T H E J O U R N A L O F C E L L B I O L O G Y
© The Rockefeller University Press $8.00
The Journal of Cell Biology, Vol. 173, No. 6, June 19, 2006 853–859
The lumen of the ER enables proteins to fold properly before
they are transported along the secretory pathway (Ellgaard and
Helenius, 2003). When proteins misfold, the ER quality con-
trol system ensures that they are retained in the ER to prevent
them from reaching their fi nal destination and/or to allow for
their refolding. Irreversibly misfolded proteins are eliminated
by retrotranslocation to the cytosol, where they are ubiqui-
tinated and degraded by the proteasome (for review see Tsai
et al., 2002). The ER factors facilitating these opposing re-
actions are largely unknown.
We used cholera toxin (CT), which is secreted by the
bacterium Vibrio cholerae, to study retrotranslocation. CT
consists of a receptor-binding homopentameric B subunit that
is noncovalently associated with a single catalytic A subunit.
Once CT is secreted from V. cholerae, the A subunit is cleaved
into the A1 toxic domain and the A2 domain, which are con-
nected by a disulfi de bond and other noncovalent interactions.
To intoxicate cells, the holotoxin is endocytosed and travels
from the plasma membrane to the ER lumen (Fujinaga et al.,
2003). In the ER, the A subunit is disguised as a misfolded pro-
tein and hijacks the retrotranslocation machinery so that the A1
chain reaches the cytosol, where it is resistant to proteasomal
degradation (Rodighiero et al., 2002), whereas the B subunit
remains in the ER (Fujinaga et al., 2003). In the cytosol, the A1
peptide activates a cAMP-dependent signal cascade that results
in chloride and water secretion, leading to diarrhea (Sears and
Kaper, 1996). Elucidating the ER–cytosol transport mecha-
nism of CT will not only clarify a decisive step in toxin traf-
fi cking but will also clarify the retrotranslocation mechanism
of misfolded proteins.
Previous in vitro analysis found that the ER oxidoreduc-
tase protein disulfi de isomerase (PDI) unfolds the A and A1
chains of CT (Tsai et al., 2001), a reaction we believe prepares
the toxin for retrotranslocation. The PDI-like protein ERp29 has
also been implicated in protein unfolding reactions (Magnuson
et al., 2005). However, PDI family proteins have also been
shown to facilitate protein folding (for review see Wilkinson and
Gilbert, 2004). Thus, it is possible that certain PDI-like proteins
are dedicated to the retention and refolding of misfolded poly-
peptides, whereas other PDI family members function to unfold
misfolded proteins in preparation for their retrotranslocation.
In this study, we developed a semipermeabilized cell sys-
tem that monitors the ER–cytosol transport of CT and found
that PDI facilitates the toxin’s retrotranslocation, whereas ERp72,
a PDI-like protein, mediates its ER retention. Furthermore,
these activities were found to operate on endogenous ER
misfolded proteins, indicating the generality of this mechanism.
These results identify PDI family members as playing opposite
roles in ER quality control and establish an assay to elucidate
the retrotranslocation process of CT.
Protein disulfi de isomerase–like proteins play
opposing roles during retrotranslocation
Michele L. Forster,1 Kelsey Sivick,2 Young-nam Park,3 Peter Arvan,3 Wayne I. Lencer,4 and Billy Tsai1
1Department of Cell and Developmental Biology, 2Department of Microbiology and Immunology, and 3Division of Metabolism, Endocrinology, and Diabetes,
University of Michigan Medical School, Ann Arbor, MI 48109
4GI Cell Biology, Children’s Hospital, Harvard Medical School, Boston, MA 02115
ER chaperones that guide these opposing processes are
largely unknown. We developed a semipermeabilized
cell system to study the retrotranslocation of cholera toxin
(CT), a toxic agent that crosses the ER membrane to reach
the cytosol during intoxication. We found that protein
disulfi de isomerase (PDI) facilitates CT retrotranslocation,
isfolded proteins in the endoplasmic reticulum (ER)
are retained in the organelle or retrotranslocated
to the cytosol for proteasomal degradation.
whereas ERp72, a PDI-like protein, mediates its ER retention.
