions and to result in the accumulation of ARF6 in the internal, tubulovesicular compartment. We previously showed that treatment of cells with
inhibitors of actin polymerization, such as cytochalasin D
(CD), prevented the AlF-induced response (Radhakrishna
et al., 1996
). In this study, we use CD treatment and mutants of ARF6 to demonstrate that ARF6 regulates through its GDP/GTP cycle the movement of PM into and out of a
novel recycling compartment.
MATERIALS AND METHODS
subunit) was detected with mouse
monoclonal anti-Tac antibodies (7G7; Rubin et al., 1985
). Rabbit antibodies to human transferrin were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). Mouse antibodies against human MHC class I,
W6/32 were kindly provided by Dr. Paul Roche (National Institutes of Health,
Bethesda, MD). Fluorescein-conjugated WGA and Oregon green-labeled
phalloidin were obtained from Molecular Probes, Inc. (Eugene, OR). Fluorescein- and rhodamine-conjugated donkey anti-mouse and donkey
anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). All other reagents, including iron-saturated human transferrin, CD, and latrunculin B, were purchased from Sigma Chemical Co. (St. Louis, MO).
). A DNA fragment from the ClaI site at 286 bp to the end of ARF6, containing a stop codon and a BglII site, was amplified by PCR and digested with ClaI/BglII. This fragment was used to
replace the corresponding fragment in the epitope-tagged T27N and
Q67L mutants of ARF6. The sequences of all constructs, including regions
generated by PCR, were confirmed by DNA sequencing (SeqWright
DNA Sequencing, Houston, TX). A modified pCDL-SR
expression vector (termed pXS; Takebe et al., 1988
) was used to express wild-type and
mutant ARF6, as well as Tac cDNAs in HeLa cells.
). Cotransfections of ARF6 and Tac were performed using 10 µg of each
plasmid in 10-cm dishes. Under these conditions, the level of ARF6 overexpression was estimated to be 10-40-fold over the endogenous protein.
). To remove anti-Tac antibody remaining at the surface, the cells were chilled to
4°C, rinsed three times quickly with 0.5% acetic acid, 0.5 M NaCl, pH 3.0 (low pH buffer), and then three times with ice-cold complete medium before fixation.
RESULTS
). To further investigate this response to CD, HeLa cells transfected with plasmid
encoding ARF6 were treated with CD for 30 min. The
cells were then fixed and immunolabeled with polyclonal
anti-ARF6 antibodies, and the actin filaments were labeled with Oregon green-conjugated phalloidin. In untreated cells, ARF6 localized along the PM at the peripheral edge of cells and in internal structures near the
nucleus. Fine tubular elements were observed emanating
out of the juxtanuclear region towards the peripheral edge
of some cells (Fig. 1, untreated). The extent to which these
tubular elements were observed in untreated cells varied
(for example, see tubular elements in Figs. 4 and 5); often
they were difficult to discern over the cell surface labeling.
Fig. 1.
CD redistributes
ARF6 to juxtanuclear tubular structures that resemble
the compartment where the
GTP-binding defective mutant of ARF6, T27N, resides.
HeLa cells transfected with
wild-type ARF6 plasmid
were either untreated (Untreated), or treated with 1 µM
CD for 30 min (CD). Other
cells were transfected with
the ARF6/T27N plasmid. The
cells were then fixed in 2%
formaldehyde and labeled with
ARF6-specific antiserum, followed by rhodamine-labeled donkey anti-rabbit IgG and
Oregon green-labeled phalloidin to visualize actin filaments.
[View Larger Version of this Image (101K GIF file)]
Fig. 4.
Endogenous MHC-I localizes to the tubular compartment with or without ARF6 overexpression. HeLa cells were
transfected with ARF6 plasmid and then incubated in the absence (Unt) or presence (CD) of 1 µM CD for 30 min at 37°C,
and they were fixed and processed for immunofluorescence localization of MHC-I using mouse anti-MHC-I antibodies and for
overexpression of ARF6 using antibody to ARF6. Anti-MHC-I
was detected with sequential FITC-labeled goat anti-mouse and
FITC-labeled donkey anti-goat antibodies, while anti-ARF6 was
detected with rhodamine-conjugated donkey anti-rabbit antibody. The tubular structures (arrowheads) radiating from the
perinuclear region, labeled with anti-MHC-I antibodies, become
more apparent after CD treatment, and are observed in both
ARF6-transfected and untransfected cells.
[View Larger Version of this Image (93K GIF file)]
Fig. 5.
Surface Tac is internalized into and colocalizes with ARF6 in the tubular
compartment, and accumulates in this compartment during CD treatment. HeLa
cells expressing both ARF6
and Tac were chilled to 4°C
and incubated with mouse
anti-Tac antibodies (7G7) for
30 min to label surface Tac.
After washing to remove excess antibodies, cells were
warmed to 37°C for 30 min in
the absence (No drug) or
presence of 1 µM CD (CD)
or AlF (AlF) to permit internalization of the bound anti-Tac antibodies, and were fixed
and processed for indirect
immunofluorescence. Tac antibodies were detected with
fluorescent donkey anti-
mouse secondary antibodies
and ARF6 was localized with
rabbit anti-ARF6 antiserum.
