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#2007 The Authors
Journal compilation #2007 Blackwell Publishing Ltd
doi: 10.1111/j.1600-0854.2007.00631.x
Traffic 2007; 8: 1644–1655
Blackwell Munksgaard
Two Human ARFGAPs Associated with
COP-I-Coated Vesicles
Gabriella Frigerio
1,2
, Neil Grimsey
1
,
Martin Dale
1
, Irina Majoul
1,3
and
Rainer Duden
1,3,
*
1
Department of Clinical Biochemistry, Cambridge
Institute for Medical Research, University of Cambridge,
Hills Road, Cambridge CB2 2XY, United Kingdom
2
European Bioinformatics Institute, Wellcome Trust
Genome Campus, Hinxton, Cambridge CB10 1SD,
United Kingdom
3
Centre for Biomedical Sciences, School of Biological
Sciences, Royal Holloway University of London,
Egham TW20 0EX, United Kingdom
*Corresponding author: Rainer Duden,
rainer.duden@rhul.ac.uk
ADP-ribosylation factors (ARFs) are critical regulators of
vesicular trafficking pathways and act at multiple intracel-
lular sites. ADP-ribosylation factor-GTPase-activating pro-
teins (ARFGAPs) are proposed to contribute to site-specific
regulation. In yeast, two distinct proteins, Glo3p and
Gcs1p, together provide overlapping, essential ARFGAP
function required for coat protein (COP)-I-dependent traf-
ficking. In mammalian cells, only the Gcs1p orthologue,
named ARFGAP1, has been characterized in detail. How-
ever, Glo3p is known to make the stronger contribution to
COP I traffic in yeast. Here, based on a conserved signature
motif close to the carboxy terminus, we identify ARFGAP2
and ARFGAP3 as the human orthologues of yeast Glo3p.
By immunofluorescence (IF), ARFGAP2 and ARFGAP3 are
closely colocalized with coatomer subunits in NRK cells in
the Golgi complex and peripheral punctate structures. In
contrast to ARFGAP1, both ARFGAP2 and ARFGAP3 are
associated with COP-I-coated vesicles generated from
Golgi membranes in the presence of GTP-g-S in vitro.
ARFGAP2 lacking its zinc finger domain directly binds to
coatomer. Expression of this truncated mutant (DN-ARF-
GAP2) inhibits COP-I-dependent Golgi-to-endoplasmic
reticulum transport of cholera toxin (CTX-K63) in vivo.
Silencing of ARFGAP1 or a combination of ARFGAP2 and
ARFGAP3 in HeLa cells does not decrease cell viability.
However, silencing all three ARFGAPs causes cell death.
Our data provide strong evidence that ARFGAP2 and
ARFGAP3 function in COP I traffic.
Key words: ARF, ARFGAP, coated vesicles, coatomer,
COP I, Golgi
Received 18 June 2007, revised and accepted for publica-
tion 24 July 2007, uncorrected manuscript published
online 27 July 2007, published online 29 August 2007
Cytoplasmic coat proteins govern the transport of proteins
between membrane-bound compartments of the secretory
and endocytic pathways by shaping the membrane of the
donor organelle into vesicular or tubular transport carriers
and selecting appropriate cargo into them (1–3). The well-
characterized minimal machinery to form coat protein (COP)-
I-coated vesicles from Golgi membranes comprises coatomer,
a stable heptameric protein complex comprising a-, b-, b’-,
g-, d-, e- and z-COP, and the small ras-like GTPase ADP-
ribosylation factor (ARF) in its GTP-bound form (4,5). The
GTP/GDP cycle of ARF proteins is regulated by guanine
nucleotide exchange factors and GTPase-activating pro-
teins (GAPs) (6). ARF inactivation by GTP hydrolysis relies
on stimulation of a low intrinsic GTPase activity by ARF-
GTPase-activating proteins (ARFGAPs). ARFGAPs form
a large family of proteins that share a conserved catalytic
domain of approximately 70 residues that includes a zinc
finger motif, but they differ in their non-catalytic domains
(for review see 6,7). In this pathway, ARFGAP activity
triggers uncoating of COP-I-coated vesicles through GTP
hydrolysis on ARF, which renders both ARF and coatomer
cytosolic (4). Interestingly, regulated GTP hydrolysis on
ARF is required not only for uncoating but also for cargo
selection into COP I vesicles because vesicles produced
in the presence of the nonhydrolysable GTP analogue,
GTP-g-S, are depleted of protein cargo (8–10).
There is good evidence that a single ARF species may have
multiple roles and been in different locations in the cell
(6,11). It is generally thought that ARFGAPs with restricted
cellular localizations will contribute to differential regula-
tion of ARF to enable it to function in this way. In yeast,
two ARFGAPs, Gcs1p and Glo3p, provide an overlapp-
ing essential function in the COP-I-mediated Golgi-to-
endoplasmic reticulum (ER) transport (12,13). Several lines
of evidence suggest that Glo3p makes the stronger
contribution to COP-I-mediated trafficking. glo3Dmutants
display a much stronger retrograde Golgi-to-ER transport
defect than gcs1Dmutants (12). Glo3p but not Gcs1p is as-
sociated with yeast COP-I-coated vesicles formed in vitro
(14). A mutant in GLO3, named ret4-1 (13), was isolated
based on a genetic selection for mutants with defects in
dilysine motif-dependent Golgi-to-ER retrieval that had
previously identified several coatomer subunits (15).
Re-use of this article is permitted in accordance with the
Creative Commons Deed, Attribution 2.5, which does not
permit commercial exploitation.
1644 www.traffic.dk
Lastly, Nakano’s laboratory has recently shown that GLO3
but not GCS1 can suppress the temperature-sensitive (ts)-
growth defect of the arf1-16 and arf1-17 mutants, which
display strong defects in retrograde trafficking for several
cargo proteins (16). These data provide compelling evi-
dence that Glo3p in yeast has a critical function in COP-I-
dependent traffic that cannot be complemented by Gcs1p.
