Proteins and lipids traffic via the retrograde route from endosomes
to the Golgi complex (Bonifacino and Rojas, 2006; Johannes and
Popoff, 2008). In mammalian cells, this pathway enables a
functional cycle for lysosomal enzyme delivery by mannose-6-
phosphate receptors (MPRs) (Duncan and Kornfeld, 1988; Snider
and Rogers, 1985), and trafficking of TGN38/46 between TGN
and plasma membrane (Reaves et al., 1993). Retrograde trafficking
is crucial for the maintenance of Golgi morphology (Ghosh et al.,
2003; Naslavsky et al., 2009; Yoshino et al., 2005), and a range of
other cellular and pathological functions depend on retrograde
transport, including the cellular entry of pathogens and pathogenic
factors (for reviews, see Bonifacino and Rojas, 2006; Johannes
and Popoff, 2008). A well-studied example is the bacterial Shiga
toxin (Johannes and Römer, 2010). After binding to its cellular
receptor, the glycosphingolipid Gb3, the toxin follows the
retrograde route to the endoplasmic reticulum from where its
catalytic A-subunit is translocated to the cytosol to inhibit protein
biosynthesis by modification of ribosomal RNA (Lord et al., 2005).
Several trafficking factors are required for retrograde Shiga
toxin transport to the TGN (Amessou et al., 2007; Mallard et al.,
2002; Popoff et al., 2009; Popoff et al., 2007; Saint-Pol et al.,
2004). In the budding step, termed retrograde sorting, the function
of clathrin and retromer are of crucial importance (Bujny et al.,
2007; Lauvrak et al., 2004; Popoff et al., 2009; Popoff et al., 2007;
Saint-Pol et al., 2004; Utskarpen et al., 2007). Upon depletion of
clathrin, Shiga toxin fails to reach the Golgi and remains in
transferrin receptor (TfR)-positive early and recycling endosomes
(Popoff et al., 2007; Saint-Pol et al., 2004). By contrast, in cells
that are depleted of Vps26, a protein of the cargo selection subunit
of retromer (Bonifacino and Hurley, 2008), Shiga toxin is
segregated from TfRs in early endosome-linked tubular structures
that have been termed retrograde tubules (Popoff et al., 2007).
With the recent demonstration of a function for Rab7 in retromer
recruitment (Rojas et al., 2008; Seaman et al., 2009), the combined
evidence suggests that the clathrin requirement in retrograde sorting
on early or maturing endosomes precedes that for retromer.
The ADP-ribosylation factor (Arf) GTPases are ~20 kDa GTP-
binding proteins that regulate membrane traffic through the
recruitment of clathrin or COPI coats, the modulation of lipid-
modifying enzyme activity, or by controlling actin dynamics at
membrane surfaces (D’Souza-Schorey and Chavrier, 2006;
Donaldson and Lippincott-Schwartz, 2000). Mammals express six
Arf isoforms, Arf1-Arf6, which are grouped into three classes
based on primary sequence and gene organization (Kahn et al.,
2006). The best-characterized Arf proteins are Arf1 and Arf6. Arf6
regulates endosomal trafficking and plasma membrane organization,
whereas Arf1 is thought to be localized specifically to the Golgi
complex (Peters et al., 1995). Recent studies have shown that Arf1
can also be recruited to endosomal membranes (Gu and Gruenberg,
2000) and the plasma membrane (Kumari and Mayor, 2008), and
that pairs of Arf1 with Arf3, Arf4 or Arf5 regulate transferrin
recycling (Volpicelli-Daley et al., 2005). ARF proteins cycle
between active GTP-bound
conformations. Hydrolysis of GTP is mediated by GTPase
activating proteins (ArfGAPs), whereas the exchange of GDP for
triphosphate nucleotide is mediated by guanine nucleotide-exchange
The function of Arf proteins and their GAPs and GEFs in
retrograde transport from early endosomes to TGN remains largely
and inactive GDP-bound
AGAP2 regulates retrograde transport between early
endosomes and the TGN
Yoko Shiba1,2, Winfried Römer1,2, Gonzalo A. Mardones3,4, Patricia V. Burgos3,4, Christophe Lamaze1,2and
1Institut Curie – Centre de Recherche, Traffic, Signaling and Delivery Laboratory, 26 rue d’Ulm, 75248 Paris Cedex 05, France
2UMR144 CNRS, France
3Cell Biology and Metabolism Program, NICHD, National Institutes of Health, Building 18T, Room 101, Bethesda, MD 20892, USA
4Department of Physiology, Universidad Austral de Chile, Valdivia 509-9200, Chile
*Author for correspondence (email@example.com)
Accepted 22 April 2010
Journal of Cell Science 123, 2381-2390
© 2010. Published by The Company of Biologists Ltd
The retrograde transport route links early endosomes and the TGN. Several endogenous and exogenous cargo proteins use this
pathway, one of which is the well-explored bacterial Shiga toxin. ADP-ribosylation factors (Arfs) are ~20 kDa GTP-binding
proteins that are required for protein traffic at the level of the Golgi complex and early endosomes. In this study, we expressed
mutants and protein fragments that bind to Arf-GTP to show that Arf1, but not Arf6 is required for transport of Shiga toxin from
early endosomes to the TGN. We depleted six Arf1-specific ARF-GTPase-activating proteins and identified AGAP2 as a crucial
regulator of retrograde transport for Shiga toxin, cholera toxin and the endogenous proteins TGN46 and mannose 6-phosphate
receptor. In AGAP2-depleted cells, Shiga toxin accumulates in transferrin-receptor-positive early endosomes, suggesting that
AGAP2 functions in the very early steps of retrograde sorting. A number of other intracellular trafficking pathways are not affected
under these conditions. These results establish that Arf1 and AGAP2 have key trafficking functions at the interface between early
endosomes and the TGN.