In vitro analysis revealed that PDI and ERp72 alter
CT’s conformation in a manner consistent with their roles
in retrotranslocation and ER retention. Moreover, we
found that PDI’s and ERp72’s opposing functions operate
on endogenous ER misfolded proteins. Thus, our data
identify PDI family proteins that play opposing roles in ER
quality control and establish an assay to further delineate
the mechanism of CT retrotranslocation.
Correspondence to Billy Tsai: email@example.com
Abbreviations used in this paper: BFA, brefeldin A; CT, cholera toxin; NEM,
N-ethylmaleimide; PDI, protein disulfi de isomerase; Tg, thyroglobulin.
JCB • VOLUME 173 • NUMBER 6 • 2006 854
Results and discussion
Retrotranslocation of CT
To study CT retrotranslocation, we developed an assay that
monitors the transport of the A and A1 subunits from the ER
into the cytosol, taking advantage of a semipermeabilized cell
assay that effi ciently separates cytosolic from ER proteins
(Le Gall et al., 2004). CT-intoxicated HeLa cells were treated
with 0.04% digitonin to permeabilize the plasma membrane
and were fractionated by centrifugation. The supernatant
should contain cytosolic proteins as well as ER–cytosol-
transported CT, whereas the pellet should contain the plasma
membrane, intracellular organelles (including the ER), and
toxin that did not undergo retrotranslocation. We tested the
purity of these fractions and found the ER resident protein
ERp57 to be entirely in the pellet (Fig. 1 A, second panel from
bottom; lanes 2, 4, and 6) and the cytosolic protein Hsp90 to
be mostly in the supernatant (Fig. 1 A, bottom; lanes 1, 3, and 5).
When cells were intoxicated with CT at 37°C, a portion of
the A1 subunit was found in the supernatant (Fig. 1 A, top;
compare lane 6 with 5), whereas the B subunit was absent in
this fraction (Fig. 1 A, second panel from top; compare odd
with even lanes) as expected. Typically, 15–30% of toxin and
<0.01% of ER resident proteins was detected in the superna-
tant (calculation not depicted), indicating that the presence of
the toxin in this fraction is not caused by ER leakage. This
range is likely caused by the variable effi ciency of the low
level of detergent used in this study in permeabilizing the
When cells were incubated with CT at 4 or at 37°C in the
presence of brefeldin A (BFA), which are conditions shown pre-
viously to block the arrival of CT to the ER (Fujinaga et al.,
2003), the A1 chain did not appear in the supernatant (Fig. 1 A,
top; compare lane 5 with 1 and 3). A fraction of the A1 peptide
in the pellet was generated after lysis, as cell permeabilization
in the presence of the alkylating reagent N-ethylmaleimide
(NEM) diminished the appearance of the A1 chain (Fig. 1 A,
compare lane 10 with 8). However, the level of A1 peptide in the
supernatant is similar regardless of whether NEM is present in
the lysis buffer (Fig. 1 A, compare lane 9 with 7). Thus, postly-
sis reduction of CT in the pellet does not trigger the release of
the A1 chain to the supernatant.
To further verify the assay, we tested the fractionation
pattern of a CT mutant whose A chain cannot be cleaved
because of a mutation at the cleavage site (R192H). Chloride
secretion triggered by the R192H mutant is attenuated
dramatically (Lencer et al., 1997); thus, we anticipated that
less of this toxin would arrive to the cytosol when compared
with the wild-type toxin. Indeed, when cells were incubated
with the R192H toxin, no toxin was found in the supernatant
(Fig. 1 B, compare lane 3 with 1).
CT-stimulated chloride secretion was previously shown to
be resistant to proteasome inactivation (Rodighiero et al., 2002),
suggesting that the toxin escapes proteasomal degradation in
the cytosol. As expected, proteasome inactivation with MG132,
which was confi rmed by the accumulation of polyubiquitinated
proteins (Fig. 1 C, compare lane 2 with 1), did not signifi cantly
alter the toxin level in the cytosol (Fig. 1 C, top; compare lane 5
with 3). The consistency of these results (Fig. 1) with previous
chloride secretion studies (Lencer et al., 1997; Rodighiero et al.,
2002) validates the use of this semipermeabilized assay as a tool
to study CT retrotranslocation directly.