In some cells, after CD treatment, Tac antibodies were
localized with secondary antibodies in the absence of detergent to detect Tac antibody remaining at the surface
(CD Surface only); ARF6
was subsequently localized in
these cells after detergent
permeabilization. Note that
Tac antibodies were internalized into the tubular compartment in the absence of
CD treatment (No Drug).
During CD treatment, both
ARF6 and the internalized
Tac antibodies accumulated
in the tubular compartment.
In contrast, AlF treatment
resulted in the sequestration
of anti-Tac antibodies and
ARF6 in surface protrusions and inhibited movement into
the tubular compartment.
[View Larger Version of this Image (84K GIF file)]
), also resulted in extensive tubular staining for ARF6 (not
shown). The effects of CD treatment on ARF6 distribution were fully reversible within 30 min after the removal
of CD (not shown). Even 0.1 µM CD was sufficient to shift
ARF6 to the tubular structures and inhibit the AlF response, although little change in the appearance of actin
filaments was evident (data not shown).
). Surface staining of fixed cells, in the absence
of permeabilization, with fluorescent WGA (not shown)
or antibodies to PM proteins (e.g., see Fig. 5) did not label
the tubular structures. Furthermore, we could demonstrate that these were internal structures that could be
loaded from the PM and thus represented an endosomal compartment (see Fig. 5). Double labeling with antibodies
to ARF6 and tubulin showed that the tubular compartment was aligned along the microtubules; the ability to observe these tubular structures during CD treatment was diminished when microtubules were disrupted by pretreatment
with nocodazole (data not shown).
). In contrast,
the GTP-binding defective mutant, ARF6/T27N, localized
exclusively to tubulovesicular structures. Since CD treatment redistributes wild-type ARF6 from the PM to tubular structures, we examined whether these structures represent the compartment where ARF6/T27N resides.
ARF6/T27N localized to tubular structures originating
from the perinuclear region as well as to vesicular structures scattered throughout the cell (Fig. 1). These observations suggest that CD treatment of cells expressing wild-type ARF6 causes a shift in the distribution of ARF6 to
the tubular compartment where ARF6/T27N localizes.
; Lippincott-Schwartz et al., 1991
; Wood et al., 1991
).
To investigate the relationship between these two endosomal compartments, we examined the distribution of ARF6
and transferrin in HeLa cells transfected with ARF6-HA.
The cells were incubated with transferrin alone or in the
presence of CD (1 µM), BFA (1 µM), or CD plus BFA for
30 min, and were then rinsed in media, fixed and processed for immunofluorescence with antibodies to HA to
detect ARF6-HA-transfected cells and with antibodies to
transferrin to detect cell-associated transferrin. In untreated cells, transferrin was localized to the perinuclear
region and to punctate structures in the periphery, but
showed little overlap with ARF6-labeled structures (Fig.
2, untreated). The distribution of transferrin in transfected
cells was indistinguishable from that observed in untransfected cells. In the presence of CD, no change in the distribution of transferrin was observed, whereas the ARF6 distribution shifted towards the tubular structures emanating out of the perinuclear region (Fig. 2, CD). After treatment
with BFA, the transferrin compartment exhibited tubular
structures, but the ARF6 compartment was unaffected
and did not colocalize with the transferrin compartment
(Fig. 2, BFA). Treatment of cells with both CD and BFA
resulted in tubulation of the transferrin endosome and enhanced tubular morphology of the ARF6 endosome, with
little overlap between the two systems (Fig. 2, CD+BFA). The ARF6 compartment also did not colocalize with
LAMP, a marker for late endosomes and lysosomes (Mellman, 1996
), nor with other organelle markers tested, including those recognizing the Golgi complex and ER (data
not shown).
Fig. 2.
The ARF6-labeled
tubular structures are distinct
from transferrin-positive endosomes. Transfected HeLa
cells expressing ARF6-HA were incubated with 30 µg/
ml iron-loaded human transferrin (Tfn) for 30 min at
37°C either in the absence
(Untreated) or presence of 1 µM CD (CD), 1 µM BFA
(BFA), or CD plus BFA
(CD+BFA). Cells were
washed, fixed, and processed
for indirect immunofluorescence. ARF6-HA was labeled with a mouse anti-HA
antibody, and transferrin was
labeled with rabbit anti-Tfn
antiserum; primary antibodies were visualized with appropriately labeled donkey
secondary antibodies. Note
that in the presence of both
CD and BFA, the ARF6 tubules are distinct from the
transferrin tubules.