Key words: Shiga toxin, Arf, ArfGAP, ARAP, Mannose 6-phosphate receptor, TGN46, Cholera toxin, Clathrin, Retromer, VSVG, Transferrin
Journal of Cell Science
unknown. When cells were treated with brefeldin A (BFA), a
fungal metabolite that inhibits ArfGEFs, exit of Shiga toxin B-
subunit (STxB) from Tf-positive tubular membranes was prevented
(Mallard et al., 1998). In addition, GBF1, a BFA-sensitive ArfGEF
was suggested to function in retrograde transport of STxB (Saenz
et al., 2009). This conclusion was based on the use of a small-
molecule compound, golgicide A, as an inhibitor of GBF1. Whether
GBF1 functions directly in endosome-to-TGN transport, or
indirectly through its effects on maintaining Golgi structure, is
currently unknown (Saenz et al., 2009).
ArfGAPs were originally considered as simple regulators of
ARFs, and it has only recently been shown that ArfGAPs
themselves can be effectors that transduce signals in cells (Inoue
and Randazzo, 2007). In Golgi-to-ER trafficking, ArfGAP1 is
necessary for COPI vesicle budding (Lanoix et al., 1999; Nickel et
al., 1998; Pepperkok et al., 2000; Yang et al., 2002). Very few
studies have addressed ArfGAP function in post-Golgi membrane
traffic. SMAP2 is an ArfGAP that binds to clathrin heavy chain
and clathrin assembly protein CALM. SMAP2 colocalizes on
endosomes with the clathrin adaptor epsinR, and the overexpression
of a SMAP2 clathrin-binding mutant inhibits retrograde transport
of murine TGN38 in COS-7 cells (Natsume et al., 2006). These
findings suggest that ArfGAPs are effectors of Arf in retrograde
Here, we have found that ARF1, but not ARF6 is required for
early-endosomes-to-TGN transport of Shiga toxin. We inhibited
the expression of six ArfGAP family members that have GAP
activity to Arf1, and found that AGAP2 is required for retrograde
transport of STxB. In AGAP2-depleted cells, Shiga toxin localizes
in endosomes that are positive for the transferrin receptor and
Rab4, and to a lesser extent with the retromer protein Vps26.
These results establish that ARF1 and AGAP2 participate in very
early steps of retrograde sorting on early endosomes.
ARF involvement in STxB transport from early endosomes
to the TGN
As a first approach to testing the function of Arf proteins in
retrograde transport to the TGN in HeLa cells, we expressed GFP-
tagged protein fragments that bind to Arf1-GTP and Arf6-GTP.
Expression of GFP alone had no effect on retrograde transport, and
STxB accumulated efficiently in perinuclear Golgi membranes
labeled by the medial-Golgi marker CTR433 (Fig. 1A). By contrast,
cells that expressed the GFP-tagged Arf-GTP binding domain
(ARFBD) of ARHGAP10 (Dubois et al., 2005) showed increased
peripheral STxB labeling (Fig. 1B), and in a small fraction of cells,
STxB did not reach Golgi membranes at all (Fig. 1H). Similar
results were observed in cells expressing the HA-tagged VHS-
GAT domain of GGA1 that also binds to ARF-GTP (data not
A permeabilized-cells approach (Amessou et al., 2006) was
used to further test the effect of ARFBD on STxB trafficking
between early endosomes and the TGN (Fig. 1C). In this approach,
a STxB variant with a tandem sulfation signal, termed STxB-Sulf2,
is used to measure arrival in the TGN. STxB-Sulf2was accumulated
in early endosomes by incubation with cells at 19.5°C. After
plasma-membrane permeabilization with SLO and removal of
endogenous cytosol, the permeabilized cells were incubated for 20
minutes at 37°C in the presence of GST or GST-ARFBD and
radioactive sulfate. Sulfation signal in the presence of exogenous
cytosol was set to a 100% for maximal TGN arrival, and the
2382 Journal of Cell Science 123 (14)
Fig. 1. ARF involvement in retrograde transport of STxB. (A,B,D-G) HeLa
cells were transfected with the indicated constructs, and then incubated with
Cy3-labeled STxB for 45 minutes at 37°C. The cells were fixed and stained
with anti-CTR433 antibody. In ARFBD- and ARF1QL-transfected cells,
strongly increased STxB localization to peripheral structures is found. Scale
bars: 10m. (C)STxB-Sulf2transport to the TGN was assayed by sulfation
analysis on permeabilized cells in the presence of the indicated concentrations
of recombinant GST or ARFBD. Sulfation signals are expressed as
percentages of signal observed under control conditions (+cytosol and 1M
GST). The means ± s.e.m. of three independent experiments are shown.
(H)Among the 50 cells that were transfected and had internalized STxB, the
percentage of cells showing colocalization of STxB and CTR433 was
determined. Independent transfections were repeated three times, and the
colocalization means ± s.e.m. were calculated.
Journal of Cell Science
background signal was determined in the absence of exogenous
cytosol (Fig. 1C). The addition of GST-ARFBD, but not of GST
alone, led to a dose-dependent inhibition of retrograde transport
(Fig. 1C), thus confirming the results obtained on intact cells (Fig.
