Báo cáo khoa học: Passage through the Golgi is necessary for Shiga toxin B subunit to reach the endoplasmic reticulum pptx

Passage through the Golgi is necessary for Shiga toxin
B subunit to reach the endoplasmic reticulum
Jenna McKenzie
1
, Ludger Johannes
2,3
, Tomohiko Taguchi
4
and David Sheff
1
1 Department of Pharmacology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
2 Institut Curie, Centre de Recherche, Laboratoire Trafic, Signalisation et Ciblage Intracellulaires, Paris, France
3 CNRS UMR144, Paris, France
4 Department of Biochemistry, Osaka University Graduate School of Medicine, Japan
Shiga toxin (Stx) is a bacterial exotoxin responsible for
an estimated 165 million annual cases of severe dysen-
tery worldwide [1]. The toxin attacks cytosolic targets
in mammalian cells. To reach these targets, the toxin
navigates a retrograde pathway that passes sequentially
through the plasma membrane, endosomes, Golgi and
endoplasmic reticulum (ER) [2–5]. Passage through the
Golgi appears to be rate limiting on this pathway,
resulting in prominent labeling of this organelle. How-
ever, such prominent labeling may be misleading.
Recycled transferrin was assumed to pass sequentially
through the early endosomes (EEs) and recycling endo-
somes (REs) based on prominent labeling of the RE at
later time points [5,6]. It later became evident that the
majority of transferrin actually bypasses the RE. The
same may be true for Golgi passage of Stx. Empirical
data supporting a requirement for passage through the

Golgi is lacking. Indeed, treatment with brefeldin A
provides protection against the holotoxin, suggesting
involvement of the Golgi. However, that protection is
incomplete, suggesting that Golgi passage may be
favored but not required [7,8]. Furthermore, other
toxins, such as diphtheria toxin, bypass the Golgi and
ER by escaping the endosomal compartment directly
into the cytosol [9]. SV40 virus is internalized into a spe-
cialized compartment which can communicate directly
Keywords
endosomes; Golgi; membrane traffic;
retrograde traffic; Shiga toxin
Correspondence
D. Sheff, Department of Pharmacology,
Carver College of Medicine, University of
Iowa, Iowa City, IA 52242-2600, USA
Fax: +1 319 335 8930
Tel: +1 319 335 7705
E-mail:
(Received 11 November 2008, revised 4
January 2009, accepted 7 January 2009)
doi:10.1111/j.1742-4658.2009.06890.x
Both Shiga holotoxin and the isolated B subunit, navigate a retrograde
pathway from the plasma membrane to the endoplasmic reticulum (ER) of
mammalian cells to deliver catalytic A subunits into the cytosol. This route
passes through early ⁄ recycling endosomes and then through the Golgi.
Although passage through the endosomes takes only 30 min, passage
through the Golgi is much slower, taking hours. This suggests that Golgi
passage is a key step in retrograde traffic. However, there is no empirical
data demonstrating that Golgi passage is required for the toxins to enter

the ER. In fact, an alternate pathway bypassing the Golgi is utilized by
SV40 virus. Here we find that blocking Shiga toxin B access to the entire
Golgi with AlF
4
)
treatment, temperature block or subcellular surgery
prevented Shiga toxin B from reaching the ER. This suggests that there is
no direct endosome to ER route available for retrograde traffic. Curiously,
when Shiga toxin B was trapped in endosomes, it entered the cytosol
directly from the endosomal compartment. Our results suggest that traffick-
ing through the Golgi apparatus is required for Shiga toxin B to reach the
ER and that diversion into the Golgi may prevent toxin escape from endo-
somes into the cytosol.
Abbreviations
BFA, brefeldin A; EE, early endosome; ER, endoplasmic reticulum; MEM, minimal Eagle’s medium; PDI, protein disulfide isomerase; RE,
recycling endosome; Stx, Shiga toxin; StxB, Shiga toxin B; Tfn, transferrin; TfnR, transferrin receptor; TGN, trans-Golgi network; WGA,
wheatgerm agglutinin.
FEBS Journal 276 (2009) 1581–1595 Journal compilation ª 2009 FEBS. No claim to original US government works 1581
with the ER, bypassing endosomes and Golgi [10].
There may even be alternative retrograde pathways
between the endosomes and ER that either include or
bypass the Golgi, where the majority of traffic nor-
mally passes through the Golgi. To investigate these
possibilities, we examined the fate of Stx where access
to the Golgi was blocked.
Stx is secreted by Shigella dysenteriae. It is highly
homologous to the Shiga-like toxins (also termed vero-
toxins) secreted by enterohemorrhagic strains of Esc-
herichia coli. Stx is a member of the A-B
5

family of
toxins, which are composed of one enzymatic A sub-
unit, noncovalently bound to a B subunit composed of
a homopentamer of B fragments [11]. The Stx A sub-
unit is an rRNA N-glycosidase, which stops protein
synthesis and causes cell death [12]. The A subunit
must be delivered to the host-cell cytosol to encounter
its ribosomal substrate. To reach this destination, it is
carried by a homopentameric B subunit (StxB) along a
retrograde pathway from the plasma membrane
through the EE ⁄ RE to the Golgi and the ER. Stx
takes advantage of trafficking through the Golgi to
facilitate cleavage and activation of the catalytic
A subunit by trans-Golgi network (TGN) resident
furin protease [13]. The catalytic domain remains
attached to the anchor domain by a disulfide bridge
that is cleaved when the complex enters the cytosol.
Entry of the catalytic A subunit into the cytosol is via
retrotranslocation [14–16]. The B subunit initially gains
entry to cells by binding the neutral glycosphingolipid,
globotriaosyl ceramide (Gb3 or CD77) at the cell
surface [17]. Bound toxin is endocytosed via both
clathrin-dependent and -independent mechanisms and
is delivered to EEs [18–20]. There is no known protein
receptor for Stx B subunit (StxB), and the mechanism
by which it is recruited into clathrin-coated pits
remains unknown. StxB binding to Gb3 at the cell sur-
face induces changes in plasma membrane topology
resulting in the formation of tubular invaginations that
facilitate internalization [21]. It remains to be deter-