[View Larger Version of this Image (108K GIF file)]
), it was
possible that ARF6 might also move from the PM via the
cytosol to the tubular compartment. However, given that
immuno-EM localization (Peters et al., 1995
) and subcellular fractionation studies (Cavenagh et al., 1996
; Song, J.,
and J. Donaldson, unpublished observations) suggest that
the majority of ARF6 in cells is membrane associated, another possibility was that ARF6 might move between the
PM and the tubular compartment by a membrane-mediated process. To begin to address this, we examined whether
other plasma membrane proteins might also redistribute
to the tubular compartment during CD treatment.
subunit, as a PM
marker. Tac has been thoroughly studied and used as a generic integral PM protein with no retention or targeting information that moves through the secretory pathway to
the PM (Leonard et al., 1984
; Weissman et al., 1986
; Bonifacino et al., 1990
; Subtil et al., 1997
). We cotransfected
HeLa cells with plasmids encoding Tac and wild-type
ARF6, and determined the distributions of the proteins after incubation in the presence or absence of CD or AlF.
Tac and ARF6 showed extensive colocalization at the
plasma membrane and with an internal, juxtanuclear compartment in untreated cells (Fig. 3). Whereas CD treatment of cells shifted the localization of Tac and ARF6 to
the tubular compartment, addition of AlF (obtained by
adding 30 mM NaF and 50 µM AlCl3) resulted in colocalization of ARF6 and Tac in surface protrusions (Fig. 3). In
HeLa cells coexpressing ARF6 and a Tac chimeric protein
containing the cytoplasmic tail of HLA-DM, a sequence
specifying clathrin-coated pit localization (Marks et al.,
1995
), the chimeric protein did not codistribute with ARF6
in either surface protrusions or the tubular compartment (not shown). These results demonstrate that PM proteins
lacking known signals for clathrin-coated pit localization
colocalize with ARF6 in the tubular compartment of HeLa
cells during CD treatment.
Fig. 3.
The PM protein Tac colocalizes with ARF6 in the tubular compartment during CD treatment and in surface protrusions
during AlF treatment. HeLa cells transfected with plasmids encoding wild-type ARF6 and the PM protein Tac (IL-2 receptor subunit) were incubated in the absence (Unt) or presence of 1 µM CD (CD) or AlF (AlF) for 30 min, and they were fixed and
processed for indirect immunofluorescence localization of ARF6
with rabbit polyclonal anti-ARF6 antibodies and of Tac with
monoclonal mouse anti-Tac antibodies, followed by the appropriate secondary antibodies.
[View Larger Version of this Image (131K GIF file)]
). We analyzed the distribution of endogenous
MHC-I in HeLa cells, some of which were overexpressing
ARF6 by transient transfection, and thus labeled with the
antibody to ARF6. In untreated cells, MHC-I localized to
the PM, the juxtanuclear region, and occasionally in fine
tubular arrays that radiated from this region (Fig. 4, arrowheads). After CD treatment, there was an apparent increase in MHC-I labeling of these tubular membranes that
extended out to the peripheral edge of the cells (Fig. 4,
CD). The distribution of MHC-I was similar whether the
cells were overexpressing ARF6 (detected with the ARF6
antibody) or were untransfected. In the transfected cells,
ARF6 colocalized with MHC-I at the PM and in the tubular structures. These observations demonstrate that the tubular compartment is normally present in HeLa cells, becomes more elaborate in the presence of CD, and is not
induced by overexpression of ARF6. Thus, we can monitor the movement of ARF6 and Tac in transfected HeLa
cells to study this novel membrane system.
).
Fig. 6.
Low pH buffer removes surface-bound Tac antibodies,
but not the internalized Tac antibodies in the tubular compartment. Tac- and ARF6-transfected HeLa cells were chilled to 4°C
and incubated with mouse anti-Tac antibodies for 30 min. One
set of cells were fixed at 4°C either before (4 °C Total) or after
(4 °C Internal) rinsing with low pH buffer (0.5% acetic acid, 0.5 M
NaCl, pH 3.0). Another set of cells were warmed to 37°C for 30 min in the presence of 1 µM CD and then fixed before (37 °C + CD Total) or after (37 °C +CD Internal) rinsing with low pH
buffer. Fixed cells were then processed for indirect immunofluorescence.
[View Larger Version of this Image (75K GIF file)]
Fig. 7.
CD treatment blocks the recycling of Tac from the tubular compartment back out to the PM. Anti-Tac antibodies
bound to the surface of cells expressing ARF6 and Tac were
loaded into the tubular compartment by incubation with 1 µM
CD at 37°C for 30 min. The cells were chilled to 4°C, rinsed with
low pH buffer to remove the remaining surface anti-Tac antibodies, and then either fixed immediately (Load) and assessed for
the Tac antibody loaded, or warmed to 37°C for 30 min in the absence or presence of CD before fixation. Tac antibody that reappeared on the cell surface was detected by incubation with fluorescently labeled secondary antibodies in the absence of detergent
permeabilization (Surface Reappearance); ARF6 was subsequently
localized in these cells after permeabilization.
[View Larger Version of this Image (90K GIF file)]
CD). Phalloidin labeling of these cells indicated that the sites of Tac reappearance were enriched in actin filaments (not shown).