These results strongly suggest the involvement of Arf proteins
in membrane trafficking at the interface between early endosomes
and the TGN. To identify the Arf isoform that is involved in this
trafficking step, we expressed in intact HeLa cells constitutively
active mutants of Arf1 and Arf6, i.e. Arf1Q71L or Arf6Q67L, or
mutants defective in nucleotide binding, Arf1N126I or Arf6N122I.
In Arf1Q71L-expressing cells, STxB remained blocked in
peripheral endosomes (Fig. 1D), and in many cells the protein
failed to reach the Golgi altogether (Fig. 1H). In cells expressing
Arf1N126I, a similar albeit weaker effect was observed (Fig.
1E,H). It should be noted that Golgi integrity was slightly affected
in these cells (Fig. 1D-E), and high expression of Arf1N126I often
led to the disruption of the Golgi (data not shown). The dispersed
Golgi fragments were not reached by STxB, however (Fig. 1D-E).
In cells expressing the different Arf6 mutants, endosomal STxB
accumulation was as low, as in control cells (Fig. 1F-G), and STxB
transport to the Golgi was not visibly altered (Fig. 1H). These
results demonstrate that Arf1, but not Arf6, is involved in STxB
transport from early endosomes to the TGN.
Identification of ArfGAP proteins that function in
Based on the finding that Arf1, but not Arf6 was required for
retrograde transport to the TGN, we performed sulfation analysis
on intact cells that were transfected with validated siRNAs pools
(except SMAP2) against six ArfGAPs with preferential GAP
activity on Arf1 (Miura et al., 2002; Natsume et al., 2006; Nie et
al., 2005; Vitale et al., 2000; Yoon et al., 2004). The depletion of
the individual proteins was not assayed, and negative results can
therefore not be interpreted. As shown in Fig. 2, the strongest
inhibition of STxB sulfation was observed in cells transfected with
a smart pool of four siRNAs against ARAP1, and a weaker
inhibition in cells transfected with siRNA against AGAP2. For
AGAP1 and SMAP2-1, inhibition was not significant and
immunofluorescence analysis revealed that STxB efficiently
accumulated in Golgi membranes (data not shown). In the case of
cells transfected with GIT2 and SMAP2-2 siRNA, sulfation levels
were increased. The significance of these findings is not clear at
this stage. For further analysis, we focused on ARAP1 and AGAP2.
ARAP1 is not required for retrograde transport to the TGN
To study ARAP1 function in retrograde transport, the sulfation
assay was repeated using the four siRNAs of the smart pool against
ARAP1 individually. All four siRNAs efficiently depleted ARAP1
protein (Fig. 3A). Sulfation levels on STxB were decreased in all
cases, most strongly with sequences 3 and 4 (Fig. 3B). Inspection
of STxB labeling by fluorescence microscopy showed that many
cells that were transfected with these siRNAs had reduced signals
of cell-associated STxB (Fig. 3C, arrows). This finding suggested
that in ARAP1-depleted cells, plasma membrane Gb3 levels were
reduced, or that Gb3 molecules were organized in a way such that
STxB could not be bound efficiently. In cells in which STxB
binding could still be detected (Fig. 3C, arrowheads), retrograde
transport to the TGN was apparently not affected. Quantification
confirmed that 68% or 70% of cells failed to bind STxB in cells
transfected with ARAP1 siRNA sequences 3 and 4, respectively,
whereas this percentage was much smaller in cells transfected with
control siRNA (7%). Dosage of Gb3 after lipid extraction and
overlay (Falguières et al., 2001) revealed that total cellular Gb3
levels were not altered in cells transfected with ARAP1 siRNA
(data not shown). ARAP1 is probably required for Gb3 transport
from the Golgi to the plasma membrane, but other interpretations
cannot be excluded at this stage.
AGAP2 functions at the interface between early
endosomes and the TGN
To study AGAP2, we generated a peptide antibody that detected
the protein by western blotting only upon overexpression (not
shown), and by immunofluorescence only when cells were fixed
in methanol. Under these fixation conditions, endogenous AGAP2
was found in the perinuclear region in good colocalization with
TGN46 (Fig. 4A), to a lesser extent with the Golgi marker giantin
(supplementary material Fig. S1A), and not with the late endosomal
or lysosomal marker Lamp-1 (supplementary material Fig. S1B).
TfR only weakly overlapped with AGAP2 (supplementary material
Fig. S1C), which for peripheral sites might be due to poor
preservation under conditions of methanol fixation. GFP-tagged
AGAP2 partially colocalized with STxB after short times of
internalization (5 minutes; Fig. 4B). These findings and other
published results (Nie et al., 2005) show that AGAP2 is localized
at the TGN and on endosomes.
As above for ARAP1, the function of AGAP2 was addressed in
sulfation experiments by depleting AGAP2 expression individually
with each of the four siRNA sequences of the smart pool. Since
our antibody did not work for western blotting, we relied on RT-
PCR (supplementary material Fig. S2A) and immunofluorescence
(see below) to confirm the efficacy of the AGAP2 siRNAs. The
STxB sulfation signal was strongly reduced with each of the four
siRNAs that were used to deplete AGAP2 (Fig. 4C). Upon
prolonged incubation (120 minutes), sulfation still remained much
lower in the depletion condition (supplementary material Fig. S2B),
suggesting that STxB failed to reach TGN membranes altogether.
Since STxB degradation was not detected in AGAP2-depleted
cells upon incubation for at least 4 hours (supplementary material
Fig. S2C), it appears likely that STxB remained in the early
endosomal membrane system (see below), as we described before
AGAP2 and retrograde trafficking
Fig. 2. Sulfation analysis in cells transfected with siRNAs to knock down
ARF GAPs. HeLa cells were transfected with the indicated siRNAs, incubated
with STxB-Sulf2for 20 minutes at 37°C and sulfated STxB was quantified.