We have previously shown that Glo3p but not Gcs1p can
interact with yeast coatomer in vitro (17,18). Using the
two-hybrid system and in vitro binding assays, we dem-
onstrated that this binding occurs via the b0- and g-COP
subunits of coatomer (17,18). All these data combined
functionally tie in Glo3p tightly with the COP I pathway.
Surprisingly, in mammalian cells only ARFGAP1, which by
sequence analysis can be clearly identified as a Gcs1p
orthologue, has been well characterized so far. ARFGAP
was the first ARF-directed GAP to be discovered (19).
ARFGAP1 shuttles between the Golgi complex and cytosol
and is involved in COP-I-dependent trafficking in vivo
(20,21). Assays with Golgi membranes and recombinant
proteins in vitro have suggested that ARFGAP1 is required
for COP I vesicle generation, and under certain in vitro
conditions ARFGAP1 can be observed as a stoichiometric
component of the COP I coat, namely in vesicles produced
in the presence of GTP rather than GTP-g-S (22,23).
Significantly, however, a Drosophila mutant that lacks the
fly orthologue of ARFGAP1 encoded by the Gap69C gene
is viable and fertile (24), consistent with the notion that it is
not an essential, key component to COP I function and that
other, presumably Glo3p-type ARFGAPs can compensate
for the loss of ARFGAP1 function in the fly. In this study,
we aimed to identify and characterize human Glo3p
orthologues.
Sixteen genes encoding ARFGAPs are found in the human
genome. Using a conserved signature motif present in
GLO3 proteins from all species, named the Glo3 motif, we
unambiguously identify ARFGAP2 and ARFGAP3 as the
two human orthologues of yeast Glo3p, and we provide
their initial cell biological characterization. ARFGAP1, ARF-
GAP2 and ARFGAP3 are coexpressed in mammalian cells
(e.g. HeLa and NRK cells). Our data strongly suggest that
ARFGAP1, ARFGAP2 and ARFGAP3 co-operate to perform
essential, overlapping functions in COP-I-mediated traf-
ficking in mammalian cells.
Results
Two human orthologues of yeast Glo3p
We wished to identify a human orthologue for the yeast
ARFGAP Glo3p, which in yeast has been demonstrated to
have a prominent role in coatomer-mediated trafficking.
Among over 16 human proteins with an ARFGAP domain,
we found human Gcs1p, which is named ARFGAP1, and
four novel proteins with a closely related ARFGAP domain.
In order to identify a true orthologue of yeast Glo3p, we
searched for proteins with sequence similarity beyond the
ARFGAP domain. Making use of a Glo3 protein from
Schizosaccharomyces pombe, we were able to identify
a small conserved signature motif, named the Glo3 motif,
found in 19 ARFGAP proteins from 14 different species.
Using this information, we found two distinct human
orthologues of yeast Glo3p, ARFGAP2 and ARFGAP3. In
summary, searching the increasing amount of data avail-
able from systematic sequencing projects, we identified
two distinct human sequence orthologues of the yeast
ARFGAP Glo3p.
The zinc finger domain and the Glo3 motif
Both Glo3p and Gcs1p share high sequence homology in
their amino terminal catalytic zinc finger domain, which is
known to engage ARF. Figure 1A shows an alignment of
Glo3p, Gcs1p and their orthologues from S. pombe,as
well as rat and human ARFGAP1, and the human ARF-
GAP2 and ARFGAP3. Note the presence of the four
conserved cysteine residues characteristic for the zinc
finger domain of ARFGAPs highlighted in yellow.
The conserved Glo3 motif, which is characteristic for Glo3
proteins from a wide variety of eukaryotic organisms
including plants, is about 45 amino acid residues in length
and is situated in the carboxy terminal part of the Glo3
proteins (see Figure 1B). It consists of two repeats of 15
residues separated by a linker of 19–23 residues, depending
on the species. Gaps immediately after some of the repeats
suggest flexibility in the exact length of the sequence in the
region (Figure 1B). Not surprisingly, the overall degree of
conservation is lowest in the parasitic amoeba Plasmodium
yoelii and the fruit fly Drosophila melanogaster.Inthese
organisms, only a single Ile-Ser-Ile tripeptide is present in
the second repeat, whereas only the first repeat is well
conserved in the nematode Caenorhabditis elegans.During
preparation of this manuscript, Nakano’s group also noted
the presence of the Glo3 motif and could demonstrate that
it is important for Glo3p function in yeast (16).
The Glo3 motif, which is always found strictly associated
with an ARFGAP domain, allowed us to identify the true
human orthologues of yeast Glo3p as ARFGAP2 and
ARFGAP3, by sequence comparison. In Figure 2, a protein
sequence alignment of yeast Glo3p, ARFGAP2 and ARF-
GAP3 is shown. ARFGAP2 is a protein of 521 residues
and had not been previously named. However, a zinc
finger protein of unknown function and designated Zfp289
had been previously identified from SCp2 mouse mam-
mary epithelial cells (25). The protein sequence of human
ARFGAP2 and mouse Zfp289 shows complete identity.
ARFGAP2 and ARFGAP3 share 49.6% protein sequence
identity. In an alignment over their entire length, Glo3p
shares only 17.6 and 19.9% identity with ARFGAP2 and
ARFGAP3, respectively. As for ARFGAP3 (516 residues),
both the presence of the ARFGAP domain and its function
as a GTPase-activating protein towards ARF in vitro had
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Two Human ARFGAPs Acting in COPI-Dependent Trafficking
been noted (26,27). However, neither ARFGAP2 nor ARF-
GAP3 have previously been recognized as Glo3p rather than
Gcs1p orthologues, and the localization of both ARFGAP2
and ARFGAP3 and their role in COP I transport has notbeen
investigated.