In contrast, incubation with CD prevented the reappearance of the internalized Tac antibody back on the cell surface (Fig. 7, +CD); ARF6 remained in the tubular compartment and colocalized with the internalized Tac antibody
in samples that were detergent permeabilized to visualize
total Tac antibody staining (not shown).
; Fig. 1 this study). Thus, we examined whether the
blocks in transport observed with these drugs on the movement of Tac between the PM and the tubular endosomal compartment could be recreated in cells expressing
the ARF6 mutant proteins.
Fig. 8.
Internalization of surface Tac into the tubular compartment is blocked in cells expressing the GTPase-defective ARF6/
Q67L mutant. HeLa cells expressing Tac and ARF6/Q67L were
incubated with anti-Tac antibodies at 4°C and either fixed at 4°C
(4°C Binding) or warmed to 37°C for 30 min in the presence of 1 µM CD. The cells were then fixed either before (CD Total) or after rinsing with low pH buffer (CD Internal) and processed for indirect immunofluorescence.
[View Larger Version of this Image (105K GIF file)]
Fig. 9.
Recycling of Tac
from the tubular compartment back out to the PM is
blocked in cells expressing
the GTP-binding-defective ARF6/T27N mutant. HeLa
cells transfected with Tac and
ARF6/T27N plasmids were
incubated with anti-Tac antibodies 4°C, and were then either fixed (4 °C Binding) or
warmed to 37°C for 30 min. These cells were then chilled,
rinsed with low pH buffer,
and either fixed (37°C Uptake) to assess the internalization of Tac antibody or
warmed again to 37°C for 30 min before fixation, and surface reappearance of Tac antibody was assessed as in Fig.
7. Note that the anti-Tac antibodies accumulated in the
tubular compartment in the
absence of CD in cells expressing ARF6/T27N.
[View Larger Version of this Image (97K GIF file)]
DISCUSSION
). We
now demonstrate that inhibitors of actin polymerization, such as CD, shift the distribution of the protein from the
PM to an internal, juxtanuclear compartment that exhibits
tubular elements extending out towards the periphery. In
HeLa cells, this compartment is distinct from the transferrin receptor endosome, and resembles the compartment
where the GTP-binding defective mutant of ARF6, T27N,
resides. Along with ARF6, "generic" PM, including membrane proteins that do not contain clathrin/AP2 localization domains such as Tac, normally moves from the PM
into this compartment and back out again. Although we
had difficulty discerning the morphology of the compartment in the juxtanuclear region, it was clear during the internalization experiments that Tac would appear in both
the juxtanuclear and tubular portion of this compartment simultaneously, suggesting that they were connected. The
two treatments that alter ARF6 distribution also result in
specific blocks in this membrane traffic pathway. CD inhibits egress of ARF6 and membrane out of this system,
whereas AlF prevents the internalization of ARF6 and
membrane into this system. These transport blocks observed pharmacologically with CD and AlF treatment were
recreated by expression of either the inactive T27N or the
active Q67L mutants, respectively. This demonstrates that
the membrane recycling pathway described here is regulated by the GTP cycle of ARF6.
). In transfected HeLa cells, we observe
that ARF6 is associated with an internal membrane compartment in addition to the PM. This compartment is accessible to loading with surface PM proteins, is transferrin negative, and does not accumulate fluid phase endocytic
tracers. Accordingly, this ARF6-associated membrane
compartment that we observe would not be detected as an
"early endosome," but rather, would likely be included in
the PM fraction isolated by Cavenagh et al. (1996)
.
Fig. 10.
Model for the ARF6-regulated PM-endosomal recycling pathway. We propose that activation of ARF6, through nucleotide exchange, occurs at the tubular endosome and triggers
the recycling of membrane to discrete sites at the PM where
ARF6-GTP stimulates the formation of protrusive structures,
rich in actin. Inactivation, through GTP hydrolysis, signals the return of ARF6 to the tubular endosome via membrane intermediates that are yet to be defined. CD treatment or expression of the
GTP-binding-defective mutant of ARF6, T27N, blocks recycling
out to the PM. In contrast, AlF treatment or expression of the
GTPase-defective mutant, Q67L, blocks membrane internalization back into the tubular endosomal compartment.
[View Larger Version of this Image (22K GIF file)]
for the transferrin cycle in
CHO cells. In their study, overexpression of either the
wild-type ARF6 or the constitutively active Q67L mutant
inhibited transferrin uptake into CHO cells, whereas expression of the T27N mutant inhibited transferrin receptor
recycling back to the cell surface. Several distinctions between the two studies, however, deserve comment. First,
the extent to which this endosomal recycling pathway regulated by ARF6 is separate and distinguishable from the
transferrin receptor endosome, as we observed, may vary among different cell types. Indeed, in CHO cells, the transferrin receptor and the T27N mutant of ARF6 colocalize
(D'Souza-Schorey et al., 1995
), whereas in HeLa cells, the
compartment where ARF6 localizes is clearly distinct in its
morphology and behavior from the transferrin receptor
compartment (Radhakrishna et al., 1996
; this study). Second, we were able to confirm the existence and behavior
of the ARF6-regulated compartment in HeLa cells in the absence of ARF6 overexpression by observing endogenous MHC-I in untransfected cells. Finally, in CHO cells,
overexpression of the wild-type ARF6 alone causes a
block in the internalization of transferrin receptors (D'Souza-
Schorey et al., 1995). The inability to reverse this effect of
the wild-type protein limits the use of this system for defining the role of the ARF6-GTP cycle in this process. By
contrast, in HeLa cells we were able to study the full PM-
endosomal recycling pathway regulated by ARF6, since overexpression of the wild-type protein, although exhibiting enhanced nascent protrusive structures at the cell surface, does not inhibit membrane movement through the
pathway. This has allowed us to identify pharmacologic
treatments that acutely and reversibly alter the distribution and movement of ARF6 and membrane proteins through this pathway.
