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INTRODUCTION |
Vibrio cholerae colonizes the surfaces of epithelial
cells of the small intestine and produces cholera toxin
(CT),1 causing severe
diarrhea. CT comprises five identical B-subunits, which bind
specifically to GM1 ganglioside on the cell membrane, and one A-subunit
(CTA) that has enzymatic activity. CTA is cleaved into amino-terminal
A1 and carboxyl-terminal A2 peptides by a bacterial protease acting at arginine 192. A1 and
A2 are linked by a disulfide bridge between cysteine 199 and cysteine 187, reduction of which releases enzymatically active
A1-subunit capable of ADP-ribosylating G
s, a stimulatory regulator of adenylyl cyclase, leading
to persistent activation of adenylyl cyclase with accumulation of
cAMP (1-3).
According to the current view, after CT binds to GM1 ganglioside in the
cell membrane, it is internalized and delivered to the Golgi and ER by
vesicular transport (4). This concept was initially derived from
reports that brefeldin A (BFA), which reversibly disrupts the Golgi
architecture (5), suppressed CT action (6, 7), suggesting that CT
action required Golgi function. Thereafter, immunofluorescence
microscopy and subcellular fractionation studies demonstrated that both
CTA and CTB are transported to Golgi, followed by CTA transfer to the
ER (8) where the enzymatically active A1-subunit is likely
to be generated (9, 10). This retrograde toxin transport is consistent
with the presence of KDEL sequences near the carboxyl terminus of CTA
(11, 12). These sequences serve as retrieval signals for soluble
resident proteins of the ER and facilitate the retrograde transport of
toxin proteins from Golgi to ER.
COP1-coated vesicles participate in retrograde traffic from Golgi to
ER. Antibodies against
COP, which is one of the components of COP1
coats, suppressed transport of CT from Golgi to ER (13). The budding of
COP1-coated vesicles requires ARF1, a 20-kDa guanine nucleotide binding
protein, which recruits COP1 coats to the membrane (14). ARF was
discovered as an activator of CT-catalyzed ADP-ribosylation of
G
s (15). ARFs belong to a multigene family, related to
the Ras GTPase superfamily. Like other GTPases, ARF with GDP-bound is
inactive, whereas the GTP-bound form is active. ARF1 activation is
regulated by specific guanine nucleotide-exchange proteins (GEPs) (16,
17), which accelerate the binding of GTP to ARF1, and thereby ARF1-GTP
binding to membranes with subsequent recruitment of COP1 coat proteins
from the cytosol.
ARF1-GTP is inactivated by an ARF1 GTPase-activating protein (GAP)
(18), one of which is recruited from cytosol to the transmembrane KDEL
receptor (Erd2). The interaction of Erd2 and GAP is enhanced by KDEL
protein (19). Inactivated ARF-GDP dissociates from the vesicle along
with bound COP proteins. This uncoating is necessary for vesicle fusion
with a target membrane. Thus, cycling of ARF1 between GDP and GTP forms
is necessary for effective vesicular trafficking.
Six mammalian ARFs are now known. They are divided into three classes,
with ARF1, 2, and 3 in class I, ARF4 and 5 in class II, and ARF6 in
class III. ARF6 is most structurally different among the ARFs. It is
localized in the plasma membrane and endosomes, where it participates
in endocytosis and recycling of the plasma membrane (20, 21). Unlike
ARF6, class I and II ARFs are associated with Golgi and ER, as well as
endosomes, suggesting overlapping functions. There are reports showing
the differences among ARFs in their regulatory proteins (22) and
effectors (23).
To investigate the role of ARF1 in CT action in intact cells, ARF1
mutants, inactive ARF1(T31N), "a GDP mutant," and constitutively active ARF1(Q71L), "a GTP mutant" (24, 25), were used. To detect CT activity, morphological changes of CHO cells were quantified. As expected for a process dependent on Golgi transport, CT-induced morphological changes in CHO cells were suppressed by BFA. The expression of either ARF1 mutant decreased CT-induced morphological changes in CHO cells. This effect appeared specific to ARF1 because ARF5 and ARF6 had no effect, suggesting that CT trafficking leading to
CHO cell morphological changes depends on cycling of ARF1, but not ARF5
or ARF6.