Sulfation levels in all conditions were expressed as the percentages of control
(means of three determinations ± s.e.m.). Pools of four different siRNAs were
used against ARFGAP1, ARAP1, AGAP1, AGAP2 and GIT2. For SMAP2, two
different siRNA sequences were designed and transfected individually. siRNA
to knock down syntaxin-16 (Synt16) was used as a positive control (Amessou
et al., 2007).
Journal of Cell Science
in cells in which retrograde transport was abolished upon BFA
treatment (Mallard et al., 1998).
The perinuclear AGAP2 labeling that was seen with the
methanol-fixation protocol in control cells (Fig. 4A and
supplementary material Fig. S3, top panel) was strongly diminished
in cells that were transfected with AGAP2 siRNAs 1 to 4
(supplementary material Fig. S3). This loss of perinuclear AGAP2
labeling was not observed in cells transfected with ARAP1 siRNA
(data not shown), confirming the specificity of the labeling. In
cells transfected with control siRNA (supplementary material Fig.
S3, top panel), perinuclear STxB labeling at the Golgi was well
preserved in the methanol-fixation protocol. In siRNA-transfected
cells, this perinuclear STxB labeling was lost (supplementary
material Fig. S3, lower panels for siRNA sequences 1 to 4; see
right column for Golgi labeling with giantin). As opposed to
ARAP1, the apparent loss of global STxB signal was in this case
not due to loss of STxB binding. Indeed, when cells that were
transfected with AGAP2 siRNA sequence 3 (Fig. 4D, lower panel)
were fixed using paraformaldehyde, STxB (red) was largely absent
from perinuclear Golgi membranes (giantin, blue), as in the
methanol-fixation condition. However, STxB could now be detected
in peripheral structures.
We noticed that Golgi morphology was somewhat affected in
AGAP2 siRNA-transfected cells (Fig. 4D and supplementary
material Fig. S3). We therefore used the permeabilized cell assay
to validate the function of AGAP2 in an experimental set-up that
allows interference with protein function without going through
prolonged depletion conditions. Recombinant glutathione-S-
transferase (GST) and tagged wild-type AGAP2 were used as
purified proteins. As shown in Fig. 4E, GST had no effect on
retrograde transport of STxB. By contrast, wild-type AGAP2
potently inhibited, confirming a function for AGAP2 in retrograde
STxB accumulates in early endosomes of AGAP2-depleted
The analysis of sites of STxB accumulation in AGAP2-depleted
cells was performed in paraformaldehyde-fixed cells. Transfection
of siRNA sequence 3 induced an efficient inhibition of retrograde
transport with minimal effects on Golgi morphology, and this
siRNA sequence was chosen for all experiments described below.
In AGAP2-depleted cells, STxB colocalized with TfR on
perinuclear and peripheral endosomes (Fig. 5A). The GFP-tagged
early endosomal marker Rab4 also decorated STxB-positive
enlarged structures under these conditions (Fig. 5B). Importantly,
GFP-Rab4 expression by itself did not affect STxB trafficking to
the TGN (data not shown). Clathrin is a crucial component for
retrograde sorting. In clathrin-depleted cells, STxB fails to reach
Golgi membranes, and remains blocked in TfR-positive early
endosomes (Popoff et al., 2007; Saint-Pol et al., 2004), similarly
to the situation described here for AGAP2-depleted cells. We also
found that clathrin was often juxtaposed to sites of STxB
accumulation (Fig. 5C). The retromer complex has been suggested
to be involved in the processing of retrograde tubules in which
STxB is taken out of TfR-positive early endosomes (Popoff et al.,
2007), and therefore appears to function consecutively to clathrin
(Johannes and Popoff, 2008). Little or no colocalization was
observed here between the retromer component Vps26 and STxB
(Fig. 5D). Taken together, the colocalization of STxB with TfR in
AGAP2-depleted cells, the proximity to clathrin and the lack of
overlap with Vps26 suggest that AGAP2 functions in very early
steps of retrograde sorting. In agreement with this hypothesis, the
recycling endosomal marker Rab11 (Fig. 5E) and the late
endosomal or lysosomal marker Lamp-1 (Fig. 5F) were not
colocalized with STxB in AGAP2-depleted cells.
Live-cell imaging was used to confirm the AGAP2 function in
very early steps of retrograde sorting. We previously showed that
depletion of the retromer protein Vps26 leads to the prolonged
appearance of STxB in retrograde tubules that although connected
to early endosomes, were devoid of Tf (Popoff et al., 2007). By
contrast, here we found that in AGAP2-depleted cells, STxB and
Tf were colocalized in early endosomal tubules (Fig. 6A and
supplementary material Movie 1), similarly to the colocalization
2384 Journal of Cell Science 123 (14)
Fig. 3. ARAP1 is not required for retrograde transport. (A)HeLa cells
were transfected with control siRNA or four different siRNAs against ARAP1.
Cell lysates were analyzed by western blotting. (B)Sulfation analysis was
performed as described in Fig. 2 with cells transfected with four different
siRNAs against ARAP1. Means ± s.e.m. of three determinations.
(C)Immunofluorescence analyses with cells transfected with ARAP1 siRNA
sequences 3 and 4. Results after a 45 minute incubation with Cy3-STxB at
37°C. Note that total Cy3-STxB signals are strongly reduced in several cells
(arrows). On other cells, Cy3-STxB still bound (arrowheads) and was
transported to the Golgi. Scale bars: 10m.