were among the
first to document the existence of extensive tubular endosomal networks observable by thick-section EM in a variety of cell types, including HeLa cells. Although in some cases tubular endosomes have been reported to contain
the transferrin receptor (van der Sluijs et al., 1992
; Hopkins et al., 1994
), the ARF6-regulated tubular endosome
that we observe does not. There are other reports of novel
endosomal compartments distinct from the well-characterized transferrin endosomal system. The autocrine motility
factor receptor localizes to a tubular compartment in
MDCK and HeLa cells that does not colocalize with transferrin receptor (Benlimame et al., 1995
). In NRK cells, expression of endotubin, a protein normally associated with
the apical early endosomes of intestinal epithelia, localizes
to a compartment that contains neither transferrin nor
fluid phase markers and is not altered by treatment with
BFA (Wilson and Colton, 1997
). It will be interesting to
determine whether either of these proteins are associated
with the ARF6-regulated membrane compartment described here.
). Although these granules do not exhibit tubular morphology, through its
GTP cycle, ARF6 may still regulate membrane movement
through such compartments.
).
The ARF6-regulated pathway might allow for bulk membrane to be internalized and then recycled to the cell surface at defined sites in such a way that it could be used to
regulate the surface area and shape of cells. Indeed, in
HeLa cells, ARF6 and membrane recycle to defined sites
along the PM often associated with actin-rich protrusions.
Additionally, we have demonstrated that recycling of
ARF6 and membrane to the edge of cells is required during cell spreading; expression of ARF6/T27N inhibits
HeLa cell spreading (Song, J., Khachikian, H. Radhakrishna,
and J. Donaldson, manuscript in preparation). These observations suggest that the ARF6-regulated membrane cycle may be involved in PM remodeling events that are initiated in response to physiological stimuli (e.g., growth
factors, chemotactic agents, and metastasis).
). It is not likely that CD is specifically inhibiting nucleotide exchange onto ARF6, analogous to the effects of
BFA on ARF1 at the Golgi (Donaldson et al., 1992b
;
Helms and Rothman, 1992
; Randazzo et al., 1993
), since
other inhibitors of actin polymerization, including cytochalasin B and latrunculin, also inhibit movement of ARF6
back to the PM. Further details of the actin dependence of
this step will require in vitro methods to analyze the nucleotide status of ARF6 and reconstitute the ARF6-mediated membrane recycling to the PM. Once at the PM, the accumulation of ARF6 and perhaps the delivery of membrane
there results in a stimulation of actin polymerization and
formation of protrusive structures. Thus, it appears that
ARF6 (localization/GTP status) depends on, but is also capable of stimulating, actin polymerization. Further studies
should elucidate the connection between this novel ARF6-regulated membrane traffic pathway and the actin cytoskeleton.
Received for publication 21 March 1997 and in revised form 20 June 1997.
Address all correspondence to Julie Donaldson, Laboratory of Cell Biology, NHLBI, National Institues of Health, Bethesda, MD 20892. Tel.: (301) 402-2907. Fax: (301) 402-1519. e-mail: jdonalds{at}helix.nih.govWe thank Drs. J. Bonifacino, M. Marks, and P. Roche for generously providing reagents used in this study. We also thank Drs. O. Al-Awar, E. Korn, J. Lippincott-Schwartz, and A. Sobota for critical reading of the manuscript.
ARF, ADP-ribosylation factor; AlF, aluminum fluoride; BFA, brefeldin A; CD, cytochalasin D; HA, hemagglutinin; MHC-I, MHC class I proteins; PM, plasma membrane.
1. | Benlimame, N., D. Simard, and I.R. Nabi. 1995. Autocrine motility factor receptor is a marker for a distinct membranous tubular organelle. J. Cell Biol. 129: 459-471 [Abstract]. |
2. | Bonifacino, J.S., C.K. Suzuki, J. Lippincott-Schwartz, A.M. Weissman, and R.D. Klausner. 1989. Pre-Golgi degradation of newly synthesized T cell antigen receptor chains: intrinsic sensitivity and the role of subunit assembly. J. Cell Biol. 10: 73-83 . |
3. | Bonifacino, J.S., P. Cosson, and R.D. Klausner. 1990. Colocalized transmembrane determinants for ER degradation and subunit assembly explain the intracellular fate of TCR chains. Cell. 63: 503-513 |
4. | Brown, H.A., S. Gutowski, C.R. Moomaw, C. Slaughter, and P.C. Sternweis. 1993. ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity. Cell. 75: 1137-1144 |
5. |
Cavenagh, M.M.,
J.A. Whitney,
K. Carroll,
C.-j. Zhang,
A.L. Boman,
A.G. Rosenwald,
I. Mellman, and
R.A. Kahn.