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MATERIALS AND METHODS |
Reagents--
8-Bromo cAMP (8Br-cAMP), brefeldin A (BFA), and
3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma. CT was
from List Biological laboratories. FuGENE6 transfection reagent was from Roche Molecular Biochemicals. Monoclonal antibody against GFP was
from Roche Molecular Biochemicals. Polyclonal rabbit anti-
COP and
anti-HA (Y-11) antibodies were from Affinity Bioreagents and Santa Cruz
Biotechnology, respectively. cy3-conjugated anti-rabbit IgG and
FITC-conjugated anti-rabbit IgG antibodies were from Sigma. FITC-conjugated anti-mouse IgG was from ICN Pharmaceutical. The cAMP
assay kit was from Cayman Chemical.
Cell Culture--
CHO cells were grown at 37 °C in Eagle's
MEM with 10% fetal bovine serum, penicillin (100 units/ml), and
streptomycin (100 µg/ml), in humidified air with 5%
CO2.
Assay of CT-induced Morphological Change in CHO
Cells--
Exponentially growing CHO cells were harvested and
transferred to 6-well plates (3 × 104 cells/well).
Eagle's MEM supplemented with 1% FCS (2 ml/well) was added with or
without CT. After incubation for 4-8 h in a CO2 incubator,
cells were fixed and inspected microscopically to determine the
percentage of cells with morphological changes. More than 100 cells
were counted in each plate. Cells overexpressing ARF were transferred
to 6-cm plates (8 × 104/plate) with coverslips
(24 × 45 mm), incubated without or with CT, fixed, and analyzed
as described.
cAMP Assay--
Freshly trypsinized cells were transferred to
6-well plates (2 × 105 cells/well) and incubated
overnight at 37 °C in Eagle's MEM with 10% FCS. Medium was changed
and BFA was added. After incubation for 30 min, 0.5 mM IBMX
was added, followed 30 min later by 10 nM CT; 2 h
later, cells were washed with PBS, and cAMP was extracted in 6%
trichloroacetic acid solution at 4 °C for 30 min. Trichloroacetic acid was removed from samples by ether extraction before assay of cAMP
according to instructions in the enzyme-linked immunosorbent assay kit
of Cayman Chemical.
Transfection of ARF and ARF Mutants--
Samples of DNA were
incubated for 15 min at room temperature with 5 µl of FuGENE6 in 100 µl of optiMEM (Life Technologies, Inc.) before addition to 50-80%
confluent cells. After incubation for approximately 16 h, cells
were used for experiments. The GFP and HA tags allowed quantification
of extent of transfection by fluorescence microscopy or by Western
analysis of samples of cell homogenates.
Construction of ARF1 and ARF1 Mutants--
Sequences of all
plasmid clones were confirmed by sequencing. cDNAs for ARF1 and
ARF1 mutants were constructed in pcDNA3.1+ (Invitrogen). To ligate
ARF1 or mutant DNA in this vector, an NheI site was
introduced upstream of the initiation codon and a BamHI site
was inserted after the termination codon. These sites were used for
ligation of all constructs into pcDNA3.1+. For ARF1, PCR was
performed with forward primer, F3
(5'-CTGTCCGCTAGCATGGGGAACATCTTCGCCAA-3') and reverse
primer, R5 (5'-AGGGGATCCTCACTTCTGGTTCCGGAGCTG-3'). The
boldface sequence in F3 is the NheI site, and in
R5 the BamHI site. The italic sequence in F3 is
the initiation codon, and in R5 the termination codon. PCR product was
ligated into pcDNA 3.1+.
For ARF1(T31N) construction, the first PCR was performed with forward
primer F3 and reverse primer R3
(5'-AAGCTTGTAGAGGATCGTGTTCTTCCCTGCAGCATCCA-3'), where the
italic T is the mutated base. The other PCR employed forward
primer F4 (5'-TGGATGCTGCAGGGAAGAACACGATCCTCTACAAGCTT-3') and
reverse R5. The italic A is the mutated base. Both PCR
products were gel-purified before use as templates for the second PCR
with primers F3 and R5 to synthesize full-length ARF1(T31N). The PCR product was ligated into pcDNA 3.1+ vector. For ARF1(Q71L), two PCR
products, one with forward primer F3 and reverse primer R4 (5'-CAGGGGCCGGATCTTGTCCAGGCCACCCACGTCCCACA-3'), and the
other PCR with forward primer F5
(5'-TGTGGGACGTGGGTGGCCTGGACAAGATCCGGCCCCTG-3') and reverse
primer R5 were prepared. Mutated bases are represented by italic
A in F3 and T in F5. Both PCR products were
gel-purified before use as templates for the second PCR using F3 and R5
to synthesize full-length ARF1(Q71L), which was ligated into pcDNA 3.1+ vector. All ARF1, ARF1(T31N), and ARF1(Q71L) constructs were transformed into DH5
-competent cells.