Down-regulation of PDI-like proteins
We next tested whether PDI, an ER chaperone that unfolds the
A and A1 chains in vitro (Tsai et al., 2001), plays a role in the
Figure 1. Retrotranslocation of the CTA1 subunit.
(A) HeLa cells were incubated with CT for 90 min at 4 or
37°C with or without BFA or NEM. Cells were permea-
bilized, centrifuged, and the supernatant and pellet
fractions were separated, subjected to nonreducing
SDS-PAGE, and immunoblotted with the indicated
antibodies. CTA, CTA1, and CTB are 28, 22, and
11 kD, respectively. (B) As in A except a CT mutant,
R192H, was used. PDI is 58 kD. (C) As in A except
where indicated, cells were treated with MG132.
Polyubiquitinated proteins range from 100 to 300 kD.
White lines indicate that intervening lanes have been
PDI-LIKE PROTEINS FUNCTION IN ER QUALITY CONTROL • FORSTER ET AL.855
ER–cytosol transport of CT. Our approach was to down- regulate
PDI and other PDI-like proteins in cells using RNAi and test
their effect on CT retrotranslocation. Of the 14 known human
PDI-like proteins in the ER, we chose to down-regulate ERp72
and ERp57 as controls because they are expressed at similar
levels as PDI (unpublished data). PDI, ERp72, and ERp57 are
characterized by the presence of a CxxC sequence within their
thioredoxin domain (Fig. 2 A). PDI (PDI−), ERp72 (ERp72−),
and ERp57 (ERp57−) were down-regulated in cells separately
(Fig. 2 B, top); in each case, the expression of other PDI-like
proteins (Fig. 2 B, bottom three panels) was unaffected. These
results indicate that PDI, ERp72, and ERp57 expression can be
reduced effi ciently and specifi cally.
We asked whether down-regulation of the PDI-like pro-
teins elicited the unfolded protein response, a cellular stress
response in which ER misfolded protein accumulation triggers
the expression of ER chaperones such as BiP to alleviate protein
misfolding. We found that BiP expression was only up- regulated
marginally in PDI−, ERp72−, and ERp57− cells compared
with wild-type cells (Fig. 2 C, compare lanes 3, 5, and 7 with 1),
indicating that the lack of these proteins did not signifi cantly
induce the unfolded protein response. Moreover, the addition
of tunicamycin, a drug that blocks N-glycosylation and, thus,
causes the accumulation of misfolded proteins, induced BiP
expression in wild-type, PDI−, ERp72−, and ERp57− cells
(Fig. 2 C, compare lane 2 with 4, 6, and 8). We conclude that
down-regulation of the PDI family proteins did not globally
disrupt ER function.
PDI and ERp72 play opposing roles during
We assessed the effect of PDI down-regulation on the transport
of CT to the cytosol by examining the toxin level in the super-
natant after cell fractionation. When cells were incubated with
CT for 45 or 90 min, we found a decreased level of both the A
and A1 chains from PDI− cells when compared with wild-type
cells (Fig. 3 A, top; compare lane 2 with 1 and lane 4 with 3;
quantifi ed as shown in the graphs). These data indicate that PDI
facilitates toxin transport from the ER to the cytosol, which is
consistent with our hypothesis that PDI-dependent unfolding of
CT mediates the toxin’s retrotranslocation (Tsai et al., 2001).
We note that a low A subunit level also appeared in the supernatant.
This is unlikely to be caused by ER leakage, as ER markers and
CTB (CT B subunit) were absent from this fraction. Thus, it is
possible that although the A1 subunit is the preferred substrate
for retrotranslocation, the A chain is also transported, albeit
with lower effi ciency.
In contrast, the A and A1 peptide level in the supernatant
increased in ERp72− cells when compared with wild-type cells
(Fig. 3 A, top; compare lane 6 with lane 5 and lane 8 with 7;
quantifi ed as shown in the graphs), suggesting that ERp72 acts
to retain CT in the ER, a reaction that opposes PDI’s function.