Journal of Cell Science
on static images that we observed in clathrin-depleted cells (Saint-
Pol et al., 2004). The live-cell imaging also confirmed that after 45
minutes of incubation with AGAP2-depleted HeLa cells, STxB
and Tf were still dynamically associated, notably in the perinuclear
area (Fig. 6B and supplementary material Movie 2, right), whereas
in control cells, STxB has efficiently reached the Golgi at this time
point, and no overlap with Tf was seen (Fig. 6C and supplementary
material Movie 2, left).
AGAP2 regulates retrograde transport of several
exogenous and endogenous cargos
The GM1-binding B-subunit of cholera toxin (CTxB) shares with
STxB some of the trafficking requirements that have been analyzed
to date (Amessou et al., 2007; Lu et al., 2004). Here, we found that
retrograde transport of CTxB to the Golgi was also inhibited in
AGAP2-depleted cells (Fig. 7A-B). Trafficking of the endogenous
retrograde cargo protein TGN46 was followed using a dynamic
antibody-uptake protocol. In control cells, the antibody efficiently
accumulated in the perinuclear region, in colocalization with the
TGN marker Golgin-97 (Fig. 7C). By contrast, the anti-TGN46
antibody remained in peripheral structures that did not colocalize
with Golgin-97 in AGAP2-depleted cells (Fig. 7D). Similarly,
STxB also failed to reach Golgin-97-positive membranes in
AGAP2-depleted cells (supplementary material Fig. S4). In these
experiments, we noticed a slight effect of AGAP2 depletion on the
distribution of Golgin-97, which appeared less compact than in
control cells, suggesting that TGN morphology was affected.
In a further experiment, the steady-state localization of cation-
independent MPR (CI-MPR) was analyzed. In our HeLa cell clone,
most CI-MPR was localized in perinuclear membranes in
colocalization with the Golgi matrix protein GM130 (Fig. 7E). In
AGAP2-depleted cells, more CI-MPR labeling was visible in
peripheral structures (Fig. 7F). Taken together, these findings
strongly suggest that AGAP2 functions in retrograde transport of
several exogenous and endogenous retrograde cargo proteins.
Effects of AGAP2 depletion on other intracellular
A number of intracellular-trafficking routes were analyzed to address
the specificity of AGAP2 function at the early-endosome-TGN
interface. Since AGAP2 depletion has a slight effect on TGN
morphology (see above), we analyzed anterograde transport along
the biosynthetic or secretory pathway in control and depletion
conditions. A temperature-sensitive version of the glycoprotein from
vesicular stomatitis virus (VSVG) was blocked in the ER at the
restrictive temperature. Upon shift to the permissive temperature for
2 hours, the protein was transported in a brefeldin A (BFA)-sensitive
manner to the cell surface. We found here that VSVG transport was
AGAP2 and retrograde trafficking
Fig. 4. AGAP2 is required for retrograde transport of STxB. (A)HeLa
cells were fixed with methanol, and stained with antibodies against AGAP2
(green) and TGN46 (red). A strong overlap between both markers was seen in
the perinuclear area. (B)HeLa cells were transfected with GFP-tagged AGAP2
(green) and incubated for 5 minutes with Cy3-STxB (red). An overlap could
be detected on peripheral structures (arrows). (C)Sulfation analysis was
performed with cells transfected with four different siRNAs against AGAP2.
Means ± s.e.m. of 3-5 determinations. (D)Immunofluorescence analysis of
HeLa cells transfected with control siRNA or with AGAP2 siRNA sequence 3.
After binding, Cy3-STxB (red) was incubated with the cells for 45 minutes at
37°C. The cells were then fixed and labeled with antibodies against AGAP2
(green) and giantin (blue). Merges show overlay of STxB and giantin. Note
that the anti-AGAP2 antibody works poorly in paraformaldehyde-fixation
conditions. Nevertheless, the loss of perinuclear labeling can be revealed in
siRNA-transfected cells. (E)STxB-Sulf2transport to the TGN was assayed by
sulfation analysis on permeabilized cells in the presence of the indicated
concentrations of recombinant GST or tagged wild-type (wt) AGAP2 at the
indicated concentrations. Sulfation signals are expressed as percentages of
signal observed under control conditions (+cytosol and 1M GST). The
means ± s.e.m. of two (no error bars) or three independent experiments are
Journal of Cell Science
40% inhibited in AGAP2-depleted cells, when compared
with siRNA-transfected control cells (Fig. 8A). Since VSVG
might also traffic via endosomes to the cell surface (Cancino
et al., 2007; Chen et al., 1998), we believe that this effect is
consistent with a post-Golgi function of AGAP2, as
described above for the retrograde toxin cargos and the
endogenous proteins TGN46 and CI-MPR. A function of
AGAP2 in anterograde transport at the TGN cannot be
excluded either. However, since STxB and anti-TGN46
antibody fail to reach the extended TGN in AGAP2-depleted
cells (see above), a direct role of AGAP2 in post-Golgi
retrograde transport remains the most likely possibility. In
quantitative biochemical assays we could finally show that
the endocytosis of STxB (Fig. 8B) and Tf (Fig. 8C) and the
recycling of Tf (Fig. 8D) were not affected by the depletion
In this study, we demonstrate the involvement of Arf1 in
retrograde transport between early endosomes and the
TGN, using quantitative biochemical tools and
morphological approaches. Furthermore, we identify the
ARF1 GAP AGAP2 as a crucial factor for retrograde
sorting on early endosomes.