EGFP fusion ARF1 and ARF1 mutants (referred to subsequently as,
e.g. GFP-ARF1) were prepared by PCR-based amplification from ARF1 and mutant cDNA in pcDNA3.1+. Forward primer was
5'-CCTGCTAGCCACCATGGGGAACATCTTCGCAAC-3' (boldface is an NheI site; initiator ATG is
underlined) and reverse primer,
5'-CAGCTCCGGAACCAGAAGGATCCCCT-3' (boldface is a BamHI site). PCR product was ligated into
pEGFP-N1(CLONTECH).
HA epitope-tagged ARF1 and ARF1 mutants were produced by PCR-based
amplification of ARF1 and ARF1 mutant cDNA in pcDNA3.1+ vector.
Forward primer was the same used to prepare GFP fusion ARF1 and reverse
primer was
5'-AGGGGATCCTAAGCGTAGTCTGGGACGTCGTATGGGTACTTCTGGTTCCGGAGCTG (italic sequence is the termination codon,
boldface is a BamHI site, and the HA-tag sequence
is underlined). PCR product was digested with
NheI and BamHI and ligated into pcDNA3.1+
vector that had been digested with NheI and
BamHI.
Construction of ARF5 and ARF5 Mutants--
GFP-ARF5 and mutant
fusion constructs were produced by PCR-based amplification from ARF5
cDNA. For GFP-ARF5, forward primer 5'-TCTGCTAGCCACCATGGGCCTCACCGTGTCCGCGCT-3' and
reverse primer 5'-AGGGGATCCTTGCGCTTTGACAGCTCGT-3' were used.
Boldface sequences in forward and reverse primers are
NheI and BamHI sites, respectively. Amplified
products were digested with NheI and BamHI and
ligated in pEGFP-N1 vector that had been digested with the same
enzymes, which was then transformed into DH5
-competent cells. To
construct HA-tagged ARF5, reverse primer was
5'-AGGGGATCCTAAGCCTAGTCTGGGACGTCGTATGGGTAGCGCTTTGAGAGCTCGT-3'. Forward primer was the same as that used to prepare GFP-ARF5. Amplified product was digested with the NheI and the
BamHI and ligated in pcDNA3.1 that had been digested
with the same enzymes, which was then transformed in DH5
competent
cells. ARF5(T31N) and ARF5(Q71L) mutants were generated by
site-specific mutagenesis using a QuikChange mutagenesis kit
(Stratagene) according to the manufacturer's instruction. The primers
used were 5'-GGATGCGGCTGGCAAGAACCACAATCCTGTACAAAC-3' and 5'-GAAAGTACAGGATTGTGTTCTTGCCAGCCGCATCC-3' for
ARF5(T31N), and 5'-GGACGTGGGAGGCCTGGACAAGATTCGGC-3' and
5'-GCCGAATCTTGTCCAGGCCTCCCACGTCC-3' for ARF5(Q71L). Mutated
bases are shown in italics.
Construction of ARF6 and ARF6 Mutants--
For HA-tag
constructs, ARF6 cDNA was amplified from ARF6pET7 (26) by PCR with
forward primer
5'-CCGGAATTCATGGGGAAGGTGCTATCCAAAATCTTC-3' and reverse
primer
5'-GGCAGATCTTTAAGCGTAATCTGGAACATCGTATGGGTAAGATTTGTAGTTAGAGGTTAAC-3'. Boldface are EcoRI and XbaI
restriction sites, respectively. HA-tag sequence is
underlined, and italics represent the starting
and termination codons, respectively. The PCR product was purified and
ligated in-frame to the EcoRI- and XbaI-digested
pXS expression vector. For GFP fusion construct, ARF6 cDNA was
amplified from ARF6pET7 by PCR with forward primer
5'-TATCTCGAGATGGGGAAGGTGCTATCCAAAATCTTC-3' and reverse
primer 5'-ATAGTCGACAGATTTGTAGTTAGAGGTTAAC-3'. Boldface sequences are XhoI and SalI
sites, respectively. Italic is the starting codon. The PCR
product was purified and ligated in-frame to the XhoI- and
SalI-digested pEGFP-N1 expression vector. ARF6(T27N) and
ARF6(Q67L) mutant were generated by site-specific mutagenesis using the
QuikChange mutagenesis kit (Stratagene) according to the
manufacturer's instructions. ARF6(T27N) was generated by PCR with
forward primer
5'-GACGCGGCCGGCAAGACTACAATCCTGTACAAG-3' and reverse
primer 5'-CTTGTACAGGATTGTAGTCTTGCCGGCCGCGTC-3'. ARF6(Q67L) was generated by PCR with forward primer
5'-TGGGATGTGGGCGGCCTGGACAAGATCCGGCCG-3' and reverse primer
5'-CGGCCGGATCTTGTCCAGGCCGCCCACATCCCA-3'.