The effects of down-regulating PDI and ERp72 on toxin trans-
port were also observed when NEM was present in the lysis
buffer (unpublished data). In ERp57− and wild-type cells, the
toxin level in the supernatant was similar (Fig. 3 A, top; com-
pare lane 10 with 9 and lane 12 with 11). We conclude that the
opposing effects of PDI and ERp72 on CT transport are specifi c
and are unlikely the result of a general disruption of ER chaper-
To show that PDI and ERp72 did not affect CT traffi cking
to the ER, we used a variant of CT harboring consensus motifs
for N-glycosylation on the B subunit (glycosylated CT [CT-GS]).
Modifi cation by N-glycosylation, detected by a molecular mass
increase in the B subunit, indicates the toxin’s arrival to the ER.
This toxin was previously used to show that CT is transported
as a holotoxin from the plasma membrane to the ER (Fujinaga
et al., 2003). Thus, N-glycosylation of the B subunit indicates
the arrival of the A subunit to the ER. When cells were incu-
bated with CT-GS at 37°C and the cell lysate was analyzed
by SDS-PAGE followed by immunoblotting with an antibody
against the B subunit, a band larger than the nonglycosylated B
subunit was observed (Fig. 3 B, lane 2). This band was shown
previously to be N-glycanase sensitive (Fujinaga et al., 2003)
and, thus, represents glycosylated B subunits. Glycosylated B
subunits were not detected in cells incubated with the toxin at
Figure 2. Down-regulation of PDI-like proteins. (A) Structural
organization of PDI, ERp72, and ERp57. (B) PDI, ERp72,
ERp57, and ERp29 protein levels were examined in wild-type
(WT), PDI−, ERp72−, and ERp57− cells. (C) Wild-type, PDI−,
ERp72−, and ERp57− cells were incubated with 2 μg/ml
tunicamycin (TM) for 8 h, and BiP expression was assessed
by SDS-PAGE and immunoblot analysis. BiP is 78 kD.
JCB • VOLUME 173 • NUMBER 6 • 2006 856
4 or 37°C in the presence of BFA (Fig. 3 B, lanes 1 and 3).
These experiments validate the use of CT-GS in monitoring
A-subunit arrival to the ER. Signifi cantly, the similar level of
glycosylated B subunits found in wild-type, PDI−, and ERp72−
cells (Fig. 3 B, lanes 4–6) indicate that PDI and ERp72 act on
the toxin only after it reaches the ER. Furthermore, PDI and
ERp72 down-regulation does not affect CT reduction (Fig. 3 C,
compare lane 2 with 1 and lane 4 with 3).
Upon reaching the cytosol, the catalytic A1 subunit ADP
ribosylates the Gαs protein, activating adenylate cyclase that, in
turn, generates cAMP. Therefore, we measured the CT-induced
cAMP response in PDI− and ERp72− cells. The CT-triggered
cAMP level in PDI− cells was 50% lower than in wild-type cells
(Fig. 3 D, left). Forskolin, which stimulates adenylate cyclase
directly, elicited a similar cAMP level in wild-type and PDI−
cells (unpublished data), indicating that PDI down-regulation
did not affect adenylate cyclase. In contrast, the CT-induced
cAMP level in ERp72− cells was 40% higher than in wild-type
cells (Fig. 3 D, right). This result was normalized against the
forskolin-induced cAMP response, as forskolin also triggered a
higher cAMP response in ERp72− cells. The higher forskolin-
induced cAMP response in these cells is likely caused by a con-
comitant increase in cell surface expression of adenylate cyclase
(unpublished data). Thus, the cAMP data are consistent with the
ER–cytosol transport assay—namely that PDI retrotranslocates
CT, whereas ERp72 retains the toxin in the ER.