mined whether this toxin-induced pathway or clathrin-
mediated endocytosis is predominant in normal cells.
In both cases, newly internalized StxB appears to be
delivered to EEs. StxB binding is not a passive process.
Binding and endocytosis of the toxin is accompanied
by activation of Syk kinase and activation of microtu-
bule networks, which facilitate transport into the cell
[22,23].
Passage of Stx through the EEs ⁄ REs is well docu-
mented and involves many proteins that are now being
identified [3,9,24]. Two Rab GTPases, Rab11a and
Rab6A¢, regulate retrograde traffic of Stx from the
EEs ⁄ REs to the Golgi, suggesting that this is a
regulated vesicular trafficking process [25,26]. In addi-
tion, components of the retromer complex, specifically
sorting nexins 1 and 2 and Vps26 are required for traf-
fic of Stx through the endosomes, but it is still unclear
if this mediates an intra-endosomal step or if they are
required for delivery to the TGN [27–29]. Delivery to
the TGN does appear to involve the GARP complex,
first identified in yeast as mediator of retrograde traffic
into the Golgi [30]. It is clear that Stx does not pass
through the late endosomes [24]. Instead, direct trans-
port to the TGN is mediated by syntaxin 5, syntaxin 6,
and syntaxin 16, a pathway that is shared by the
endogenous protein TGN38, which cycles between
Golgi and plasma membrane via REs [31,32]. Unlike
TGN38, traffic of StxB from endosomes to Golgi is
dependent upon the Golgin, GCC185 [31,33–35].
Here we examine the traffic of StxB which follows

the same route as the holotoxin through the retrograde
trafficking pathway [15,36]. We perturbed access to the
Golgi by an AlF
4
)
treatment, temperature block and
subcellular surgery to examine whether there exit
routes for StxB to bypass the Golgi while trafficking
from endosomes to ER. Using these systems, we deter-
mined that Golgi transit is required for trafficking to
the ER.
Results
StxB co-localizes with transferrin-positive
endosomes
We first sought to establish a time-line for retrograde
traffic of StxB in green monkey kidney BSC-1 cells.
These cells were selected due to their distinct endo-
somal and Golgi morphologies that allow ready visual
identification. Like HeLa cells, different strains of
BSC-1 cells show different affinities for StxB. Our lab-
oratory strain (a gift from I. Mellman) binds StxB
readily. Another strain reported by Spooner et al. [37]
does not. For co-localization studies, cells were
infected with adenovirus containing human transferrin
receptor (TfnR), a well-studied marker of the endo-
cytic recycling pathway [5,38]. This infection did not
alter the morphology of internalized StxB observed in
uninfected cells (not shown). Cells were labeled on ice
with both Cy3–StxB and Alexa 488–transferrin (Tfn)
for 30 min. Internalization of both labels was per-

formed at 37 °C in label-free medium for the indicated
times (Fig. 1A). After 5 min, Tfn was in peripheral
puncta representing EEs (Fig. 1A; 5 min) [5]. StxB
co-localized with Tfn throughout the EEs. This
suggested that although internalization of StxB may be
through clathrin-dependent or -independent mechanisms,
Shiga toxin in the Golgi J. McKenzie et al.
1582 FEBS Journal 276 (2009) 1581–1595 Journal compilation ª 2009 FEBS. No claim to original US government works
they converge on the EEs [39,40]. After 10 min, StxB
and Tfn co-localized in both the peripheral EEs and a
perinuclear organelle, identified by Tfn pulses as the
RE (Fig. 1A; 10 min) [41]. After 30 min, Tfn primarily
labeled the endosomes, although the signal was weaker
due to recycling of Tfn into the media, whereas StxB
had entered a separate perinuclear structure (Fig. 1A;
30 min). This structure had the appearance of a Golgi
ribbon in these cells. The difference in localization was
more obvious after 45 min (Fig. 1A; 45 min arrow
indicates transferrin-containing endosomes). At the
later times, Tfn had recycled out of the endosomes and
was no longer clearly visible although StxB remained
in Golgi morphology (Fig. 1A; 60, 120 min) [38,42].
At 180 min, the internalized StxB took on a lacy
appearance typical of the ER (Fig. 1A), suggesting
that a substantial amount of the toxin had been deliv-
ered to the ER [43]. Thus, endocytosed StxB was deliv-
ered into the endocytic recycling pathway within
5 min, was transferred to perinuclear endosomes
within 10–20 min, and then was delivered to the Golgi
within 30–45 min of internalization.

A
B
C
Fig. 1. Trafficking of StxB in BSC-1 cells. Cy-3 StxB was bound to BSC-1 cells on ice and internalized at 37 °C for the times shown. (A) StxB
passes through Tfn-positive endosomes. Alexa 488 Tfn and StxB bound to BSC-1 cells expressing human Tfn receptor on ice and then
warmed for times shown. Both co-localized up to 20 min. By 45 min Tfn (green) and StxB (red) had separated. Arrow indicates perinuclear
endosome. StxB remained in a Golgi-like ribbon for the remainder of the Tfn ⁄ StxB time-course 60–180 min. (B) Internalized StxB (red) with
cells fixed and immunolabeled for Golgi marker GM130 (green). Note co-localization (yellow). (C) Internalized StxB (red) with cells fixed and
immunolabeled for ER marker PDI (green). Note co-localization at 240 min. Inset is indicated area magnified. Bars = 10 l
M.
J. McKenzie et al. Shiga toxin in the Golgi
FEBS Journal 276 (2009) 1581–1595 Journal compilation ª 2009 FEBS. No claim to original US government works 1583
StxB is delayed in the Golgi before entering the ER
We next characterized the passage of StxB through the
Golgi of BSC-1 cells under normal cell culture condi-
tions (Fig. 1B,C). The distribution of StxB at various
time points was compared with that of the cis ⁄ medial
Golgi marker GM130, or the ER marker protein disul-
fide isomerase (PDI) [44,45]. Cells were labeled with
StxB as before and fixed for immunofluorescence. StxB
initially partly co-localized with the cis ⁄ medial Golgi
marker GM130 after 20 min (Fig. 1B), and co-localiza-
tion increased up to 120 min (Fig. 1B). This confirmed
that StxB passes from the transferrin-positive endo-
somes to the Golgi rather than to another compart-
ment such as late endosomes [24]. Passage through the
Golgi was slow, as observed elsewhere [2]. To deter-
mine how long it took for StxB to enter the ER, we
internalized StxB for up to 4 h and labeled the cells
for the ER marker, PDI (Fig. 1C). StxB remained in a