Italicized sequences indicate mutated bases.
Indirect Immunofluorescence--
Cells were fixed with 3%
paraformaldehyde in PBS for 20 min, incubated with 50 mM
NH4Cl for 10 min, and permeabilized with 0.1% Triton X-100
in PBS for 4 min. Then cells were treated with 3% bovine serum albumin
in PBS for 1 h for blocking. To identify the HA epitope, cells
were incubated with polyclonal anti-HA antibody followed by
cy3-conjugated sheep anti-rabbit IgG antibody. For double staining of
HA-tagged ARF and
COP, cells were incubated with monoclonal anti-HA
antibody (1:400) as a primary antibody and then FITC-conjugated goat
anti-mouse IgG (1:3000) for 1 h as a secondary antibody. To
identify
COP, polyclonal rabbit anti-
COP antibody (1:2000) was
the primary antibody and cy3-conjugated sheep anti-rabbit IgG (1:3000)
was used as the secondary antibody. All procedures used were performed
at room temperature, and, following each step, cells were washed with
PBS three times.
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RESULTS |
CT-induced Morphological Changes in CHO Cells--
When CHO cells,
which had been detached with trypsin, were incubated with 10 nM CT, 60-80% of the cells became bi-polar in shape or
less frequently showed polymorphic shape changes. Most of the untreated
cells were round even after overnight culture (usually fewer than 5%
of cells changed their morphology) (Fig. 1). Similar morphological changes were
induced by treatment with dibutyryl cAMP, 8Br-cAMP, or forskolin, a
direct activator of adenylyl cyclase (data not shown). Appearance of
the morphological changes was about 1 h later with CT than with
8Br-cAMP (Fig. 2), corresponding to the
time needed for CT internalization and activation (27).

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Fig. 1.
Morphological changes of CHO cells induced by
CT. Freshly trypsinized CHO cells in Eagle's MEM
containing 1% FCS were plated (3 × 104/well) in
6-well plates with (B) or without (A) 10 nM CT. After 6 h of incubation at 37 °C, cells were
fixed with methanol and stained with Giemsa.
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Fig. 2.
Effect of CT and 8Br-cAMP on CHO cell
morphology. Cells were incubated in the presence of 10 nM CT (open square) or 0.5 mM
8Br-cAMP (closed square). At the indicated time, cells were
fixed and morphology was evaluated microscopically. More than 100 cells
were examined at each time to calculate percentage of morphologically
changed cells. The data are average of two plates.
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BFA Suppressed CT-induced Morphological Changes in CHO
Cells--
BFA was used to determine whether an intact Golgi complex
was necessary for CT action on CHO cells. BFA is a fungal metabolite that reversibly disrupts Golgi structure by stabilizing an
ARF1GEP·ARF1-GDP complex, thereby preventing ARF1 activation
(28). In the presence of BFA, CT-induced morphological changes were
reduced (Fig. 3A). BFA
treatment also suppressed the accumulation of cAMP (Fig.
3B). These data suggested that ARF1 activation is necessary
for CT action on CHO cells. To confirm this, we examined the effects of
overexpression of ARF1 dominant negative and constitutively active
mutants on the CT-induced morphological changes in CHO cells.

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Fig. 3.
Effect of BFA on CT action on CHO cells.