Opposing effects of PDI and ERp72
on CT conformation
PDI’s ability to unfold CT (Tsai et al., 2001) is consistent with
its role in toxin retrotranslocation. ERp72’s role in facilitating
ER retention suggests that it may recognize CT as a misfolded
protein and attempt to “refold” the toxin’s structure. Incubation
of purifi ed PDI but not the control protein BSA with CTA
(CT A subunit) was shown previously to render the A and A1
chains sensitive to trypsin digestion (Fig. 4 B, compare lane
2 with 1; Tsai et al., 2001), indicating that PDI unfolded the
toxin. Purifi ed ERp72 (Fig. 4 A) did not cause the toxin to
Figure 3. PDI and ERp72 exert opposing effects on
CT retrotranslocation. (A, top) Wild-type (WT), PDI−,
ERp72−, and ERp57− cells were incubated with CT
for 45 or 90 min, permeabilized, and the supernatant
fraction was analyzed as in Fig. 1. (bottom) The inten-
sities of the CTA and CTA1 bands in the supernatant
were quantifi ed. Graphs show the mean ± SD (error
bars) of two to four experiments. (B) Wild-type, PDI−,
and ERp72− cells were incubated with CT-GS, and the
cell lysate was subjected to SDS-PAGE followed by
immunoblotting with an anti-CTB antibody. Nonglyco-
sylated and glycosylated CTB are 17 and 21 kD, re-
spectively. (C) Whole cell lysates from CT-intoxicated
wild-type, PDI−, and ERp72− cells were prepared in
the presence of NEM and subjected to immunoblot-
ting with CTA antibody. (D) Wild-type, PDI−, and
ERp72− cells were incubated with CT for 45 or 90 min,
and the cAMP level was measured by a cAMP
Biotrak Enzyme Immunoassay System (GE Healthcare).
Means ± SD of two to four experiments are shown.
White lines indicate that intervening lanes have been
PDI-LIKE PROTEINS FUNCTION IN ER QUALITY CONTROL • FORSTER ET AL.857
become protease sensitive (not depicted). We now fi nd that
regardless of PDI’s presence, a higher trypsin concentration
rendered the A and A1 chains sensitive to degradation (Fig. 4 B,
compare lanes 3 and 4 with 1). However, under this condi-
tion, ERp72 protected the toxin from degradation (Fig. 4 B,
compare lane 5 with 3 and 4), indicating that ERp72 alters
CT’s conformation to render it more folded and compact.
Alternatively, it is possible that ERp72 protects the toxin against
protease digestion by either stabilizing the native conformation
of the toxin or interacting with CT in a similar manner as PDI
but with reduced effi ciency. Nonetheless, PDI and ERp72’s
opposing effects on CT’s conformation likely refl ect their roles
in facilitating retrotranslocation and ER retention during ER
A general role of PDI and ERp72 in quality
control of ER misfolded proteins
Do the opposing functions of PDI and ERp72 represent a gen-
eral mechanism operating in the ER? We developed a method
to measure the bulk arrival of misfolded proteins from the ER
to the cytosol. Upon reaching the cytosolic surface of the ER
membrane, ER misfolded polypeptides are polyubiquitinated
before being targeted for proteasomal degradation (for review
see Tsai et al., 2002). Consequently, proteasome inactivation
ought to lead to the accumulation of polyubiquitinated ER mis-
folded proteins as well as cytosolic proteins. The effect of ER
resident protein down-regulation on the accumulation of total
polyubiquitinated proteins should, therefore, refl ect their role in
the ER–cytosol transport process. We measured the level of
Figure 4. Opposing effects of PDI and ERp72 on the confor-
mation of CT. (A) His-tagged ERp72 protein was purifi ed from
bacteria and analyzed by SDS-PAGE followed by Coomassie
staining or immunoblotting with an antibody against ERp72.
(B) CTA was incubated with BSA, PDI, or ERp72 followed by
the addition of trypsin. Samples were subjected to SDS-PAGE
followed by immunoblotting with an anti-CTA antibody.
Figure 5. A general role of PDI and ERp72 in
the retrotranslocation of ER misfolded proteins.
(A, top) Wild-type (WT), PDI−, or ERp72− cells
were treated with MG132, and the cell lysate
was subjected to SDS-PAGE followed by immuno-
blotting with the indicated antibodies. (bottom)
The total ubiquitin signal intensity was measured.