ARFGAP2 and ARFGAP3 are found in the Golgi
complex as well as in pre-Golgi punctate structures
In order to confirm that these two human Glo3p ortho-
logues are involved in COP I transport, we raised mono-
specific polyclonal antibodies against them (see Materials
and Methods) and determined their intracellular localiza-
tion. By IF studies on methanol–acetone-fixed NRK cells,
we find the majority of ARFGAP2 as well as ARFGAP3
associated with a juxtanuclear densely packed structure
typical for the mammalian Golgi complex (Figures 3 and 4).
The remaining protein is associated with small punc-
tate structures scattered throughout the cytoplasm.
Double-labelling experiments reveal that these structures
labelled with antibodies against ARFGAP2 as well as
ARFGAP3 are largely identical to those labelled with anti-
bodies against subunits of the coatomer complex, identi-
fying them as ER-Golgi intermediate compartment
(ERGIC)/vesicular-tubular compartment (VTC) structures.
To extend these results, we performed double-labelling
experiments with a number of well-established marker
proteins. The tethering protein p115 is known to be
associated with the early Golgi complex as well as with
a punctate pre-Golgi compartment (28). Although
because of its exceptional quality, the monoclonal anti-
body against p115 labels a considerably larger number of
peripheral structures, the majority of ARFGAP2- or ARF-
GAP3-positive structures are p115 positive as well.
Within the Golgi compartment, there are areas where
closer inspection of the patterns suggests that the
mammalian ARFGAP2 and ARFGAP3, and p115, respect-
ively, are localized in a closely associated but not
identical pattern. This is in clear contrast to these proteins
Figure 1: ARFGAP domain and
conserved motif of a representa-
tive subset of Glo3 proteins. A)
The N-terminal ARFGAP domains of
Glo3 and Gcs1 proteins from Sac-
charomyces cerevisiae,Schizosac-
charomyces pombe and Homo
sapiens were aligned using the CLUS-
TAL V algorithm in the MEGALIGN pro-
gram (DNASTAR package). Note the
conserved cysteine residues high-
lighted in yellow and the overall high
degree of sequence conservation in
this domain. B). The Glo3 motif
(highlighted in orange) consists of a
repeat of 15 residues separated by
11–17 residues. It is a signature
motif that allows unambiguous
identification of Glo3 orthologues.
Genbank accession numbers: cere-
visiae: 6320969 (S. cerevisiae);
Plasmodium 23478251 (P. yoelii);
worm: 25153991 (Caenorhabditis
elegans); fly: 24668642 (Drosophila
melanogaster); plant A: 7487780,
plant B: 18403775, plant C 15237500
(Arabidopsis thaliana); fish: assem-
bled from several ESTs: 12148139,
12171549, 12158629 and 12265392
(Danio rerio); frog: 27695479 (Xenopus
laevis) and human A: 21263420 and
human B: 31543983 (H. sapiens).
1646 Traffic 2007; 8: 1644–1655
Frigerio et al.
in comparison with subunits of the coatomer complex,
where most features within the Golgi complex are
labelled in a remarkably identical way.
Again, this is not the case for ARFGAP2 and ARFGAP3
when compared with the well-characterized early Golgi
marker GM130 (29) (see Figures 3 and 4). In this case, the
proteins are localized in a closely associated but not
identical fashion in the region of the Golgi complex. As
a control, we analysed the localization of ARFGAP2 and
ARFGAP3 in comparison to g-adaptin, a subunit of the
clathrin adaptor complex AP-1, which is involved in traffic
between the trans Golgi network (TGN) and endosomal
compartments (30). As expected, we found no significant
overlap in the extensive punctate peripheral label as well
as the juxtanuclear label associated with the Golgi complex
(Figures 3 and 4). We conclude that the localization of
ARFGAP2 and ARFGAP3 is consistent with a function in
COP I trafficking.
ARFGAP2 and ARFGAP3 are associated with
COP-I-coated vesicles generated in vitro
We next wished to test whether ARFGAP2 and ARFGAP3
are associated with COP I vesicles produced in vitro. For
this we employed the ‘classic’ budding assay developed by
the Rothman/Wieland labs (31,32), using purified rat liver
Golgi and pig brain cytosol. The budding reaction was
performed in the presence of the nonhydrolysable GTP
analogue, GTP-g-S, which locks ARF on the membrane of
vesicles and thus prevents uncoating. Vesicles and Golgi
donor membranes were separated on a linear sucrose
Figure 2: Sequence alignment of
yeast Glo3p and human ARF-
GAP2 and ARFGAP3. ARFGAP2
and ARFGAP3 share 49.6% protein
sequence identity. ARFGAP2 and
ARFGAP3 share 17.6 and 19.9%
sequence identity with Glo3p,
respectively.
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Two Human ARFGAPs Acting in COPI-Dependent Trafficking
gradient by overnight centrifugation (see Materials and
Methods, and 31), and proteins in the fractions obtained
were analysed by immunoblotting and silver staining of
bands after SDS–PAGE (Figure 5). The characteristic set of
coatomer bands (a-, b’-, b-, g- and d-COP) were found
enriched in the expected positions for COP-I-coated
vesicles in the gradient (fractions 8 þ9; corresponding to
40–43% sucrose) where also the blot signals for g-COP
(Figure 5A,B) and b-COP (data not shown) showed a
corresponding major peak. ARFGAP1 was predominantly
detected in the donor Golgi fractions, but only small
amounts were found in the fractions containing COP I
vesicles (Figure 5B). This is consistent with the previously
reported findings from the Hsu lab that ARFGAP1 is
depleted from COP I vesicles formed in the presence of
GTP-g-S (23). On the other hand, ARFGAP2 and ARFGAP3
showed a strong peak in the COP I vesicle fractions
(Figure 5B), indicating that these novel Glo3-type ARFGAPs
can be actively recruited into budding COP I vesicles even in
the presence of GTP-g-S. Clathrin heavy chain and the
g-subunit of the AP-1 adaptor complex used as controls
were absent from the COP I vesicle fractions, as expected.