Arf proteins and Arf1-sensitive coatomer have
previously been involved in trafficking to the late endocytic
pathway (Aniento et al., 1996; Daro et al., 1997; Gu and
Gruenberg, 2000; Whitney et al., 1995). Our finding that
Arf1 is also required for retrograde sorting adds further
complexity to the endosomal coat network. Indeed,
clathrin, the clathrin adaptors epsinR, AP-1 and OCRL,
and the clathrin uncoating ATPase Hsc70 and its early
endosomal adaptor RME-8 are also required for efficient
retrograde sorting of endogenous and exogenous cargo
proteins (Choudhury et al., 2005; Folsch et al., 2001;
Lauvrak et al., 2004; Popoff et al., 2009; Saint-Pol et al.,
2004; Shi et al., 2009). How exactly Arf1 links into this
sorting machinery still remains to be determined. Our
unpublished evidence suggests that coatomer I proteins
(COPI) are not required for retrograde transport of Shiga
toxin to the TGN, and it therefore appears likely that Arf1
rather interacts with the clathrin machinery, as it has been
found at the TGN (Puertollano et al., 2001; Traub et al.,
A likely link between Arf1 and the clathrin machinery
might operate via AGAP2. A previous study had shown
that AGAP2 is localized on endosomes and interacts with
the clathrin adaptor AP-1 (Nie et al., 2005). We found
here that depletion of AGAP2 leads to an inhibition of
retrograde transport of Shiga toxin in a way such that
2386 Journal of Cell Science 123 (14)
Fig. 5. Detailed analysis of STxB localization in AGAP2-
depleted cells. HeLa cells were transfected for 3 days with AGAP2
siRNA sequence 3 and after binding was incubated with Cy3-STxB
for 45 minutes at 37°C. The cells were then labeled with antibodies
against TfR (A), clathrin heavy chain (CHC) (C), Vps26 (D) and
Lamp-1 (F). Alternatively, siRNA-treated cells were transfected for
24 hours with GFP-Rab4 (B) or GFP-Rab11 (E). Note that in GFP-
Rab4-expressing cells, enlarged endosomes were observed. Scale
Journal of Cell Science
AGAP2 and retrograde trafficking
Fig. 6. Live-cell-imaging analysis. HeLa cells were transfected with AGAP2 siRNA (A,B) or control siRNA (C) and incubated with Alexa-Fluor 488-STxB and
Alexa Fluor 568-Tf on ice for 30 minutes. After washing, cells were shifted to 19.5°C for 60 minutes, then subjected to imaging at 37°C. After 5 minutes, STxB
colocalized with Tf in the same tubules (A, arrows) and this colocalization persisted up to 60 minutes in the perinuclear region (B). In control cells, the majority of
STxB is localized at the Golgi by 60 minutes and only a remnant of Tf is observed in recycling endosomes (C, arrows). Time in minutes is shown on bottom left of
Journal of Cell Science
STxB accumulates in colocalization with TfR in Rab-positive
endosomes that are close to clathrin patches. This phenotype is
similar to the one observed upon clathrin depletion (Saint-Pol et
al., 2004), and different from the retromer protein Vps26 depletion
condition, in which STxB moves away from TfR-positive
membranes (Popoff et al., 2007). Our body of data therefore
suggest a function for Arf1/AGAP2 in clathrin-dependent
retrograde tubule formation on early endosomes. Since our
previous studies had shown that AP-1 was not required for
retrograde transport of STxB (Saint-Pol et al., 2004), one must
assume that AGAP2 can also interact with the clathrin machinery
via other molecules.
The inhibition of retrograde transport by AGAP2 depletion
might at first sight appear surprising, since one might expect Arf1
to be preferentially in the active GTP-bound conformation under
these conditions and thereby be stimulating the trafficking step.
However, in the COPI-dependent vesicle-formation model, it is
now well established that cargo sorting is inhibited when the
2388 Journal of Cell Science 123 (14)
Fig. 7. AGAP2 is required for retrograde transport of various exogenous
and endogenous cargos. HeLa cells were transfected with control or AGAP2
siRNA sequence 3 for 3 days. (A,B)Fluorescently labeled cholera toxin B-
subunit (CTxB, red) was incubated with HeLa cells for 45 minutes. Note that
CTxB fails to reach Golgi membranes (giantin, green) in AGAP2-depleted
cells. Arrows indicate CTxB-positive structures that are positive or negative
for giantin (A or B, respectively). (C,D)Anti-TGN46 antibody (red) was
incubated with HeLa cells for 6 hours. In AGAP2-depleted cells, the antibody
fails to reach the distended TGN, labeled with Golgin-97 (green). (E,F)Cells
labeled with anti-CIMPR (red) and anti-GM130 (green, arrow) antibodies. The
presence of CIMPR at peripheral sites is increased in AGAP2-depleted cells.
Fig. 8. The effects of AGAP2 depletion on other trafficking pathways.
(A)VSVG-tsO45-GFP was transfected into HeLa cells that were previously
treated with AGAP2 siRNA sequence 3. VSVG levels at the plasma membrane
were measured by FACS after a trafficking pulse of 0 or 2 hours. BFA was
used as a positive control. Means ± s.e.m. of three determinations.
(B,C)Endocytosis assays. After binding, STxB-S-S-biotin (B) or Tf-S-S-biotin
(C) was incubated for the indicated times with cells transfected with control
siRNA or AGAP2 siRNA. The percentages of cell-surface-inaccessible
(internalized) STxB or Tf were determined. Means ± s.e.m. of three
determinations. (D)Tf recycling assay. Tf-S-S-biotin was incubated with HeLa
cells for 40 minutes at 37°C. After washing, cells were incubated for the
indicated times in the presence of an excess of non-biotinylated Tf. Residual
cell associated Tf was determined at each time point. A representative of two
determinations is shown.