perinuclear ribbon (Golgi, as shown by co-localization
in Fig 1B) up to 120 min. StxB began to co-localize
with PDI at 150 min (not shown) and 180 min (not
shown). By 240 min (Fig 1C), StxB was localized to
the ER as shown by co-localization with the ER resi-
dent, PDI. These data support the observation that
passage through the Golgi is the slowest step in the
retrograde pathway, requiring up to 120 min [46].
Taken together, Fig. 1A–C established a normal time-
course of StxB traffic in BSC-1 cells. We used this
time-course as a basis for our further experiments.
Passage through the Golgi is required for StxB to
reach the ER in cytoplasts
We wished to test directly if passage through the Gol-
gi ⁄ TGN was required for StxB entry into the ER. To
accomplish this, we made use of subcellular surgery to
create cytoplasts lacking a Golgi apparatus [47].
Peripheral extensions of adherent BSC-1 cells were
cleaved using a glass micro-pipette to create cytoplasts
(peripheral areas lacking a nucleus) and karyoplasts,
containing the nucleus, the Golgi apparatus and the
REs [48]. Cytoplasts generated in this manner lack a
Golgi apparatus, and importantly, cannot regenerate
one [47]. By contrast, cytoplasts can regenerate func-
tional REs from peripheral EEs, as we have previously
demonstrated. Recycling of Tfn in cytoplasts is com-
plete and follows the same kinetics in cytoplasts as in
whole cells [6]. Cytoplasts and karyoplasts were labeled
with StxB and Tfn for 5 min at 37 °C (rather than on
ice to avoid releasing the cytoplast from the coverslip)

and both ligands were chased into the cytoplasts for
various times (Fig. 2A). After 10 min, StxB co-local-
ized with Tfn in endosomal structures (Fig. 2A; 10 min
yellow arrow). After 30 min, Tfn and StxB continued to
co-localize with Tfn in endosomes (compare Fig. 2A;
30 min to Fig. 1A). Because Tfn recycles out of
cytoplasts at longer StxB internalization times
(120 min), it was necessary to add Tfn to the media
for 5 min and chase in unlabeled media for the final
25 min of the assay before fixation to illuminate the
endocytic pathway. Surprisingly, after 120 min,
although the majority StxB (red arrows) remained
inside the cytoplasts, it did not co-localize with endo-
somal structures (labeled with Tfn, green arrow).
Rather, it appeared in a diffuse cytosolic-like pattern
(red arrows, Fig. 2A; 120 min). To ensure that Golgi
was not inadvertently included in the cytoplasts, we
immunolabeled cytoplasts for GM130 and found it to
be absent from the cytoplast, but readily visible in the
karyoplast (Fig. 2B). To identify which compartment
the StxB had entered, we chased StxB into cytoplasts
for 120 min and labeled the plasma membrane (wheat-
germ agglutinin, WGA; Fig. 2C), ER (PDI; Fig. 2D),
and cytosol (Rho GDI; Fig. 2E). StxB (red arrows)
did not co-localize with WGA (green arrows; Fig. 2C)
and thus had not recycled to the plasma membrane.
Nor did it co-localize with the ER marker, even when
allowing 240 min for co-localization with PDI (green
arrows; Fig. 2D). However, StxB did co-localize with
cytosolic GDI (yellow arrow; Fig. 2E), suggesting that

StxB was in the cytosol. The GDI immunolabel
required methanol fixation, which causes a grainy cast
to cytosolic proteins. Cytosolic depletion using SLO or
saponin proved unfeasible as treated cytoplasts
detached from the coverslip. Together, these results
suggest that when the Golgi was absent, StxB did not
enter the ER. Furthermore, under these conditions the
toxin was able to escape the endosomes directly into
the cytosol. At no time was an ER morphology or
co-localization with PDI of StxB observed. While it is
possible that some remnant Golgi, below the threshold
of visualization, was present in the cytoplast, it was
clearly insufficient to mediate StxB traffic to the ER.
This phenomenon may occur to some extent during
normal transit of the endosomes, although the amount
of toxin available for escape may be minimal as the
toxin passes rapidly through the endosomes to the
Golgi. It may, however, correspond to the brefel-
din A-resistant toxicity reported elsewhere [8].
Aluminum fluoride traps StxB and Tfn in
perinuclear endosomes
We wished to confirm the requirement for Golgi pas-
sage and to quantify escape of StxB from endosomes
Shiga toxin in the Golgi J. McKenzie et al.
1584 FEBS Journal 276 (2009) 1581–1595 Journal compilation ª 2009 FEBS. No claim to original US government works
into the cytosol. However, cytoplasts are extremely
small, and must be made individually, making frac-
tionation impossible. Therefore, we used a pharmaco-
logical approach. Aluminum fluoride (AlF
4

)
)isan
activator of small GTPases and is well-documented to
block recycling of Tfn from the RE [5,49]. Although
the precise target of AlF
4
)
at the RE is not known,
the effect of this drug on Tfn recycling is immediate
and remarkably specific to recycling out of the RE in
nonpolar cells and to basolateral recycling from the
RE in polarized cells [5]. Treatment for > 1 h results
in dispersal of the Golgi although both the TGN and
the ER remain functional [50,51]. Because StxB
co-localized extensively with Tfn in perinuclear REs,
we suspected that AlF
4
)
might also block retrograde
StxB from the endosomes to the TGN just as it did
for recycling traffic to the plasma membrane. Fortu-
itously, both StxB and Tfn were trapped together in
the REs following AlF
4
)
treatment (Fig. 3). This was
especially apparent after 60 min, when Tfn would
normally have recycled out of the cell, and StxB would
normally have moved to the Golgi. Both remained in
the endosomes of treated cells after 60 and even