Graphs show the mean ± SD (error bars)
of three to fi ve experiments. (B) Wild-type,
PDI−, or ERp72− cells were treated with TNFα,
and the lysate was subjected to SDS-PAGE
followed by immunoblotting with an antibody
against IκBα. IκBα is 39 kD. (C) CHO cells
stably expressing cogTg (CHO-P) and PDI
(CHO-PDI) or ERp72 (CHO-ERp72) were pulse
labeled with [35S]methionine and chased for
the times indicated. CogTg was immunopre-
cipitated from the cell lysate with anti-Tg anti-
body and analyzed by SDS-PAGE. Data show
quantifi cation of the radioactive cogTg band
intensity. (D) Diagram depicting the similarity
between the quality control system in the ER
and cytosol. See last paragraph of Results and
discussion. White lines indicate that interven-
ing lanes have been spliced out.
JCB • VOLUME 173 • NUMBER 6 • 2006 858
polyubiquitinated proteins and found that incubation of cells
with the proteasome inhibitor MG132 resulted in an increase of
polyubiquitinated proteins (Fig. 5 A, compare lane 3 with 1 and
lane 7 with 5). However, in PDI− cells, less MG132-induced
polyubiquitinated proteins appeared when compared with wild-
type cells (Fig. 5 A, compare lane 4 with 3; quantifi ed as shown
in the graphs), whereas in ERp72− cells, more polyubiquitinated
proteins were present (Fig. 5 A, compare lane 8 with 7; quanti-
fi ed as shown in the graphs). Although the source of the poly-
ubiquitinated proteins is unclear, they likely originated from the
ER. Quantifi cation showed that although PDI down-regulation
reduced the total level of polyubiquitinated proteins by ?40%,
a signifi cant portion (?60%) was unaffected; the unaffected
polyubiquitinated proteins are presumably cytosolic proteins.
We then asked whether cytosolic ubiquitination reactions
are affected nonspecifi cally by PDI and ERp72 down- regulation.
IκBα is a cytosolic protein that is polyubiquitinated in response
to TNFα stimulation before being degraded by the proteasome
(for review see Karin and Ben-Neriah, 2000). Indeed, we found
that IκBα was degraded with similar effi ciency upon TNFα
treatment in wild-type, PDI−, and ERp72− cells (Fig. 5 B, com-
pare lanes 4–6 with 1–3), suggesting that IκBα ubiquitination
was not disrupted by PDI and ERp72 down-regulation. These
fi ndings indicate that PDI facilitates the retrotranslocation
of some ER misfolded proteins, whereas ERp72 mediates their
We further characterized this effect on a specifi c ER mis-
folded substrate. Thyroglobulin (Tg), the thyroid prohormone,
is synthesized and folded in the ER before being secreted. A
mutant form of Tg, cogTg, was shown previously to undergo
retrotranslocation and proteasomal degradation (Tokunaga et al.,
2000). PDI overexpression in CHO cells stably expressing
cogTg (CHO-PDI) stimulated the rate of cogTg degradation
when compared with parental CHO cells expressing cogTg
(CHO-P; Fig. 5 C, compare open with closed squares). In con-
trast, ERp72 overexpression in CHO cells (CHO-ERp72) de-
creased the rate of cogTg degradation (Fig. 5 C, compare open
squares with circles). These results suggest that PDI stimulates
the ER–cytosol transport of cogTg, whereas ERp72 retains it
in the ER. We conclude that PDI and ERp72’s opposing roles
operate not only on CT retrotranslocation but more generally
for ER misfolded proteins.
The role of PDI and ERp72
in retrotranslocation and ER retention
How do chaperones that belong to the same family serve oppo-
site functions? PDI possesses two thioredoxin domains contain-
ing the redox-active CxxC motif (Fig. 2 A, CxxC box) and two
thioredoxin-like domains without the CxxC motif (Fig. 2 A,
white rectangles), whereas ERp72 contains three redox-active
and two redox-inactive domains (Fig. 2 B). Their major differ-
ence is that PDI contains an additional c domain that is absent
in ERp72. It is possible that this domain participates in the un-
folding of polypeptides. Interestingly, PDI but not ERp72 was
implicated in the unfolding of the nonnative structure of bovine
pancreatic trypsin inhibitor during disulfi de bond rearrange-
ment (Weissman and Kim, 1993; Satoh et al., 2005), supporting
our conclusion that PDI specifi cally unfolds misfolded substrates.