Journal of Cell Science
reaction is performed in the presence of the non-hydrolysable GTP
analogue GTPS (Lanoix et al., 1999; Nickel et al., 1998;
Pepperkok et al., 2000), suggesting that inhibition of GAP activity
would also lead to a blockage.
AGAP2 has been localized to focal adhesions, interacts with
focal adhesion kinase, and by interfering with AGAP2 function
impacts the integrity of focal adhesions (Zhu et al., 2009). Put in
perspective with our current study on the involvement of AGAP2
in retrograde sorting, the possibility arises of a molecular link
between retrograde trafficking and focal-adhesion dynamics. Such
a link could be achieved through trafficking of focal-adhesion
proteins via the retrograde route, which has, however, not yet been
described. Alternatively, a link might exist between retrograde
sorting and recycling machineries. Focal-adhesion dynamics clearly
depends on recycling from Rab4-positive early endosomes (Roberts
et al., 2001), and we found here that in AGAP2-depleted cells,
STxB is trapped in the Rab4 compartment. Cholera toxin, which
similarly to Shiga toxin, follows the retrograde route, has been
found to alternate between retrograde sorting and recycling,
depending on cell adhesion (Balasubramanian et al., 2007). These
data imply that the machinery of retrograde transport could have
an additional role in the recycling pathway under certain conditions.
The mechanisms by which recycling and retrograde pathways are
linked should be the subject of future studies.
Materials and Methods
Recombinant proteins, antibodies, siRNAs and other reagents
STxB-Sulf2 was purified as described (Mallard and Johannes, 2003). Briefly,
periplasmic extracts were loaded on a QHP column (GE Healthcare) and eluted in
a linear NaCl gradient (25 mM Bis-Tris-HCl, pH 6). STxB-Sulf2eluted from the
column at about 500 mM. Wild-type STxB was purified as above, with a NaCl
gradient at pH 8. STxB eluted around 150-250 mM, and was dialyzed against
coupling buffer (20 mM HEPES-KOH, pH 7.4, 150 mM NaCl), and subjected to
coupling with Cy3 mono-reactive Dye Pack (GE Healthcare). The Cy3-coupled
STxB was purified by PD-10 column (GE Healthcare), quickly frozen and stored
at –80°C. Streptolysin-O (SLO) was purchased from Sucharit Bhakdi, Institute of
Medical Microbiology and Hygiene, Mainz, Germany. The monoclonal anti-STxB
antibody 13C4 was purified with Protein-A-Sepharose from culture medium of the
corresponding mouse hybridoma cells (ATCC). Glutathione S-transferase (GST)
and GST-ARFBD were purified on glutathione Sepharose beads, according to the
manufacturer’s instructions (GE Healthcare). The polyclonal antibody against
AGAP2 was raised in rabbits against the following two peptides:
LNRLRKLAERVDDP and TPSITATPSPRRR. The antiserum recognizes
overexpressed AGAP2 by western blotting, and endogenous AGAP2 by
immunofluorescence. The specificity of the antibody was verified on siRNA-
transfected cells. The monoclonal antibodies CTR433 and anti-VSVG were generous
gifts from Michel Bornens and Franck Perez (Institut Curie, Paris, France). The
rabbit polyclonal antibody against ARAP1 was the generous gift from Paul Randazzo
(NIH, Bethesda, MD). The monoclonal antibodies against giantin (Abcam), Golgin-
97 (Invitrogen) and Lamp-1 (BD Bioscience) were purchased from the indicated
suppliers. The cDNA constructs for all ARF proteins, GFP-Rab4, GFP-Rab5, GFP-
Rab11, GFP-ARFBD, GFP-RHOGAP and GST-ARFBD were generous gifts from
Kazuhisa Nakayama (Kyoto University, Kyoto, Japan), Jean Salamero, Bruno Goud
and Philippe Chavrier (Institut Curie). The pools of four siRNAs against ArfGAPs
that target different sequences from the same mRNAs were purchased from
Dharmacon, and individual siRNA from Sigma. The control siRNA sequence was
gacagaaccagaacgccaTT. For SMAP2, the following sequences were chosen: SMAP2-
1 aacctcgaccagtggactcaaTT and SMAP2-2 gaagacccacagctacctcTT. The siRNA
sequences against AGAP2 were as follows: AGAP2#1 aaacagagcuuccuacuaaTT,
AGAP2#2 gagaaacgaagcuuggauaTT, AGAP2#3 uuaacgggcucgucaaggaTT, AGAP2#4
gagcgcgagucguggauucTT. Within the ArfGAP family, all four sequences are perfectly
complementary only with AGAP2 (Kahn et al., 2008). By BLAST search, AGAP2#4
also matches hCG2014417. The other three sequences have no match in the human
mRNA database except with AGAP2.
For siRNA experiments, 1.2?105HeLa cells were seeded in 24-well dishes 1 day
before the experiment. The cells were incubated for 90 minutes at 37°C in DMEM
without sulfate, and then for 30 minutes on ice with 1 M STxB-Sulf2in DMEM
without sulfate. After washing, the cells were shifted for 20 minutes to 37°C in
DMEM without sulfate containing 480 Ci/ml [35S]sulfate (Perkin-Elmer). For the
permeabilized cell assay, 2.4?105cells were seeded. On the day of the experiment,
the cells were sulfate starved and incubated on ice with STxB-Sulf2, as described
above. The assay was performed essentially as described (Amessou et al., 2006).