120 min (Fig. 3; 60 min, 120 min, yellow arrows).
Although this result is based on the fortunate effects
of AlF
4
)
treatment on these two pathways, it does not
necessarily imply that the same drug target is involved
in both retrograde and recycling pathways. It does,
however, present a unique opportunity. As in the
cytoplast, StxB is prevented from reaching the Golgi,
and it is trapped inside of the endosomes. This allowed
us to quantify the consequences of trapping StxB in
endosomes.
StxB leaks into the cytosol when trapped at
endosomes
StxB trapped in the endosomes of AlF
4
)
-treated cells
took on a diffuse cytosolic appearance at later time
points following internalization (Fig. 3; 120 min, red
A B
E D C
Fig. 2. StxB cannot access the ER in BSC-1 cytoplasts. BSC-1 cells were manually cut with a glass needle to create karyoplasts (k) contain-
ing both the nucleus and Golgi and cytoplasts (c). All cytoplasts and karyoplasts were labeled with Cy-3 StxB (red) that was internalized for
times shown. (A) Shiga and Tfn (green) internalized together for 10 min then chased for 10 or 30 min. For 120 min, Tfn was internalized for
the final 25 min. (B) Cytoplast with StxB (red) immunolabeled for Golgi marker GM130 (green). (C) Cytoplast stained for plasma membrane
with wheat germ agglutinin (green). Note that the cytoplast has moved next to the karyoplast but the two remain separate. (D) Cytoplast
labeled for ER marker PDI (green), at various times of StxB (red) internalization. Note exclusion of StxB from ER. (E) Cytoplast labeled
for cytosolic marker GDI (green) note co-localization (yellow) with StxB (red). Insets are cytoplasts presented in single channels with larger

inset showing a magnified view of the combined channels. Red arrows indicate StxB, green arrows indicate other compartment markers as
indicated. Bars = 10 l
M.
J. McKenzie et al. Shiga toxin in the Golgi
FEBS Journal 276 (2009) 1581–1595 Journal compilation ª 2009 FEBS. No claim to original US government works 1585
arrows). RE-associated versus peripheral fluorescence
was measured using NIH image in 20 cells; 20 ± 7%
was found in the periphery. The diffuse material did
not have an ER morphology, however, AlF
4
)
is
known to disperse the medial Golgi in some cells after
extended treatment (> 120 min) [50]. To determine
which cellular compartment StxB had entered, we per-
formed a series of co-localizations (Fig. 4). The distri-
bution of StxB was compared to the cis ⁄ medial Golgi
marker GM130 in BSC-1 cells in the presence of
AlF
4
)
. Although dispersed, the Golgi remained clearly
visible as structures surrounding StxB-labeled endo-
somes at times up to 240 min (Fig. 4A). GM130 did
not co-localize with the StxB in punctate (endosomal)
structures nor did it co-localize with the diffuse StxB
(Fig. 4A). This confirmed both that AlF
4
)
treatment

prevented access to the Golgi and that StxB was not in
the fragmented Golgi.
It was possible that AlF
4
)
treatment may have
allowed StxB to bypass the Golgi and enter the ER.
However, despite changes in ER morphology (Fig. 4B),
StxB did not co-localize with the ER marker PDI,
even at 240 min (Fig. 4B). As with the cytoplasts, StxB
prevented from reaching the Golgi by AlF
4
)
did not
access the ER. A fraction of the internalized StxB
appeared to escape the endosomes as had occurred in
the cytoplasts. This suggested that StxB alone could
escape endosomes if it was not sequestered into the
Golgi. It was not completely clear if the extra-endoso-
mal StxB was in a membrane or cytosolic fraction.
It was also unclear if endosomal escape was specific
for StxB or resulted from AlF
4
)
treatment altering the
endosomal membranes to allow escape of all cargo.
To differentiate between these possibilities, we used
a cell fractionation approach to separate cytosol from
membrane-bound organelles. Iodinated StxB was
bound to BSC-1 cells on ice, washed, then warmed in

the presence or absence of AlF
4
)
to initiate internaliza-
tion. Cells were harvested after 120 min of chase, then
homogenized in a ball-bearing cell homogenizer so as
to recover intact organelles. Membrane and cytosolic
fractions were separated via Opti-prep step-gradients.
In these experiments, (Fig. 5A) cytosol was collected
from the top of the gradient, and all membranes were
collected from an Optiprep cushion at the bottom of
the gradient. As a control for rupture of endosomes,
125
I-labeled Tfn was bound to the cell surface and then
chased into the endosomal compartments of control
cells. Because Tfn normally recycles rapidly out of the
cell, it was chased for 20 min in control cells or for
120 min in AlF
4
)
-treated cells. This ensured that Tfn
would be in the REs [5,6]. Because Tfn is released
Fig. 3. Aluminum fluoride traps Stx in endosomes. Both StxB (red) and Tfn (green) were bound to BSC-1 cells on ice. Both were internalized
at 37 °C for times shown in the presence of AlF
4
)
. Yellow arrows indicate where both Tfn and StxB have been trapped in a perinuclear
endosome. Red arrows indicate StxB in diffuse distribution. Inset is magnification of indicated area. Bar = 10 l
M.
Shiga toxin in the Golgi J. McKenzie et al.

1586 FEBS Journal 276 (2009) 1581–1595 Journal compilation ª 2009 FEBS. No claim to original US government works
A
B
Fig. 4. Aluminum fluoride traps StxB in endosomes. (A) StxB (red) bound to BSC-1 cells and internalized for times shown in the presence of
AlF
4
)
. Cells were stained for Golgi marker GM130 (green). Note diffuse StxB. Red arrows indicate StxB in endosomal structure, Green
arrows indicate the Golgi. (B) Cells labeled as in A but stained for ER marker PDI (green). Green arrows indicate ER structures. ER morphol-
ogy is altered (compare with Fig. 1). Bar = 10 l
M.
AB
Fig. 5. StxB trapped in the endosomes leaks into the cytosol. (A) Quantification of StxB in cytosol.
125
I-labeled StxB or
125
I-labeled Tfn inter-
nalized into BSC-1 cells expressing transferrin receptor for 120 min. Cells were harvested and homogenized. Total cytosol was separated
from total membranes using an Optiprep 0 ⁄ 8 ⁄ 25% step gradient. Average values for the percent of each ligand in the cytosol with and with-
out AlF
4
)
are shown. Error bars are SD. *Significant change. n = 15 for StxB conditions, n = 9 for Tfn conditions. (B) Cell fractionation of
BSC-1 organelles to identify those containing internalized ligands on preformed linear 8–25% Optiprep gradients. Top of gradient is to the
left. (I)
125
I-labeled StxB internalized in the absence (dark red, closed circles) or presence (orange, open circles) of AlF
4
)
for 1 h. Bar 1 indi-