We find that the dilysine motif-bearing protein ERGIC-53
was also not included into the COP I vesicles. ADP-
ribosylation factor-1, which is involved in many different
transport steps within the Golgi complex, was found
both in the donor Golgi fractions and in the COP I vesicle
fractions, as expected. Our data show that both novel
ARFGAPs, ARFGAP2 and ARFGAP3 are associated with
the COP-I-coated vesicles produced in vitro in the
presence of GTPgS, whereas ARFGAP1 is not or much
less so.
Figure 3: ARFGAP2 colocalizes with coatomer in NRK cells. Methanol–acetone-fixed NRK cells were stained with affinity-purified
ARFGAP2 antibodies and monoclonal antibodies against b-COP, p115, GM130 or g-adaptin. Cy3-coupled anti-rabbit and Alexa488-coupled
anti-mouse secondary antibodies were used. Images were acquired using a Zeiss Axioplan Fluorescent Microscope equipped with a charge-
coupled device camera. The individual channels are shown in monochrome; the composite overlay is shown to the right (green channel,
ARFGAP2; red channel, b-COP, p115, GM130 and gAP, respectively). Bar ¼10 mm.
1648 Traffic 2007; 8: 1644–1655
Frigerio et al.
The catalytic domain on Glo3p or ARFGAP2 is not
required for interaction with coatomer
Yeast Glo3p interacts with g-COP as well as b0-COP in the
two-hybrid system (17). This strong, direct interaction was
confirmed by pull-down experiments using recombinant
His-tagged Glo3p and yeast cytosol (17). Here we show
that the catalytic domain of yeast Glo3p is not required
for this direct interaction with coatomer. Deletion of the
N-terminal 96 amino acids including the Zn finger domain do es
not reduce the interaction of tagged Glo3p with coatomer in
vitro (Figure 6A). In this type of experiment, Gcs1p does not
bind coatomer above background levels defined by recombin-
ant GTP dissociation inhibitor (GDI) as a negative control
(Figure 6A). In order to test whether mammalian ARFGAP2
interacts with coatomer in a way comparable to yeast Glo3p,
glutathione S-transferase (GST)-tagged ARFGAP2 lacking its
ARFGAP domain was used for pull-down experiments from
rat liver cytosol. Indeed, coatomer binding from rat liver
cytosol to GST-tagged ARFGAP2 present on glutathione
beads was readily detectable by silver stain (Figure 6B),
and immunoblots using anti-peptide antibodies against a-
and b-COP demonstrated enrichment of these coatomer
subunits (data not shown). Thus, the catalytic domain of
Glo3-type ARFGAP proteins from yeast and mammals is not
required for in vitro interaction with coatomer.
Expression of DN-ARFGAP2–cyan fluorescent protein
inhibits CTX transport
Full-length ARFGAP2 was tagged at the carboxy terminus
with cyan fluorescent protein (CFP) and expressed at low
Figure 4: ARFGAP3 colocalizes with coatomer. Methanol–acetone fixed NRK cells were labelled with affinity-purified ARFGAP3
antibodies and mAbs against b-COP, p115, GM130 or g-adaptin (see Figure 3 for other details). The individual channels are shown in
monochrome; the composite overlay is shown to the right (green channel, ARFGAP3; red channel, b-COP, p115, GM130 and gAP,
respectively). Bar ¼10 mm.
Traffic 2007; 8: 1644–1655 1649
Two Human ARFGAPs Acting in COPI-Dependent Trafficking
level in Vero cells. This CFP fusion protein localized to the
Golgi complex [as identified using the Golgi enzyme mar-
ker galactosyl transferase tagged with yellow fluorescent
protein (GalT-YFP)] and also localized to punctate structures
scattered through the cytoplasm (Figure 7, upper panel).
This localization pattern is reminiscent of the localization
observed with antibodies against ARFGAP2 (compare with
Figure 3).
To assay COP-I-dependent transport in vivo, we employed
a model cargo protein previously described by us (21,33),
the non-toxic mutant version of cholera toxin fluorescently
labelled with the dye Cy3. After 3 h of internalization, CTX-
K63-Cy3 had been endocytosed from the plasma mem-
brane and was prominently present in the Golgi complex
and in the ER network (Figure 7, upper panel). In cells
expressing higher levels of ARFGAP2–CFP, a strong cyto-
plasmic staining appeared in addition to the Golgi pattern,
perhaps suggesting a limited number of binding sites on
the Golgi. Importantly, however, transport of CTX-K63-Cy3
still proceeded with kinetics very similar to control cells
(data not shown).
To test whether ARFGAP2 may be involved in COP-I-
dependent transport in vivo, we generated a DN-ARFGAP2–
CFP mutant. At low expression level, this protein was
detected at the Golgi complex when expressed in Vero
cells (not shown). At elevated expression levels (12 h after
transfection), a strong cytoplasmic pattern was seen in
addition to the Golgi pattern (Figure 7, middle panel).
Importantly, in such cells, 3 h after addition, CTX-K63-Cy3
was not observed in the ER but remained restricted to the
Golgi complex and punctate structures scattered through
the cytoplasm (Figure 7, middle panel). These data indi-
cate that expression of DN-ARFGAP2–CFP interferes with
COP-I-dependent transport of CTX-K63-Cy3 to the ER.