1996.
Intracellular distribution of
Arf proteins in mammalian cells.
J. Biol. Chem.
271:
21767-21774
|
6. | Cockroft, S., G.M.H. Thomas, A. Fensome, B. Geny, E. Cunningham, I. Gout, I. Hiles, N.F. Totty, O. Truong, and J.J. Hsuan. 1994. Phospholipase D: a downstream effector of ARF in granulocytes. Science (Wash. DC). 263: 523-526 |
7. | D'Souza-Schorey, C., G. Li, M.I. Colombo, and P.D. Stahl. 1995. A regulatory role for ARF6 in receptor-mediated endocytosis. Science (Wash. DC). 267: 1175-1178 |
8. |
Dascher, C., and
W.E. Balch.
1994.
Dominant inhibitory mutants of ARF1
block endoplasmic reticulum to Golgi transport and trigger disassembly of
the Golgi apparatus.
J. Biol. Chem.
269:
1437-1448
|
9. | Donaldson, J.G., J. Lippincott-Schwartz, and R.D. Klausner. 1991. Guanine nucleotides modulate the effects of brefeldin A in semipermeable cells: regulation of the association of a 110-kD peripheral membrane protein with the Golgi apparatus. J. Cell Biol. 112: 579-588 [Abstract]. |
10. |
Donaldson, J.G.,
D. Cassel,
R.A. Kahn, and
R.D. Klausner.
1992a.
ADP-ribosylation factor, a small GTP-binding protein, is required for binding of the
coatomer protein ![]() |
11. | Donaldson, J.G., D. Finazzi, and R.D. Klausner. 1992b. Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein. Nature (Lond.). 360: 350-352 |
12. | Donaldson, J.G., and R.D. Klausner. 1994. ARF: a key regulatory switch in membrane traffic and organelle structure. Curr. Opin. Cell Biol. 6: 527-532 |
13. |
Finazzi, D.,
D. Cassel,
J.G. Donaldson, and
R.D. Klausner.
1994.
Aluminum
fluoride acts on the reversibility of ARF-dependent coat protein binding to
Golgi membranes.
J. Biol. Chem.
269:
13325-13330
|
14. |
Galas, M.-C.,
J.B. Helms,
N. Vitale,
D. Thierse,
D. Aunis, and
M.-F. Bader.
1997.
Regulated exocytosis in chromaffin cells: a potential role for a secretory granule-associated ARF6 protein.
J. Biol. Chem.
272:
2788-2793
|
15. | Gruenberg, J., and F.R. Maxfield. 1995. Membrane transport in the endocytic pathway. Curr. Opin. Cell Biol. 7: 552-563 |
16. |
Hammond, S.M.,
Y.M. Altshuller,
T. Sung,
S.A. Rudge,
K. Rose,
J. Engebrecht,
A.J. Morris, and
M.A. Frohman.
1995.
Human ADP-ribosylation factor-activated phosphatidylcholine-specific phospholipase D defines a new and
highly conserved gene family.
J. Biol. Chem.
270:
29640-29643
|
17. | Helms, J.B., and J.E. Rothman. 1992. Inhibition by brefeldin A of a Golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF. Nature (Lond.). 360: 352-354 |
18. | Hopkins, C.R., and I.S. Trowbridge. 1983. Internalization and processing of transferrin and transferrin receptor in human carcinoma cells. J. Cell Biol. 97: 508-521 [Abstract]. |
19. | Hopkins, C.R., A. Gibson, M. Shipman, D.K. Strickland, and I.S. Trowbridge. 1994. In migrating fibroblasts, recycling receptors are concentrated in narrow tubules in the pericentriolar area, and then routed to the PM of the leading lamella. J. Cell Biol. 125: 1265-1274 [Abstract]. |
20. | Hunziker, W., J.A. Whitney, and I. Mellman. 1991. Selective inhibition of transcytosis by brefeldin A in MDCK cells. Cell. 67: 617-627 |
21. |
Kahn, R.A., and
A.G. Gilman.
1986.
The protein cofactor necessary for ADP-ribosylation of Gs by cholera toxin is itself a GTP-binding protein.
J. Biol.
Chem.
261:
7906-7911
|
22. |
Kahn, R.A.,
F.G. Kern,
J. Clark,
E.P. Gelmann, and
C. Rulka.
1991.
Human
ADP-ribosylation factors. A functionally conserved family of GTP binding
proteins.