cates cytosolic fractions. Bars 2 and 3 indicate endosome or Golgi associated peaks in treated cells. Bar 4 indicates a peak of membrane
bound StxB found only in treated cells. (II) Positions in the gradient of cytosol (red), plasma membrane (orange), lysosomes (yellow), REs
(green), Golgi (light blue), ER (dark blue) and cell debris (purple). Supporting data are given in Fig. S1. (III)
125
I-labeled Tfn internalized for
25 min in control cells to locate RE (at 1 h it is recycled out of the cell), Bar 6 (dark blue closed squares) and 1 h in AlF
4
)
treated cells (treat-
ment prevents recycling) (light blue, open squares). Note that some Tfn remains at the plasma membrane Bar 5 (n = 1, results typical).
J. McKenzie et al. Shiga toxin in the Golgi
FEBS Journal 276 (2009) 1581–1595 Journal compilation ª 2009 FEBS. No claim to original US government works 1587
from its receptor at the neutral pH of the gradient, it
acts as a sensitive indicator of endosomal rupture dur-
ing handling. It also serves as a specific indicator of
changes in endosomal fragility due to AlF
4
)
treatment.
Figure 5A shows that there was no difference in cyto-
solic transferrin between control and AlF
4
)
-treated
cells (Fig. 5A; Tfn and Tfn + AlF
4
)
). Thus, endo-
somes were not made more fragile by the drug treat-
ment. By contrast, the amount of StxB found in the

cytosol was significantly larger in the presence of
AlF
4
)
(Fig. 5A; StxB vs. StxB + AlF
4
)
). The differ-
ence was statistically significant with P < 0.0001 (Stu-
dent’s t-test). This cytosolic escape was not observed
when StxB was internalized for only 20 min (data not
shown), a time at which StxB remained in endosomes
and was not visualized in the cytosol in intact cells.
Cytosolic StxB accounted for 12% of the radiola-
beled StxB, but analysis of cell fluorescence had found
that 25% of internalized StxB was not in the endo-
somes. To resolve this difference, we utilized a differ-
ent density gradient protocol to fractionate organelles
within the cell.
125
I-labeled StxB cell homogenates
from control and AlF
4
)
-treated cells were applied to
preformed linear 8–25% Optiprep gradients. We have
previously described the use of these gradients for the
fractionation of cellular organelles [5]. Gradients were
characterized by locating fractions containing alkaline
phosphodiesterase activity (plasma membrane), B-hex-

osaminidase activity (lysosomes) radiolabeled Tfn
internalized for 25 min (REs), added phenol red (cyto-
sol), GM130 (Golgi) and PDI (ER). The position of
the peak activity for each organelle is shown in the
color-coded bar in Fig. 5B (II), and in Fig. S1. StxB
internalized for 60 min in control cells co-localized
with Golgi and REs (which could not be readily distin-
guished in this gradient). However, AlF
4
)
treatment
shifted the distribution of both StxB and Tfn into a
doublet of peaks at, and just below, the density of
REs (Fig. 5B). In this representative experiment, 9%
of StxB was observed in the cytosol in AlF-treated
cells compared with 3% in control cells. Also, 24% of
StxB was observed in a dense fraction (compared with
8% in control cells) that did not co-segregate with any
of the characterized organelles. Although this could
not be identified, we speculate that it may represent
transport vesicles derived from the endosomes, unable
to reach the Golgi. This fraction would account for
the difference between non-endosomal StxB observed
microscopically and that observed in the cytosolic frac-
tion of step gradients above.
These results suggest that AlF
4
)
can block retro-
grade traffic at the endosomes and that StxB is able to

escape the endosomes to the cytosol. Taken together
with the cytoplast results, this suggests that when StxB
is trapped within the endosomes, it can ‘escape’ into
the cytosol, as previously suggested for dendritic cells
and macrophages [52,53]. A similar escape has also
been observed for the Stx A subunit [8].
A temperature block separates StxB and Tfn
It remained possible that StxB was equally capable of
entering the cytosol from any organelle along the ret-
rograde pathway. We tested this possibility by using a
temperature block to trap StxB within the TGN for a
time. In HeLa cells, it has been reported that maintain-
ing the cells at 20 °C traps StxB along with Tfn in the
endosomes [24]. We too observed this effect in HeLa
cells (Fig. S2). However, reducing the temperature to
20 °C in BSC-1 cells had a surprising and useful effect.
Both Tfn and StxB were bound to BSC-1 cells and
internalized at 20 °C for various lengths of time
(Fig. 6A). As expected, Tfn did not recycle out of the
endosomes, but remained in the perinuclear region.
After 30 min, StxB appeared to co-localize with Tfn in
the majority of cells. However, after 60 min, StxB sep-
arated into another perinuclear structure that did not
co-localize with Tfn. This distribution was maintained
up to 180 min. We suspected that this other structure
might be part of the Golgi due to the ribbon-like
appearance. Fortunately, in BSC-1 cells, the TGN and
cis ⁄ medial Golgi can be discriminated visually
(although there is slight overlap) using the TGN
marker TGN46 and the cis ⁄ medial marker GM130

(Fig. 6B). We therefore compared the localization of
StxB with that of TGN46 and GM130 after 120 and
180 min of internalization at 20 °C. There was striking
co-localization of StxB with the TGN marker at both
time points (Fig. 6C) suggesting that at 20 °C in BSC-1
cells, StxB was trapped in the TGN. This was very dif-
ferent from the situation at 37 °C (Fig. 6D) where
StxB co-localized with both TGN and cis ⁄ medial Golgi
in as little as 90 min.
We wished to confirm that we were seeing co-locali-
zation in the TGN and not at ER exit sites. StxB inter-
nalized at 20 °C co-localized with TGN46 but clearly
did not co-localize with the ER exit site marker Sec31,
again suggesting that StxB was trapped specifically at
the TGN at this temperature (Fig. 6E).
These results suggested that BSC-1 cells, unlike
HeLa cells, hold StxB in the TGN at 20 °C. This
difference between HeLa and BSC-1 cells provided a
natural experiment in BSC-1 cells to test if StxB could
escape into the cytosol when it was held in the TGN
instead of the endosomes. Notably, no escape of StxB
into the cytosol was seen in the cells held at 20 °C
Shiga toxin in the Golgi J. McKenzie et al.
1588 FEBS Journal 276 (2009) 1581–1595 Journal compilation ª 2009 FEBS. No claim to original US government works
even after 180 min (Fig. 6C; red box and E). Taken
together these results suggest that StxB cannot escape
the TGN into the cytosol, and that escape may be
dependent upon some property of the endosomes such
as low pH found in EEs or membrane composition
[54,55].