At even longer time-points after transfection (16 h),
cells expressing DN-ARFGAP2–CFP displayed a strikingly
higher frequency of having two or even three nuclei (indi-
cated by numbers in Figure 8, lower panel). In such cells, as
judged by GalT-YFP, usually only one large Golgi complex
was present to which DN-ARFGAP2–CFP localized (Fig-
ure 7, lower panel). In such cells, transport of CTX-K63-Cy3
was again severely inhibited, with the toxin not arriving at
the ER network even after 3 h. Instead, CTX-K63-Cy3 was
present in the Golgi complex and large cytoplasmic struc-
tures (Figure 7, lower panel). Our data strongly suggest that
ARFGAP2 is involved in the COP-I-dependent trafficking
of cholera toxin from the Golgi to the ER.
Silencing of all three ARFGAPs is lethal
In order to further investigate the role of ARFGAP2 and
ARFGAP3 in COP-I-mediated transport, we analysed NRK
and HeLa cells upon knock-down of individual ARFGAP
transcripts. Protein levels were monitored by immunoblots
of total cell extracts from NRK cells 72 h after transfection
with appropriate small interfering RNA oligonucleotides
(siRNAs). As shown in Figure 8, we indeed found oligos
that allowed knock-down of the individual ARFGAPs in
Figure 5: ARFGAP2 and ARFGAP3 are
associated with COP I vesicles gener-
ated in vitro.Vesicle budding reactions
were performed according to the protocol
developed by (31), using the non-hydro-
lysable GTP analogue, GTP-g-S. Vesicles
produced in the reaction were separated
from the Golgi donor membranes on
sucrose density gradients and peaked at
40–43% sucrose. Fractions were analysed
by silver stain (upper panel) and immuno-
blotting with antibodies as indicated
(lower panel). The positions of coatomer
bands are indicated on the left and also by
asterisks. Note the abundant presence of
ARFGAP2 and ARFGAP3 in the vesicle
fractions defined by the presence of coat-
omer subunits (fractions 8 þ9) but the
absence of ARFGAP1.
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Frigerio et al.
transfections with a single RNAi oligo. Antibodies against
g-COP and b-tubulin were used to confirm equal loading of
the samples. In yeast, deletion of either the GLO3 or GCS1
gene results in a relatively mild phenotype. However,
deletion of both genes is lethal. Therefore, we tested the
effect on cell survival of an RNAi-oligo-mediated knock-
down of ARFGAP1, ARFGAP2 and ARFGAP3 either in
pairs or as a combined triple knock-down (see Materials
and Methods for details). No significant cell death was
observed in single knock-downs or in any pairwise knock-
down, compared with cells transfected with a control
oligo. However, a triple knock-down of ARFGAP1, ARF-
GAP2 and ARFGAP3 in HeLa cells resulted in strongly
significant cell death. More than 75% cell death was
routinely observed after three sets of siRNA transfections
at the 72-h time-point after the last transfection. Very
similar results were observed in NRK cells (data not
shown). We conclude that at least one of the three
ARFGAPs is required to maintain cell viability.
Discussion
We wish to understand the role of ARFGAPs in the
regulation of COP-I-dependent trafficking. In yeast, the
two ARFGAPs Glo3p and Gcs1p together provide an
overlapping, essential function in COP-I-dependent mem-
brane traffic. In mammalian cells, only the Gcs1p ortho-
logue, ARFGAP1 has been characterized so far. In this
study, we provide an initial cell biological characterization
of two recently identified human ARFGAPs, ARFGAP2 and
ARFGAP3. We unambiguously demonstrate by sequence
analysis that they are the human orthologues of yeast
Glo3p, through the presence of a conserved ‘signature’
motif at the carboxy terminus, the Glo3 motif. Using novel
antibodies, we demonstrate that they are localized to the
Golgi complex and peripheral punctate structures scat-
tered throughout the cytoplasm in NRK cells. Both ARF-
GAP2 and ARFGAP3 significantly colocalize with COP I
subunits in both locations, strongly suggesting that they
are associated with COP I on both ERGIC/VTC structures
and at the Golgi complex. Here we further show that both
ARFGAP2 and ARFGAP3 are associated with COP I
vesicles produced in vitro in the presence of the non-
hydrolysable GTP analogue, GTP-g-S.
The Golgi and VTC localization of ARFGAP2 and ARFGAP3
appears to be dynamic, as treatment with brefeldin A
(BFA) rapidly renders their localization completely diffuse-
cytoplasmic within 2–5 min of adding the drug, with
kinetics very similar to those of b-COP and ARFGAP1
(unpublished data). Similarly, re-recruitment of ARFGAP2
and ARFGAP3 to Golgi membranes occurs rapidly upon
washout of the drug. Future experiments will need to
address the dynamics of ARFGAP2/3 using live cell
imaging approaches, similar to detailed analysis that has
been reported for ARFGAP1 (20).
The Nakano group has recently reported that Glo3p but not
Gcs1p can suppress arf-Ts mutants with a severe defect in
Golgi-to-ER retrieval in an allele-specific manner (16). They
found that the Glo3 motif is required for suppression of the
growth defect of arf1-16 and arf1-17, suggesting that the
motif is important for Glo3p function (16).
We had previously shown that ARFGAP2, and more
weakly ARFGAP3, can interact with recombinant carboxy
terminal g-COP ‘ear’ domain in pull-down experiments
from rat liver cytosol (18). Furthermore, we could show
this interaction is likely to be physiologically important, as
plasmid-driven overexpression of the g-COP ‘ear’ domain
in NRK cells disrupted the Golgi localization of ARFGAP2,
whereas a point mutant version of the g-COP ‘ear’ domain
(W776S) in which the binding site to ARFGAP2 is abro-
gated had no effect (18). Here we show that the catalytic
domain of ARFGAP2 and ARFGAP3 is not required for
in vitro interactions with mammalian coatomer. The same
phenomenon was observed for their yeast counterpart
Glo3p in binding experiments using yeast cytosol. It has
Figure 6: Direct interaction of yeast and human Glo3 with
coatomer. His6- or GST-tagged proteins were used to pull down
coatomer from yeast or rat liver cytosol, under conditions
described by us previously (17). A) His6-tagged Glo3p lacking its
amino terminus was compared with full-length Glo3p as well as
full-length Gcs1p in its ability to bind coatomer from yeast cytosol.