A, freshly trypsinized CHO cells were plated as in Fig. 1
and incubated with the indicated concentration of BFA for 30 min before
addition of 10 nM CT, at which time, the cells were still
floating. After mixing well and incubation for 7 h more, cells
were fixed and the morphology was examined as in Fig. 2. B,
CHO cells (2 × 105) were incubated in 3-cm plates
overnight, then for 30 min with BFA at the indicated concentration,
followed by addition of 50 mM IBMX for another 30 min. 10 nM CT was then added, and 2 h later, cells were washed
twice with PBS and extracted with 500 µl of 6% trichloroacetic acid
for assay of cAMP. Data are means of values from duplicate
samples.
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Transient Expression of Wild Type ARF1 and ARF1 Mutant Proteins in
CHO Cells--
CHO cells were transfected with pEGFP expression vector
and GFP fusion wild type or mutant ARF1 constructs. Expression was observed initially at 8 h, and the percentage of cells expressing the GFP fusion proteins gradually increased to a maximum at 37 h,
where it remained for at least 50 h (data not shown). Transfection efficiency was 2~5% in CHO cells. Western blotting of cell
homogenate proteins demonstrated expression of ARF1 and ARF1 mutants
using anti-GFP antibody (Fig. 4). The
localization of ARF1 and ARF1 mutants in CHO cells was examined by
fluorescence microscopy. ARF1 localized to the cytosol and Golgi region
whereas ARF1(T31N) (GDP-bound) was chiefly cytosolic. On the other
hand, ARF1(Q71L) (GTP-bound) was concentrated in the Golgi region, as
expected if ARF-GTP were membrane-associated (data not shown).

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Fig. 4.
Expression of wild-type ARFs and ARF
mutants. CHO cells were transfected overnight with pEGFP vector
containing ARF, GDP-bound ARF, or GTP-bound ARF construct or
pcDNA3.1 vector containing HA epitope-tagged ARF and its mutant
constructs. The cells were collected from the plates and washed once
with PBS and disrupted with sonication. Samples (20 µg) of the
homogenate protein were used for SDS-polyacrylamide gel electrophoresis
in a 14% gel, followed by transfer to nitrocellulose filters.
Expression of GFP fusion proteins or HA-tagged proteins was quantified
with monoclonal anti-GFP antibody or polyclonal anti-HA antibody,
respectively, followed by appropriate horseradish peroxidase-conjugated
anti-mouse or anti-rabbit antibodies. Horseradish peroxidase was
detected by ECM reagent (Amersham Pharmacia Biotech). GFP fusion ARFs
and HA-tagged ARFs migrated as proteins of ~50 and ~20 kDa,
respectively.
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Time Course of CT-induced Morphological Changes in ARF1-expressing
Cells--
Effects of GFP-ARF1 on CT-induced morphological changes
were examined. After CT addition, cells were fixed at the indicated times, and morphology was evaluated by fluorescence microscopy for
comparison with that of control cells (FuGENE6-treated). As shown in
Fig. 5, ARF1 overexpression did not
change the rate of appearance of CT-induced morphological changes.

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Fig. 5.
Effect of GFP-ARF1 expression on time course
of CT-induced morphological changes in CHO cells. Cells
transfected with GFP-ARF1 construct (closed circle) or
incubated without DNA with FuGENE6 (open circle), the
reagent used for transfection for 16 h, were used to determine
CT-induced morphological changes. Percentage of morphologically changed
cells is reported. Values are means of duplicate samples.
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CT-induced Morphological Changes in Cells Expressing ARF1
Mutants--
GFP fusion ARF1 mutants were transfected to evaluate the
importance of ARF1 function in CT action. CT-induced changes in
morphology of ARF1-expressing cells were similar to control cells
(FuGENE 6-treated) and to cells transfected with vector alone (Fig.
6A). In cells expressing
ARF1(T31N) or ARF1(Q71L) mutants, CT-induced morphological changes were
significantly suppressed. To confirm that this effect was not related
to GFP, HA epitope-tagged ARF1 and ARF1 mutants were expressed in CHO
cells and CT action was determined. In this experiment, expression was
detected by indirect immunostaining method using anti-HA rabbit
antibody and then cy3-conjugated anti-rabbit IgG antibody. The
localization of expressed proteins was similar to that of GFP-ARF
fusion proteins. Transfection efficiency was less than 1%, much lower
than that with GFP fusion proteins. Western blotting results are shown
in Fig. 4. CT effects on cells expressing mutant proteins were less
than on cells expressing ARF1; the results are similar to those
obtained with GFP fusion proteins (Fig. 6B).