A
B
D
E
C
Fig. 6. A20°C temperature block traps Stx in the TGN in BSC-1 cells. (A) StxB (red) and Tfn (green) were internalized for times shown at
20 °C. Yellow arrows indicate co-localization at earlier times, red arrows indicate StxB differently distributed than Tfn. (B) anti-GM130 for
cis ⁄ medial Golgi (green) and anti-TGN46 for TGN (red) are visually resolved, two BSC-1 cells are shown. (C) StxB (red) co-localizes with
TGN46 (blue) but not with GM130 (green) when internalized at 20 °C. Purple arrows indicate co-localization of StxB and TGN46. Red bor-
dered inset shows magnification of cytosol in a cell adjacent to the labeled Golgi. Note lack of cytosolic StxB. (D) Same as (C) but internal-
ized at 37 °C. Yellow arrow indicates partial co-localization of GM130 and StxB. (E) StxB (red) co-localizes with TGN46 (blue) but not with ER
exit point marker Sec31 (green) when internalized at 20 °C. Upper insets are enlarged versions of regions indicated. Lower insets are Sec31
(green), StxB (red) and a merge of only Sec31 and StxB. Purple arrows indicate co-localization of StxB and TGN46. Green and red arrows
indicate locations of ER exit sites and StxB respectively. Bar = 10 l
M.
J. McKenzie et al. Shiga toxin in the Golgi
FEBS Journal 276 (2009) 1581–1595 Journal compilation ª 2009 FEBS. No claim to original US government works 1589
Discussion
Stx is an A–B
5
toxin that binds to the plasma mem-
brane lipid Gb3. In this respect it is like cholera toxin
and SV40 virus, both of which bind to the ganglioside
GM1 [56,57]. Both toxins, and the virus are trans-
ported to the ER after internalization [58]. However,
whereas cholera toxin appears to pass through endo-
somes and the Golgi, SV40 bypasses both the normal
endocytic organelles and the Golgi, trafficking directly
from a specialized endocytic population to the ER
[10]. These examples demonstrate that binding to a

glycolipid receptor, and even trafficking to the ER do
not guarantee passage through the Golgi. Further-
more, even if passage through the Golgi is a normal
component of the retrograde pathway, it does not
automatically follow that this pathway is exclusive of
other routes.
We sought to determine whether retrograde traffic
of StxB required passage through the Golgi to reach
the ER. A morphological examination of retrograde
traffic, as performed here, suggests that StxB appears
to progress sequentially from the plasma membrane to
the endosomes to the Golgi and then to the ER. How-
ever, just as the majority of Tfn actually bypasses the
RE, a fraction of StxB may actually bypass the Golgi
[5]. Alternatively, the Golgi transit route could be pre-
ferred and mask a lower flux endosome to ER route.
We used cytoplasts to physically separate endosomes
and ER from the Golgi in an isolated piece of living
cells. We had previously determined that these cytop-
lasts are able to regenerate a fully functional endocytic
system with both EEs and REs [6]. They also contain
ER, as demonstrated here. However, they are unable
to regenerate a Golgi, which allowed us to test directly
whether Golgi transit was required for StxB to pro-
gress from endosomes to the ER [47]. In the absence
of a Golgi, StxB was unable to access the ER in cytop-
lasts. Had a slower or lower flux pathway connected
the endosomes directly to the ER, we would have seen
StxB in the ER of the cytoplasts. Although it remains
possible that such a pathway exists for some endo-

genous proteins, it is clearly not accessed by StxB.
Our findings raise the question of why StxB should
take such a circuitous path from plasma membrane to
cytosol. Other toxins such as diphtheria toxin and
anthrax toxin take advantage of low endosomal pH to
penetrate the endosomal membrane and enter the cyto-
sol directly [59]. Our results may shed some light on the
host–pathogen interaction that has developed around
the retrograde traffic of StxB. In cytoplasts, StxB was
trapped at the endosomes. There are several possible
alternative routes that would be available out of the
EE compartments. The most obvious would be to
recycle out of the endosomes and return to the plasma
membrane along with Tfn. However, StxB did not
reappear at the plasma membrane in any visible
amount. A second possibility would be for StxB to be
shunted into late endosomes (also found in cytoplasts)
[6]. However, we did not see co-localization with lyso-
somal proteins (J. McKenzie & D. Sheff, unpublished
observations). To our surprise, StxB appeared to enter
the cytosol directly from the endosomes in cytoplasts.
Because cytoplasts are extremely small and must be
formed manually, we were unable to perform biochemi-
cal analysis or cell fractionation on these preparations.
However, we were fortunate in finding that AlF
4
)
treat-
ment blocked exit of StxB from the endosomes. Despite
being a generalized inhibitor of GTPase function in

the endocytic recycling pathway, AlF
4
)
is specific for
egress from REs [5]. Using AlF
4
)
, we were able to con-
firm that 12% of internalized StxB accessed the cytosol
directly from the endosomes. This result is inconsistent
with prior findings that a small percentage of Stx
A subunit (5%) can effectively reach its target even
when traffic along the postendosomal retrograde path-
way is impaired by disruption of the Golgi with brefel-
din A [8]. It is also consistent with the prior finding
that 10% of cell-associated StxB reaches the cytosol in
human monocyte derived macrophages [53]. This is
particularly relevant because monocytes-derived macro-
phages internalize StxB, but do not support retrograde
traffic of StxB from endosomes to the Golgi [52]. Fur-
thermore, our finding that StxB trapped in the TGN
could not enter the cytosol, suggests that endocytic
membranes are particularly susceptible to penetration
by StxB. Thus toxicity in the monocyte-derived macro-
phages may paradoxically result from the inability of
the cell to transport toxin out of the endosomes and
into the Golgi (provided that the toxin would then not
be able to exit the Golgi).
Penetration of the endosomal membrane by StxB
was surprising in light of the normal retrograde path-