Protein bound to Ni-NTA agarose beads was resolved by SDS–
PAGE, followed by Western blotting and incubation with anti-
yeast coatomer antibodies detected by ECL. His-tagged Glo3p
lacking its ARFGAP domain (DN96) still binds coatomer from yeast
cytosol in vitro, whereas Gcs1p does not. GTP dissociation
inhibitor is used as a negative control here. B) A GST-fusion
protein harbouring DN96-ARFGAP2 was used for a pull-down from
a centrifugation-cleared TX-100 extract of pig brain crude micro-
somal membranes. Glutathione S-transferase was used as the
negative control. Note absence of binding in the negative control
and the presence of the characteristic coatomer bands in the pull-
down involving the DN96-ARFGAP2 GST fusion as the fishing
hook. Enrichment of a- and b-COP in the bound fraction was
confirmed using antipeptide antibodies (data not shown).
Traffic 2007; 8: 1644–1655 1651
Two Human ARFGAPs Acting in COPI-Dependent Trafficking
recently been shown that ARFGAP1 can also directly
interact, albeit more weakly, with coatomer. However,
this interaction involves two distinct binding sites on
ARFGAP1, one in its catalytic domain and the other in
the non-catalytic region (see 23). Using the two-hybrid
system, we were unable to detect interactions of Gcs1 or
ARFGAP1 with yeast or mammalian coatomer subunits,
respectively, whereas we observed strong interactions of
Glo3p as well as ARFGAP2 and ARFGAP3 with coatomer
subunits (17, unpublished data). We show that in the
presence of GTP-g-S, ARFGAP2 and ARFGAP3 are able
to associate with COP-I-coated vesicles whereas ARF-
GAP1 is barely detectable.
In most mammalian cells there are six ARFs, of which
ARF1 is the most extensively studied. It has been impli-
cated in Golgi-to-ER transport, function of the Golgi, trans-
port from the trans-Golgi network, transport in the
endocytic pathway and recruitment of paxillin to focal
adhesions (7). Becausein mammalian genomes the number
of proteins with an ARFGAP domain is considerably larger
than the number of ARF proteins, it has been suggested
that a number of GAPs might regulate the activities of
ARF1 in a location-dependent manner.
The observed interaction of Glo3p-type ARFGAPs, namely
Glo3p in yeast and ARFGAP2 and ARFGAP3 in mammals,
Figure 7: ARFGAP2 is involved in
COP-I-dependent trafficking in Vero
cells. Vero cells coexpressing full-length
ARFGAP2–CFP and the Golgi marker
GalT-YFP were treated with CTX-K63-
Cy3 (upper panel). After 3 h of internal-
ization, CTX-K63-Cy3, the Cy3-labelled
A-subunit, is prominently present in fine
ER structures (including the nuclear
envelope; arrow) and in the Golgi com-
plex. ARFGAP2–CFP colocalizes well
with GalT-YFP in the Golgi. Additionally,
ARFGAP2–CFP is present in scattered
punctate structures, most likely interme-
diate compartment. In contrast, in Vero
cells expressing DN-ARFGAP2–CFP2
transport of CTX-K63-Cy3 to the ER is
inhibited (middle panel). DN-ARFGAP2–
CFP2 localizes to the Golgi complex.
Vero cells overexpressing DN-ARF-
GAP2–CFP2 for extended periods often
display two to three nuclei and show
severe inhibition of toxin transport
(lower panel). The bottom panel is the
characterization of the Cy3-labelled CTX-
K63 (non-toxic AB5 holotoxin) used for
the experiments (for details see methods).
Mostly the A-subunit of CTX-K63 is
labelled, with traces of B present. Scale
bar: 10 mm.
1652 Traffic 2007; 8: 1644–1655
Frigerio et al.
with coatomer subunits (17,18; this study) may thus
enable regulation of aspects of the COP I vesicle cycle,
such as cargo sorting, coat formation, membrane defor-
mation or uncoating, which may enable regulation of traffic
at different intracellular sites and at specific intracellular
sites. It is perhaps surprising, given the strong direct
interactions of ARFGAP2 and ARFGAP3 with coatomer,
that these proteins were not previously identified in purified
COP I vesicle populations. Apparently, in isolated COP-I-
coated vesicles produced in the presence of GTP-g-S
(31; this study), neither ARFGAP2 nor ARFGAP3 are
stoichiometric coat components.
There are precedents for specific interactions of ARFGAPs
with other coat proteins. For example, it has been shown
that AGAP1 can directly interact with the Golgi/endosome-
localized AP-3 adaptor but not with the closely related AP-2
adaptor involved in endocytosis from the plasma mem-
brane (34,35). The related AGAP2 interacts specifically
with the AP-1 adaptor to regulate trafficking from recycling
endosomes (35) but not with AP-2. The functional signifi-
cance of the direct interactions of ARFGAP2 and ARFGAP3
with coatomer with regard to protein sorting into COP I
vesicles and the regulation of uncoating once the vesicles
are fully assembled needs to be addressed in future
experiments.
In yeast, Glo3p and Gcs1p provide an essential, over-
lapping function for COP-I-mediated transport (12).
However, growing evidence suggests that while Gcs1p
is able to maintain ARF1-regulated membrane traffic in
several transport steps, Age2p and Glo3p are special-
ized for a transport step out of the TGN (36) and COP-I-
mediated transport, respectively (12). Our in vivo data
demonstrate that ARFGAP2 is involved in the COP-I-
dependent Golgi-to-ER transport of a model cargo,
cholera toxin.