J. Biol. Chem.
266:
2606-2614
|
23. |
Klausner, R.D.,
J. Van Renswoude,
G. Ashwell,
C. Kempf,
A.N. Schechter,
A. Dean, and
K.R. Bridges.
1983.
Receptor-mediated endocytosis of transferrin
in K562 cells.
J. Biol. Chem.
258:
4715-4724
|
24. | Koval, M., and R.E. Pagano. 1989. Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts. J. Cell Biol. 108: 2169-2181 [Abstract]. |
25. | Ktistakis, N.T., H.A. Brown, M.G. Waters, P.C. Sternweis, and M.G. Roth. 1996. Evidence that phospholipase D mediates ADP ribosylation factor- dependent formation of Golgi coated vesicles. J. Cell Biol. 134: 295-306 [Abstract]. |
26. | Lamaze, C., and S.L. Schmid. 1995. The emergence of clathrin-independent pinocytic pathways. Curr. Opin. Cell Biol. 7: 573-580 |
27. | Leonard, W.J., J.M. Depper, G.R. Crabtree, S. Rudikoff, J. Pumphrey, R.J. Robb, M. Kronke, P.B. Svetlik, N.J. Peffer, T.A. Waldmann, and W.C. Greene. 1984. Molecular cloning and expression of cDNAs for the human interleukin-2 receptor. Nature (Lond.). 311: 626-631 |
28. | Lippincott-Schwartz, J., L.C. Yuan, C. Tipper, and R.D. Klausner. 1991. Brefeldin A's effects on endosomes, lysosomes, and TGN suggest a general mechanism for regulating organelle structure and membrane trafficking. Cell. 67: 601-616 |
29. |
Marks, M.S.,
P.A. Roche,
E. Van Donselaar,
L. Woodruff,
P.J. Peters, and
J.S. Bonifacino.
1995.
A lysosomal targeting signal in the cytoplasmic tail of the
![]() |
30. |
Massenburg, D.,
J.-S. Han,
M. Liyanage,
W.A. Patton,
S.G. Rhee,
J. Moss, and
M. Vaughan.
1994.
Activation of rat brain phospholipase D by ADP-ribosylation factors 1, 5, and 6: separation of ADP-ribosylation factor-dependent
and oleate-dependent enzymes.
Proc. Natl. Acad. Sci. USA.
91:
11718-11722
|
31. | Mayor, S., J.F. Presley, and F.R. Maxfield. 1993. Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process. J. Cell Biol. 121: 1257-1269 [Abstract]. |
32. | Melançon, P., B.S. Glick, V. Malhotra, P.J. Weidman, T. Serafini, M.L. Gleason, L. Orci, and J.E. Rothman. 1987. Involvement of the GTP-binding "G" proteins in transport through the Golgi stack. Cell. 58: 329-336 . |
33. | Mellman, I.. 1996. Endocytosis and molecular sorting. Annu. Rev. Cell Dev. Biol. 12: 575-625 . |
34. |
Moss, J., and
M. Vaughan.
1995.
Structure and function of ARF proteins: activators of cholera toxin and critical components of intracellular vesicular
transport processes.
J. Biol. Chem.
270:
12327-12330
|
35. | Neefjes, J.J., V. Stollorz, P.J. Peters, H.J. Geuze, and H.L. Ploegh. 1990. The biosynthetic pathway of MHC class II but not class I molecules intersects the endocytic route. Cell. 61: 171-183 |
36. |
Palmer, D.J.,
J.B. Helms,
C.J.M. Beckers,
L. Orci, and
J.E. Rothman.
1993.
Binding of coatomer to Golgi membranes requires ADP-ribosylation factor.
J. Biol. Chem.
268:
12083-12089
|
37. | Peters, P.J., V.W. Hsu, C.E. Ooi, D. Finazzi, S.B. Teal, V. Oorschot, J.G. Donaldson, and R.D. Klausner. 1995. Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments. J. Cell Biol. 128: 1003-1017 [Abstract]. |
38. | Radhakrishna, H., R.D. Klausner, and J.G. Donaldson. 1996. Aluminum fluoride stimulates surface protrusions in cells overexpressing the ARF6 GTPase. J. Cell Biol. 134: 935-947 [Abstract]. |
39. |
Randazzo, P.A.,
Y.C. Yang,
C. Rulka, and
R.A. Kahn.
1993.
Activation of
ADP-ribosylation factor by Golgi membranes: evidence for a brefeldin A
and protease sensitive activating factor on Golgi membranes.
J. Biol. Chem.
268:
9555-9563
|
40. | Robinson, M.S., and T.E. Kreis. 1992. Recruitment of coat proteins onto Golgi membranes in intact and permeabilized cells: effects of brefeldin A and G protein activators. Cell. 69: 129-138 |
41. | Rubin, L.A., C.C. Kurman, W.E. Biddison, N.D. Goldman, and D.L. Nelson. 1985. A monoclonal antibody 7G7/B6 binds to an epitope of the human interleukin-2 (IL-2) receptor that is distinct from that recognized by IL-2 or anti-Tac. Hybridoma. 4: 91-102 |
42. | Sandvig, K., and B. van Deurs. 1994. Endocytosis without clathrin. Trends Cell Biol. 4: 275-277 . |
43. |
Spector, I.,
N.R. Shochet,
D. Blasberger, and
Y. Kashman.
1989.