way taken by StxB. However, endosomal escape is
known not only for bacterial toxins such as diphtheria
toxin, but also for fibroblast growth factor following
endocytosis [54, 60–62]. Such translocation may repre-
sent another physiological pathway that is subverted
by StxB when the Golgi pathway is unavailable.
Clearly StxB cannot be present in the cytosol while
membrane bound. This suggested that the toxin was
able to dissociate from Gb3 while inside the endosome.
Such dissociation is unlikely to be a result of low pH
because acid wash of bound StxB does not remove it
from the plasma membrane. However, StxB can bind
up to 15 Gb3 molecules, mediated through three
Shiga toxin in the Golgi J. McKenzie et al.
1590 FEBS Journal 276 (2009) 1581–1595 Journal compilation ª 2009 FEBS. No claim to original US government works
different sites on each B subunit of the pentamer [63].
The overall K
d
of the StxB ⁄ Gb3 complex is 10
)9
m. This
results from the combined associations of all sites with
Gb3 molecules in the membrane. However, binding to
site II formed by the cleft between monomers is much
weaker that that of site I [63]. Thus if Gb3 is present at
lower concentrations in the endosomes than at the
plasma membrane, one would expect dissociation of the
cleft-bound Gb3 from the complex, and dissociation of
the entire molecule when Gb3 concentrations become
sufficiently low. Recently, it has been demonstrated that

decreasing the concentration of Gb3 available at the
plasma membrane in Vero cells results in a precipitous
decline in StxB binding [64]. We would suggest that the
concentration of Gb3 may be significantly lower within
endosomes than at the plasma membrane, providing the
possibility for some StxB to no longer be bound to the
receptor [53]. Further examination of this effect is
beyond the scope of this study.
The ability of StxB to penetrate the endosomal
membrane raised the important question of whether
the endosomal membrane was uniquely permeable to
StxB. If StxB can cross any membrane, then it would
be expected that it could directly penetrate the plasma
membrane, and this is not the case, as demonstrated
by cell lines lacking Gb3 which are resistant to the
toxin [65]. Our use of BSC-1 cells allowed use to take
advantage of a unique feature of the 20 °C tempera-
ture block in these cells. Rather that trapping StxB at
the endosomes as in HeLa cells, StxB was able to traf-
fic to the TGN in these cells. Although all trafficking
is slowed (or stopped in some cases) at this tempera-
ture, this would still allow for escape of the StxB into
the cytosol at long incubation times. We did not
observe StxB entering the cytosol under these condi-
tions, suggesting that the TGN membrane is less
permeable to StxB than that of the endosomes.
One tempting possibility is that the difference in
membrane permeability has driven host–pathogen
interactions over the course of evolution. Some toxins,
such as anthrax toxin have evolved mechanisms to

avoid recycling out of the endosomes by rapidly enter-
ing the cytosol. Our data suggest that while StxB can
accomplish this penetration, the process is slow and
inefficient. Host cells may have acquired the ability to
sequester the toxin in the Golgi, thereby preserving the
cell so that over time the toxin may be degraded. Such
a possibility is supported by the rapid and near quanti-
tative diversion of StxB from the endocytic pathway
into the Golgi. The slow passage of the toxin through
the Golgi also argues in favor of this possibility. The
response of the pathogen to this sequestration appears
to have been to develop a mechanism to exploit retro-
grade transport out of the Golgi into the ER. While
this process is also slow, it is quantitative, and results
in delivery of the bulk of the toxin to the ER where
the A subunit can retrotranslocate into the cytosol by
exploiting the translocon [16,66]. In this case, the cur-
rent retrograde transport of StxB and of holotoxin
may have developed as a series of host–pathogen inter-
actions and adaptive responses. Although our data
cannot be conclusive in this respect it is at the least
supportive of the possibility.
Materials and methods
Cells and reagents
BSC-1 cells clone CCL-26 from ATCC (Manassas, VA,
USA) were cultured in minimal Eagle’s medium (MEM) sup-
plemented with 10% fetal bovine serum, 1% nonessential
amino acids, 1% sodium pyruvate, 100 unitsÆmL
)1
penicillin

and 100 lgÆmL
)1
streptomycin in 5% CO
2
, 95% air. NaF
and AlCl
3
were obtained from Fisher (Fair Lawn, NJ, USA).
Recombinant wild-type StxB derived from S. dysenteriae
was produced from clone in pSU108. The protein was
induced and purified as previously described [24,67]. Purified
StxB was labeled using Alexa-dye protein labeling kits from
Invitrogen (Carlsbad, CA, USA) according to manufacturers
directions. Polyclonal rabbit anti-Sec31 serum (kind gift
from the Warren Lab, Max F. Perutz Laboratories, Vienna,
Austria); mouse anti-GM130 mAb from BD Biosciences
(San Jose, CA, USA); mouse anti-PDI mAb from Nventa
(San Diego, CA, USA); polyclonal sheep anti-TGN46 from
Serotec (Raleigh, NC, USA); polyclonal rabbit anti-(Rho
GDI clone K-21) from Santa Cruz (Santa Cruz, CA, USA);
Alexa 488-labeled WGA, Alexa488 human holotransferrin,
Alexa 488 goat anti-mouse IgG, Alexa 488 goat anti-rabbit
IgG and Alexa 633 donkey anti-sheep IgG were obtained
from Invitrogen (Carlsbad, CA, USA).
125
I-conjugated Tfn
and StxB were made using Iodo-Gen (Pierce ⁄ Thermo Scien-
tific, Rockford, IL, USA) as previously described for Tfn [5].
StxB and Tfn labeling
BSC-1 cells were grown on glass coverslips. For cytoplasts,