We show here that a combined knock-down of ARFGAP1,
ARFGAP2 and ARFGAP3 using siRNAs is lethal in HeLa
and NRK cells. Thus, our data strongly suggest that, similar
to the situation yeast, Gcs1p-type and Glo3p-type ARF-
GAPs together provide an essential function for COP-I-
mediated transport. In the future, it will be important to
analyse in detail the cellular phenotypes resulting from
RNAi-mediated gene silencing of individual ARFGAPs and
use in vivo and in vitro assays to unravel the individual
contributions of ARFGAP1, ARFGAP2 and ARFGAP3 to
COP-I-mediated membrane traffic.
Materials and Methods
DNA cloning, sequencing and computer analysis
Escherichia coli strain DH5awas used for plasmid isolation, and polymerase
chain reaction reactions using a combination of AmpliTaq and TaqExtender
or Vent DNA polymerase, restriction enzyme digests and ligations were
performed by standard methods. All constructs were verified by DNA
sequencing. Database searches were performed using the BLAST and CBLAST
servers at National Institutes of Health (NIH). Multiple alignments were
performed with the program MEGALIGN using the CLUSTAL V algorithm.
Plasmids and IMAGE clones
Full-length clones for both human Glo3 proteins were obtained from the
IMAGE consortium. A hypothetical intron–exon structure established for the
ARFGAP2 locus on chromosome II allowed to explain a number of splice
variants and artefacts found in the expressed sequence tag (EST) database.
Splice variants most closely related to yeast Glo3p were chosen for further
experiments: IMAGE clone 6500690 for ARFGAP3 and a combination of IMAGE
clone 209610498 (ATG to NcoI site) and clone 209860241 (NcoI to TGA) for
ARFGAP2. The coding region of several IMAGE clones with full-length inserts
were sequenced.
Figure 8: Silencing of all three ARFGAPs is lethal in HeLa cells. Silencing of ARFGAP1 or a combination of ARFGAP2 and ARFGAP3
failed to cause lethality in HeLa cells, as well as in NRK cells (data not shown). A) The depletion of ARFGAP1, ARFGAP2 or ARFGAP3 in
single knock-downs in HeLa cells was verified by immunoblotting. Antibodies against tubulin and g-COP were used to verify equal protein
loading. B and C) Cell counting was performed from DAPI-stained HeLa cells grown on coverslips. Note the strong reduction in cell
numbers in the ARFGAP triple knock-down compared with a double knock-down involving ARFGAP2 and ARFGAP3 combined with
a control oligo. For details of the oligos, see Materials and Methods. D) Quantification of the reduction in cell numbers from the above.
More than 75% cell death was routinely observed after three sets of siRNA transfections at the 72-h time-point after the last transfection
(in 10 independent experiments). Bar ¼100 mm.
Traffic 2007; 8: 1644–1655 1653
Two Human ARFGAPs Acting in COPI-Dependent Trafficking
Cloning of ARFGAP2 and ARFGAP3, expression
plasmids and raising antibodies
To obtain His6-tagged recombinant proteins as antigen and for binding
experiments, DNAs encoding full-length ARFGAP2 and ARFGAP3 were
cloned into pET21d (Novagen) and expressed in lDE3 lysogens of strain
BL21 and purified by Ni-NTA chromatography as described (17). Purified
His6-tagged ARFGAP2 and ARFGAP3 were used for raising antibodies in
rabbits. Antibodies were affinity purified over immobilized GST-tagged
ARFGAP2 and ARFGAP3 protein lacking the ARFGAP domain to reduce
the risk of cross-reactivity of these antibodies between the two proteins
and characterized by immunoblotting and IF. Immunoblots on total protein
extracts from NRK and HeLa cells revealed that our antibodies recognized
the human and rat proteins with high affinity, that there is a small difference
between the two proteins in their apparent molecular weight in SDS-
polyacrylamide gels and that both proteins are expressed at readily detect-
able levels (data not shown).
Cell lines and commercial antibodies
HeLa cells, NRK and Vero (green monkey kidney fibroblast) cells were
maintained in DMEM medium þ10% bovine calf serum, 2 mMglutamine
and antibiotics penicillin and streptomycin (100 U/mL and 100 mg/mL,
respectively). In some experiments, BFA was added to the cell culture
medium at a concentration of 5 mg/mL. A stock solution of BFA (2 mg/mL in
ethanol; Epicentre Technologies) was stored at 208C.
For colocalization analysis by IF, we used monoclonal antibodies against
GM130 (clone NN 2C10; Abcam 1299), p115 (clone 5D6; Sigma P3118),
g-adaptin (clone 100/3; Sigma4200) and b-COP (clone mAD; Abcam 6323).
IF and confocal fluorescence microscopy
NRK or HeLa cells grown on glass coverslips were fixed in methanol at
208C for 4 min followed by fixation for 30 seconds with 208C acetone.
Cells were mounted in 50% glycerol on glass slides and epifluorescence
microscopy was performed on a Zeiss Axioplan microscope with a 63,1.4
oil immersion objective, with images taken using a CoolSnap CCD camera.
Pull-down experiments and immunoblot analysis
His6- or GST-tagged proteins were used to pull down coatomer from yeast
or rat liver cytosol, under conditions described by us previously (17). His6-
tagged Glo3p lacking its amino terminus was compared with full-length
Glo3p as well as full-length Gcs1p in its ability to bind coatomer from yeast
cytosol. Protein bound to Ni-NTA agarose beads was resolved by SDS–
PAGE, followed by Western blotting and incubation with anti-yeast coat-
omer antibodies detected by enhanced chemiluminescence (ECL). A
recombinant GST-fusion protein harbouring DN96-ARFGAP2 was used for
pull-downs from rat liver cytosol. Glutathione S-transferase alone or
recombinant GST-tagged GDI (a guanine nucleotide dissociation inhibitor
acting on rab proteins) were used as negative controls. SDS–PAGE and
immunoblotting analysis using ECL were performed as described (17).