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Fig. 6.
Suppression of CT-induced morphological
changes in cells expressing ARF1 mutants. A, ARF or
ARF1(T31N) or ARF1(Q71L) was expressed as a GFP fusion protein in CHO
cells. Cells were transfected with ARF1 or ARF1 mutant plasmid DNA and
FuGENE6 overnight before detachment from plates and incubation with CT
for 4.5 h. Using fluorescence microscopy to detect GFP, morphology
of expressing cells was evaluated and percentage of morphologically
changed cells is reported. More than 100 cells were examined for each
value. Data are means ± S.D. of values from five experiments.
B, ARF and ARF mutants were expressed as HA-tagged protein.
CHO cells were transfected with HA-tagged ARF or ARF mutant plasmid DNA
overnight before cells were detached from the plates and used for assay
of CT-induced morphological changes. Incubation time with CT was
4.5 h. Cells were stained with polyclonal anti-HA antibody and
cy3-conjugated anti-rabbit antibody. Data are representative of two
similar experiments.
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Effect of ARF6 on the Morphological Changes Induced by CT--
In
mammalian cells, there are six ARFs. Among them, ARF6 is the most
divergent and is grouped in class III. ARF6 localizes to the plasma
membrane and endosomes and does not participate in Golgi to ER
trafficking. Therefore, ARF6 seems not to be involved in CT action. To
confirm this, the effects of wild type ARF6, ARF6(Q67L)(GTP-bound), and
ARF6(T27N)(GDP-bound) mutants were evaluated. ARF6 and ARF6(T27N)
and ARF6(Q67L) mutants were expressed as GFP fusion proteins and
HA-tagged proteins. The rate of CT-induced morphological changes in
cells expressing GFP-ARF6 fusion protein was similar to that of cells
expressing vector (Fig. 7A).
No effect of ARF6(Q67L) was observed. Furthermore, the ARF6(T27N)
mutant did not suppress CT action on CHO cells, although clear
suppression was observed in ARF1 mutants expressing cells (Fig.
7B). HA-tagged ARF6 was also used to examine the CT effect,
and a similar result was obtained (data not shown). These data
suggested that ARF6 did not participate in CT action on CHO cells.

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Fig. 7.
Morphological changes induced by CT were
unaffected by expression of ARF6 or ARF6 mutants. A,
time course of CT-induced morphological changes in cells expressing
GFP-ARF6 or GFP-ARF6(Q67L) or vector. Cells were transfected with
GFP-ARF6 or GFP-ARF6(Q67L) construct or pEGFP vector for 16 h.
CT-induced morphological changes were examined at the indicated times.
Data are representative of two different experiments. B,
GFP-ARF6 and its mutants did not suppress CT-induced morphological
changes. This experiment was performed as described for Fig.
6A. Data are representative of four similar
experiments.
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Effect of ARF5 on the Morphological Changes Induced by CT--
To
examine whether ARF5 affected CT action in CHO cells, GFP fusion and
HA-tagged wild type ARF5 constructs, and ARF5(T31N)(GDP-bound) and
ARF5(Q71L)(GTP-bound) mutants were investigated. Expression was
confirmed by Western blotting and shown in Fig. 4. Cellular localization of GFP fusion ARF5 protein was compared with GFP fusion
ARF1 and HA epitope-tagged ARF6 (Fig. 8).
ARF5 localized to perinuclear region and cytosol similar to ARF1. Some
ARF5 colocalized with
COP. Q71L mutants of ARF1 and ARF5 were
localized to the perinuclear region, and ARF1(Q71L) was mostly
colocalized with
COP, but ARF5(Q71L) less so (Fig.
9). HA-tagged ARF6 and ARF6(Q67L) did not
colocalize with
COP and were in part associated with plasma
membrane. ARF5(T31N) was more sparsely distributed throughout cells
than was wild type ARF5 (data not shown).

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Fig. 8.
Localization of transiently expressed ARF1,
5, and 6 proteins in CHO cells. CHO cells expressing GFP-ARF1 or
GFP-ARF5 constructs or HA-tagged ARF6 were fixed with paraformaldehyde,
stained with rabbit anti- COP antibody, and cy3-conjugated
anti-rabbit IgG. HA-epitope of ARF6 was detected using monoclonal HA
antibody and FITC-conjugated anti-mouse antibody. Cells were inspected
by confocal microscopy.