Latrunculins![]() |
44. | Steinman, R.M., I.S. Mellman, W.A. Muller, and Z.A. Cohn. 1983. Endocytosis and the recycling of plasma membrane. J. Cell Biol. 96: 1-27 |
45. | Sternweis, P.C., and A.G. Gilman. 1982. Aluminum: a requirement for activation of the regulatory component of adenylate cyclase by fluoride. Proc. Natl. Acad. Sci. USA. 79: 4888-4891 [Abstract]. |
46. |
Subtil, A.,
M. Delepierre, and
A. Dautry-Varsat.
1997.
An ![]() ![]() |
47. | Swanson, J.A., and C. Watts. 1995. Macropinocytosis. Trends Cell Biol. 5: 424-428 . |
48. | Swanson, J.A., and S.C. Baer. 1995. Phagocytosis by zippers and triggers. Trends Cell Biol. 5: 89-93 . |
49. |
Takebe, Y.,
M. Seiki,
J.I. Fujisawa,
P. Hoy,
K. Yokota,
K.I. Arai,
M. Yoshida, and
N. Arai.
1988.
SR![]() |
50. | Tanigawa, G., L. Orci, M. Amherd, M. Ravazzola, J.B. Helms, and J.E. Rothman. 1993. Hydrolysis of bound GTP by ARF protein triggers uncoating of Golgi-derived COP-coated vesicles. J. Cell Biol. 123: 1365-1371 [Abstract]. |
51. |
Teal, S.B.,
V.W. Hsu,
P.J. Peters,
R.D. Klausner, and
J.G. Donaldson.
1994.
An
activating mutation in ARF1 stabilizes coatomer binding to Golgi membranes.
J. Biol. Chem.
269:
3135-3138
|
52. | Titus, M.A.. 1997. Unconventional myosins: new frontiers in actin-based motors. Trends Cell Biol. 7: 119-123 . |
53. | Tooze, J., and M. Hollinshead. 1991. Tubular early endosomal networks in AtT20 and other cells. J. Cell Biol. 115: 635-653 [Abstract]. |
54. | Traub, L.M., J.A. Ostrom, and S. Kornfeld. 1993. Biochemical dissection of AP-1 recruitment onto Golgi membranes. J. Cell Biol. 123: 561-573 [Abstract]. |
55. |
Tsuchiya, M.,
S.R. Price,
S.-C. Tsai,
J. Moss, and
M. Vaughan.
1991.
Molecular
identification of ADP-ribosylation factor mRNAs and their expression in
mammalian cells.
J. Biol. Chem.
266:
2772-2777
|
56. | Ullrich, O., S. Reinsch, S. Urbé, M. Zerial, and R.G. Parton. 1996. Rab11 regulates recycling through the pericentriolar recycling endosome. J. Cell Biol. 135: 913-924 [Abstract]. |
57. | van der Sluijs, P., M. Hull, P. Webster, P. Mâle, B. Goud, and I. Mellman. 1992. The small GTP-binding protein rab4 controls an early sorting event on the endocytic pathway. Cell. 70: 729-740 |
58. |
van Deurs, B.,
F. Von Bülow,
F. Vilhardt,
P. Kaae,
Holm, and
K. Sandvig.
1996.
Destabilization of plasma membrane structure by prevention of actin polymerization: microtubule-dependent tubulation of the plasma membrane.
J.
Cell Sci.
109:
1655-1665
|
59. | Weissman, A.M., J.B. Harford, P.B. Svetlik, W.J. Leonard, J.H. Depper, T.A. Waldmann, W.C. Greene, and R.D. Klausner. 1986. Only high affinity receptors for interleukin-2 mediate internalization of ligand. Proc. Natl. Acad. Sci. USA. 83: 1463-1466 [Abstract]. |
60. |
Wilson, J.M., and
T.L. Colton.
1997.
Targeting of an intestinal apical endosomal
protein to endosomes in nonpolarized cells.
J. Cell Biol.
136:
319-330
|
61. | Wood, S.A., J.E. Park, and W.J. Brown. 1991. Brefeldin A causes a microtubule-mediated fusion of the trans-Golgi network and early endosomes. Cell. 67: 591-600 |
62. | Yamashiro, D.J., B. Tycko, S.R. Fluss, and F.R. Maxfield. 1984. Segregation of transferrin to a mildly acidic (pH 6.4) para-Golgi compartment in the recycling pathway. Cell. 37: 789-800 |
63. | Zhang, C., A.G. Rosenwald, M.C. Willingham, S. Skuntz, J. Clark, and R.A. Kahn. 1994. Expression of a dominant allele of human ARF1 inhibits membrane traffic in vivo. J. Cell Biol. 124: 289-300 [Abstract]. |