square gridded coverslips (Belco) were used and cells for
live images were grown on glass-bottom 35 mm dishes. For
Tfn uptake experiments, cells were preincubated in serum-
free media for 30 min at 37 °C to clear Tfn from the cell.
Cells were chilled on ice and labeled with a 1 : 200 dilution
of 0.22 mgÆ mL
)1
Cy3 StxB and a 1 : 100 dilution of
5mgÆmL
)1
Alexa 488–Tfn in NaCl ⁄ P
i
. Internalization was
performed by placing the cells in 37 ° C MEM for indicated
times. Tfn was omitted as indicated. When noted, 50 lm
aluminum chloride ⁄ 30 mm sodium fluoride was included
during internalization. For 20 °C block studies, cells were
J. McKenzie et al. Shiga toxin in the Golgi
FEBS Journal 276 (2009) 1581–1595 Journal compilation ª 2009 FEBS. No claim to original US government works 1591
labeled in MEM (without fetal bovine serum) at 20 °C
instead of on ice, for 5 min, media was then replaced with
MEM. Cytoplasts are sensitive to cold and so were labeled
for 5 min at 37 °C in MEM followed by unlabeled MEM
at 37 °C.
Cytoplast creation
BSC-1 cells were grown on gridded coverslips for 2 days to
allow cells to spread out. Glass needles were prepared using
1.0 mm o.d., 0.50 mm i.d., 10 cm length capillaries from
Sutter Instruments (Novato, CA, USA) on a Sutter Instru-
ments P-97 micropipette puller. For microsurgery, cells

were transferred to bicarbonate-free MEM buffered with
10 mm Hepes pH 7.4. Cellular microsurgery was performed
using the glass needle mounted in a Sutter Instruments
MP-285 micromanipulator with a joystick controller under
manual control. Cuts took an average of 5 min. Cytoplasts
were allowed to recover for at least 1 h after microsurgery
prior to labeling. Cytoplasts were fixed as for whole cells.
Membrane and cytosol fractionation
BSC-1 cells were grown to near confluency in 35 mm
dishes. For Tfn labeling, BSC-1 cells were infected with
human Tfn receptor expressing adenovirus at an MOI of
> 50, 24 h prior to use. Cells were serum starved for
30 min then labeled on ice with
125
I-labeled Tfn or StxB for
30 min. Cells were then rinsed with chilled NaCl ⁄ P
i
and
warmed in MEM to 37 °C in presence or absence of AlF
4
)
.
For control cells (Tfn, no AlF), the radiolabeled Tfn was
taken up warm for 5 min then chased for 20 min, because
it would otherwise recycle back out to the cell surface. Cells
were then harvested and homogenized via a ball-bearing
cell homogenizer. Post nuclear supernatants were then gen-
erated by centrifugation at 1000 g for 5 min. Two different
fractionations were performed. To separate cytosol from
membranes, the supernatant was overlaid onto a layer of

8% Optiprep which was in turn overlaid onto a layer of
25% OPTI-Prep (diluted with ICT buffer: 78 mm KCl,
4mm MgCl
2
, 8.37 mm CaCl
2
10 mm EGTA, 50 mm
Hepes ⁄ KOH pH 7.0) [6,68]. Cytosol remained in the top
fraction, while membranes and organelles were recovered
from the 8 ⁄ 25% interface after centrifugation at 100 000 g
for 120 min at 4 °C. CPM in each fraction was counted in
a gamma counter for 5 min and the data compiled. Data
were normalized to the total counts in each tube. Each con-
dition was repeated at least four times. For density gradient
organelle fractionation, the supernatant was overlaid onto
an 8–25% preformed linear gradient of Optiprep created
using a Gradient master from Biocomp (Toronto, Canada)
and centrifuged at 100 000 g for 20 h to allow density
equilibration of organelles in the gradient. Cytosol was
identified visually using phenol red. Plasma membrane,
lysosomes and Golgi were identified by enzyme assay as
described previously [5]. ER was detected by western
blot for PDI. Endosomes were identified by the presence of
125
I-labeled Tfn internalized for 25 min.
Immunofluorescence
Cells were washed with NaCl ⁄ P
i
, fixed in 3% PFA at room
temperature for 15 min. Cells were permeabilized with

0.05% (w ⁄ v) saponin in NaCl ⁄ P
i
with 3% BSA for 1 h
then washed three times in blocking buffer (0.05% saponin,
3% BSA, NaCl ⁄ P
i
) for 5 min. For PDI labeling, permeabi-
lization was performed by 0.04% Triton X-100 in NaCl ⁄ P
i
for 4 min then washed three times in blocking buffer (3%
BSA, NaCl ⁄ P
i
) for 5min. For GDI labeling, cells were
rinsed with cold NaCl ⁄ P
i
then fixed for 5 min in 100%
methanol at )20 °C. For immunolabeling, cells were incu-
bated with a 1 : 200 dilution of antibodies (except PDI,
1 : 500) at room temperature for 45 min. Cells were visual-
ized with appropriate Alexa-conjugated secondary anti-
bodies at 1 : 200 dilution for 30 min at room temperature,
washed and mounted.
Image analysis
All images were acquired on a Zeiss 200M inverted micro-
scope equipped with a Hamamatsu ER camera operated by
Openlab from Improvision (Coventry, UK) on a Macintosh
G4 computer from Apple (Cupertino, CA, USA). Low
exposure and high exposure images of all cells were
obtained. For cytoplasts, low exposures are shown for kar-
yoplast and cytoplast, and the inset is of the higher expo-

sure due to the low levels of signal in cytoplasts. Contrast
in images was optimized using photoshop from Adobe
(San Jose, CA, USA) on an Apple G5 computer.
Acknowledgements
This work supported by a grant to JM from the
American Heart Association #081365G as well as to
DS from American Heart Association #035078N.
Support for TT was provided by 21st Century Center
of Excellence Program from the Ministry of Educa-
tion, Culture, Sports, Science and Technology of
Japan, The Core Research for Evolutional Science
and Technology (CREST), JSPS (Japan Society for
the Promotion of Science) Core-to-Core Program
Grants-in-aid for Scientific Research (18050019), and
Senri Life Science Foundation Grants. Many thanks
to Graham Warren for discussions about the Golgi
and to Lawrence Pelletier for discussions about
cytoplasts and Stx. We would also like to thank
Mark Stamnes, Heidi Hehnly, Naava Naslavsky and
Steve Caplan for their thoughtful discussions and
suggestions.
Shiga toxin in the Golgi J. McKenzie et al.
1592 FEBS Journal 276 (2009) 1581–1595 Journal compilation ª 2009 FEBS. No claim to original US government works
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Supporting information
The following supplementary material is available:
Fig. S1. Fractionation of BSC-1 organelles on an
8–25% Optiprep gradient.
Fig. S2. A20°C temperature block in HeLa cells.
This supplementary material can be found in the
online version of this article.
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