Briefly, the cells were washed with ICT/DTT (50 mM HEPES-KOH, pH 7.4, 8.37
mM CaCl2, 78 mM KCl, 4 mM MgCl2, 10 mM EGTA, 1 mM DTT), incubated for
10 minutes on ice with 2 g/ml SLO in ICT/DTT, washed, and incubated for
plasma membrane permeabilization for 10 minutes at 37°C with ICT/DTT containing
the indicated molecular tools. The permeabilized cells were then incubated for 30
minutes at 37°C with 12 mg/ml of HeLa cell cytosol (Mallard et al., 2002)
containing 960 Ci/ml [35S] sulfate in the continued presence of the molecular
tools. The cells were lysed with 900 l of RIPA buffer (1% NP-40, 0.5%
deoxycholate, 0.5% SDS in PBS), and STxB-Sulf2was immunoprecipitated with
12 g/ml of 13C4 and 40 l of protein-G-Sepharose beads (GE Healthcare). After
end-over-end rotation for 90 minutes at 4°C, the beads were collected and the
supernatant was precipitated using TCA. The beads were washed with buffer IV (50
mM Tris-HCl, pH 8), dried with Exmire microsyringe (ITO corporation), boiled in
sample buffer, and eluates were loaded on Tris-Tricine gels. The gels were fixed
and dried, and analyzed by autoradiography using PhosphorImager (Molecular
Dynamics). The radioactive bands were quantified by ImageQuant (Molecular
Dynamics). For total sulfation analysis, the TCA precipitated proteins from the
supernatant were glass fiber filtered (Whatman), and radioactivity was quantified
in a scintillation counter. Variations of total sulfation counts were within 10% of
Transfection and immunofluorescence
HeLa cells were transfected with siRNAs at 200 nM for 72 hours using oligofectamine
(Invitrogen), or for 6-24 hours with cDNAs using FuGene (Roche). The cells were
incubated with DMEM containing Cy3-STxB for 3 minutes at 37°C, washed, chased
in DMEM for 45 minutes at 37°C, fixed with 4% paraformaldehyde, quenched by
50 mM NH4Cl, and permeabilized with saponin buffer (0.2% saponin, 2% BSA in
PBS). Primary and secondary antibodies were diluted with saponin buffer. After
treatment with secondary antibody, the cells were washed in water and mounted with
Mowiol. Images were acquired on a Leica SP2 confocal microscope.
106HeLa cells were transfected with GFP-VSVG-ts045 plasmid using calcium
phosphate (Invitrogen). After 4 hours, the cells were incubated overnight at 40°C,
treated or not for 30 minutes at 40°C with brefeldin A (BFA) at 5 g/ml, detached
with trypsin, diluted with culture medium, incubated for 2 hours at 32°C in 15 ml
tubes in the presence or absence of BFA, centrifuged at 0.5 g for 3 minutes and
washed with 3% FCS in PBS. Anti-VSVG antibody was diluted with 3% FCS in
PBS, and incubated with the cells for 30 minutes. The cells were washed twice with
3% FCS in PBS and incubated with anti-mouse Cy5 in 3% FCS in PBS for 30
minutes. After washing, transfected cells were gated by GFP signal with FACScalibur
and the fluorescence of Cy5 in gated cells was measured.
Endocytosis and recycling assay
The endocytosis assay was performed as described previously (Saint-Pol et al.,
2004). Briefly, STxB and human diferric transferrin were biotinylated using NHS-
SS-biotin (Pierce). HeLa cells were serum starved for 1 hour and detached with 2
mM EDTA in PBS. Detached cells were incubated on ice for 30 minutes with 2.5
g/ml biotinylated STxB and 20 g/ml biotinylated transferrin. After washing with
5 mM glucose, 0.2% BSA in PBS++, the cells were divided into 2?105cells per data
point and incubated at 30°C for indicated times. Endocytosis was terminated by
placing cells on ice. Biotin on cell-surface-exposed STxB or transferrin was cleaved
by incubation on ice for 30 minutes with 200 mM MesNa in TNB buffer (50 mM
Tris-HCl, pH 8.6, 100 mM NaCl, 0.2% BSA). The reaction was quenched for 30
minutes with 300 mM iodoacetamid in TNB buffer, cells were lysed in blocking
buffer (10 mM Tris-HCl, pH7.4, 50 mM NaCl, 1 mM EDTA, 0.2% BSA, 0.1% SDS,
1% Triton X-100) before loading on ELISA plates coated either with anti-STxB
(13C4) or anti-Tf antibodies. Biotinylated STxB or transferrin was detected using
streptavidin-HRP (Roche). For transferrin recycling experiments, cells were incubated
for 40 minutes at 37°C with 80 g/ml biotinylated transferrin. After washing, cells
were placed for indicated times at 37°C in the presence of a 100-fold molar excess
of non-biotinylated transferrin. The cells were directly lysed in blocking buffer and
transferrin was quantified by ELISA.
We would like to thank Philippe Chavrier, Kazuhisa Nakayama,
Jean Salamero, and Franck Perez, Michel Bornens, and Paul Randazzo
for reagents. The work was supported by grants from Association pour
la Recherche Contre le Cancer (3143) and Agence Nationale pour la
Recherche (Programme blanc), the intramural program of NICHD
(NIH), and by a fellowship from Association pour la Recherche sur le
Cancer to Y.S. Deposited in PMC for release after 12 months.
Supplementary material available online at
AGAP2 and retrograde trafficking
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Journal of Cell Science