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Fig. 9.
Subcellular localization of GTP-bound
ARFs. Expression constructs of GFP-ARF1(Q71L) or GFP-ARF5(Q71L) or
HA-ARF6(Q67L) were transfected into CHO cells, and 16 h later,
colocalization with COP was determined.
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Morphological changes induced by CT in cells expressing GFP-ARF5(T31N)
were slightly suppressed, but less so than with ARF1(T31N) (Fig.
10). No suppression was observed in
ARF5(Q71L) mutant-expressing cells, although clear suppression was
observed in ARF1(Q71L)-expressing cells. These data were similar to
those obtained with HA epitope-tagged ARF5 (data not shown).

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Fig. 10.
Effects of ARF5 and its mutants on
CT-induced morphological changes. CHO cells were transfected with
GFP-ARF1 or GFP-ARF5 or their mutants for 16 h, before CT-induced
morphological changes were evaluated. Data are means ± S.D. of
values from three experiments.
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8Br-cAMP Induced Morphological Changes in Cells Expressing ARF1
Mutants--
To determine whether the ARF1 effect on CT-induced
morphological changes was specific for CT action and was not effected
by cAMP, the effect of 8Br-cAMP was examined on cells expressing ARF1
mutants. As shown in Fig. 11,
8Br-cAMP-induced morphological changes were observed in cells
expressing ARF1(T31N) and ARF1(Q71L) mutants to the same extent as
observed in cells expressing wild-type ARF1. In the same experiment,
morphological changes induced by CT were suppressed, consistent with
ARF1 mutants preventing the action of CT, but not that of cAMP.

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Fig. 11.
8Br-cAMP-induced morphological changes in
CHO cells expressing ARF1 mutants. Cells were incubated with 10 nM CT or 0.5 mM 8Br-cAMP for 4 h before
morphology was evaluated by fluorescence microscopy. This figure is
representative of three similar experiments.
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DISCUSSION |
To determine the effect of ARF1 function in CT action in
vivo, we used the dominant negative and constitutively active
mutants of ARF1, ARF1(T31N) and ARF1(Q71L). Overexpression of wild type ARF1 did not accelerate the appearance of CT-induced morphological changes. Expression of either mutant of ARF1 suppressed
morphological changes induced in CHO cells by CT more than did vector
alone or wild type ARF1. Thus ARF1 cycling is necessary for CT action. The suppression, however, was not complete and some percentage of cells
always showed changes in morphology, with a different percentage in
each experiment. This may be the result of differences in levels of
expression in different cells, with some cells not expressing mutant
protein sufficient to suppress endogenous ARF1 function.
Suppressive effects of class II and class III ARF mutants were
not found. Reports of the functional overlap of class I and II ARFs
include the inhibition of ER to Golgi transport by amino-terminal peptides of ARF1 and ARF4 (29) and rescue of ARF-deleted yeast by
expression of ARF1 and ARF4 (30). The peptides in the former study,
however, are probably not specific inhibitors of ARF function (31, 32).
Mutants of ARF5, which like ARF4 belongs to class II, did not clearly
suppress CT action, suggesting that ARF5 cycling is not necessary in CT
action. Indirect immunofluorescence microscopy did show differences
between the GTP forms of ARF1 and ARF5 in localization with
COP,
consistent with their participation different pathways of vesicular
transport. ARF6 (class III) did not affect CT action.
ARFs are activators of CT-catalyzed ADP-ribosylation of
G
s. In vitro, all ARFs are effective in this
reaction. It is not known which ARF, or whether any ARF, is necessary
for CT activation in intact cells. ADP-ribosylation of
G
s by CT in vitro requires GTP and
phosphatidylcholine (33), suggesting roles for ARF activation and a
membrane environment. If ADP-ribosylation of G
s occurs at the cytoplasmic face of the plasma membrane, near its effector adenylyl cyclase, ARF6 might be a good candidate because of its localization. Other ARFs are not localized at the plasma membrane. In
our experiment, the GTP-bound form of ARF6 did not affect CT action,
suggesting that it did not participate in activation of the toxin.
It appears that all available data are most consistent with the notion
that ARF1 influences CT action in cells by its critical physiological
function in the required transport of CT from plasma membrane through
the Golgi and ER.