In vitro budding reactions
In vitro budding of COP-I Golgi-derived vesicles from purified rat liver Golgi
membranes was performed in the presence of pig brain cytosol as a source
of coatomer, ATP and the nonhydrolysable GTP analogue, GTP-g-S as
described by the Rothman lab (31,32). Pig brain cytosol was prepared by
the method as described by (37). Donor Golgi membranes were prepared
according to a protocol established by the Graham Warren lab (38). Vesicles
were separated from Golgi donor membranes by sedimentation to equilib-
rium in a sucrose density gradient. Following trichloroacetic acid precipitation
and SDS–PAGE on 10% gels, proteins in fractions were analysed by silver
stain and ECL immunoblot using specific antibodies as indicated.
Cell transfections and application of cholera toxin
CTX-K63 and CFP- and YFP-fusion proteins
Vero cells were transfected by electroporation as described by us earlier
(21). Unless otherwise mentioned, cells were tested for expression of
fluorescent fusion proteins and cholera toxin binding 6 h after trans-
fection. For experiments, cells were treated in a pulse-like manner with
CTX-K63, as described before (21,33). CTX-K63 is a nontoxic mutant of
cholera toxin, which has no ADP-ribosylating activity as a result of a Ser
63
/
Lys
63
point mutation in the A-subunit (21,33). Following treatment with
CTX-K63, cells remained in the CO
2
incubator until they were transferred
to the thermostated microscope chamber. Internalization of CTX-K63-
Cy3, labelled as described below, was performed for 3 h at 378C before
imaging.
An expression clone for an YFP-tagged version of the Golgi-resident en-
zyme galactosyl transferase (GalT-YFP) was a kind gift from J. Lippincott-
Schwartz (NIH). We constructed CFP-tagged full-length or DN96-ARFGAP2
expression clones, with the CFP moiety at the carboxy terminus of the
proteins, using standard cloning procedures in the vector pECFP-C1
(ClonTech).
Cy3 labelling of CTX-K63
As described by us (21), CTX-K63 mutant toxin was labelled with Cy3,
resolved by 10% SDS–PAGE and scanned using a Typhoon 8600 Imager
with an excitation wavelength of 532 nm and standard emission filter
560LP. Pixel-by-pixel resolved fluorescence measurement revealed prefer-
ential labelling of the A-subunit in the sample that was used in the
experiments described in Figure 7. The absorption spectrum of the CTX-
K63-Cy3 sample was acquired with a Fluoromax-3 spectrofluorimeter. The
wavelength ratio of 550 nm/280 nm, that is, of dye (550 nm) to protein
(280 nm), was used to measure the concentration of proteins in the sample
and to estimate the labelling ratio, calculated using Beer’s law and the dye
extinction coefficient of Cy3 150 000 M
1
cm
1
. From this presence of
approximately 1.5–2 dye molecules per A-subunit were determined.
siRNA
Sequences were designed following the parameters set out by Dharmacon
by analysing the human genomic sequence for each protein. The following
sequences were selected: ARFGAP1, AAG GUG GUC GCU CUG GCC GAA
G (siACE-RNAi OPTION C DUPLEX); ARFGAP2, AAG CUA UGG GGU GUU
UCU CUG (siACE-RNAi OPTION C DUPLEX); and ARFGAP3, AAC CUA
UGG AGU GUU CCU UUG (siACE-RNA OPTION C DUPLEX). All oligos
were custom synthesized by Dharmacon (http://www.Dharmacon.com/).
A green fluorescent protein duplex was used as a control oligo for all
experiments (Dharmacon, D-001300-01-20).
RNAi-mediated knock-downs of ARFGAP1, 2 and 3
in HeLa cells
HeLa M cells were maintained in DMEM medium supplemented with 10%
foetal bovine serum, 2 mML-glutamine, penicillin (100 U/mL) and strepto-
mycin (100 mg/mL). siRNAs were used at a final concentration of 20 nMin
all transfection experiments. Triple knock-down of all three ARFGAPs and
combinations of these with controls were performed in a staggered 10-day
protocol, using oligofectamine. For double transfections, paired siRNA
oligos were added to 15 nMfinal concentrations. Cells were seeded on
day 0 and transfected on day 1 with either control, single ARFGAP or
ARFGAP2 and 3 oligos. On day 2, cells were split into new wells, allowed to
adhere to the dish for 6 h, then transfected with either control or ARFGAP1
oligo. On day 3, the media was changed to remove transfection reagents.
On day 5, cells were split, allowed to adhere for 6 h and transfected as on
day 1. On day 6, the media were refreshed, and on day 7, cells were
transfected as on day 2. On day 8, cells were split (some cells were
reseeded onto coverslips for 40-60-diamidino-2-phenylindole (DAPI) staining.
On day 9, the cells rested, and on day 10, cells were collected for analysis.
For immunoblot samples, cells washed with PBS, then lysed directly in the
plate by addition of 3Laemmli sample buffer, pipetted out and immedi-
ately boiled at 958C for 5 min. DNA was sheared by passing the samples
through a narrow gauge needle and vortexing at maximum speed. Samples
were then loaded onto SDS–PAGE gels. Cells growing on coverslips for
DAPI staining were washed three times in PBS, then methanol–acetone
fixed and mounted on to glass slides in DAPI-containing mounting medium
1654 Traffic 2007; 8: 1644–1655
Frigerio et al.
(10 mg/mL DAPI, 1 mg/mL p-phenylenediamine in 90% glycerol, 10% PBS,
pH 8.0). A similar protocol was used for NRK cells, yielding almost
identical results.
Acknowledgments
We are grateful to Drs J. Lippincott-Schwartz, M.S. Robinson and Paul
Luzio for generously sharing antibodies and plasmids. Thi s work was
supported by The Wellcome Trust (Senior Research Fellowship; grant
047578 to R. D.).
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