PDI has also been shown to facilitate the proteasomal degradation
of ER misfolded proteins in yeast (Gillece et al., 1999), a process
that presumably requires substrate unfolding and retrotrans-
location. Our fi ndings that ERp72 mediates the ER retention
of CT and misfolded proteins are consistent with recent data
demonstrating that ER retention of a misfolded secretory
(Cotterill et al., 2005) and a transmembrane protein (Sorensen
et al., 2005) is coincident with binding to ERp72.
ERp72 and PDI’s dedicated roles in ER quality control
appear analogous to the cytosolic chaperone system that func-
tions in protein folding and degradation (Fig. 5 D; McClellan
et al., 2005). In this case, the chaperones Hsp70 and TriC assist
in protein folding, whereas the Hsp70–Hsp90–Sti1–Sse1 com-
plex mediates protein degradation. As PDI can promote protein
folding and unfolding reactions, it is possible that PDI cofactors
exist to control these opposing processes, which is similar to
Materials and methods
Antibodies against PDI, BiP, and Hsp90 were purchased from Santa Cruz
Biotechnology, Inc. Antibodies against ERp72 were obtained from Stress-
Gen Biotechnologies, antibodies against ubiquitin were purchased from
Zymed Laboratories, and antibodies against IκBα were obtained from
Biolegend. CT A and B antibodies and CT-GS were provided by the Lencer
laboratory, the Tg antibodies were obtained from the Arvan laboratory,
and the CT R192H mutant was provided by R. Holmes (University of Colo-
rado, Boulder, CO). Hsp27, ERp57, and ERp29 antibodies were gifts from
M. Welsh (University of Michigan, Ann Arbor, MI), S. High (University of
Manchester, Manchester, England), and S. Mkrtchian (Karolinska Institutet,
Stockholm, Sweden), respectively. CT, CTA, and PDI were purchased from
Calbiochem. Mouse ERp72 cDNA was subcloned into pQE30, and the
protein was purifi ed using a Ni–nitrilotriacetic acid agarose column.
PDI-specifi c (5′-G A C C T C C C C T T C A A A G T T G T T -3′) siRNA was synthesized
by Ambion, and ERp72- (5′-C A A G C G U U C U C C U C C A A U U T T -3′) and
ERp57-specifi c (5′-U G A A G G U G G C C G UGAAUUATT-3′) siRNAs were syn-
thesized by Invitrogen. 10 nM duplexed siRNA was transfected into HeLa
cells using Oligofectamine (Invitrogen) according to the manufacturer’s
protocol. Protein expression was assessed by SDS-PAGE and immunoblot
analysis. Experiments were initiated 48 (ERp57) or 72 h (PDI and ERp72)
Cells were incubated with 10 nM CT in HBSS at 37°C for 45 or 90 min.
For permeabilization, 2 × 106 cells were resuspended in 100 μl of 0.04%
digitonin in HCN buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 2 mM
CaCl2, and protease inhibitors) with or without 10 mM NEM, incubated on
ice for 10 min, and centrifuged at 16,000 g for 10 min. The supernatant
was removed, and the pellet was washed with PBS and resuspended in
100 μl of the original buffer. Fractions were analyzed by nonreducing
SDS-PAGE and immunoblot analysis. Where indicated, cells were treated
with 2 μM MG132 for 90 min.
N-glycosylation of CTB
HeLa cells were incubated with 50 nM CT-GS in HBSS for 3 h at 4 or 37°C
in the presence or absence of 5 μg/ml BFA. Cells were harvested in TN
lysis buffer (1% Triton X-100, 1.75% n-octyl-B-/d-glucopyranoside, 10 mM
Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, and protease inhibitors), and
the lysates were analyzed for the presence of glycosylated CTB.
A cAMP enzyme immunoassay system (GE Healthcare) was used to quan-
tify cAMP synthesis induced by 10 nM CT or 50 μM forskolin in HBSS.
Samples were assayed in duplicate, and the mean optical density was
PDI-LIKE PROTEINS FUNCTION IN ER QUALITY CONTROL • FORSTER ET AL.859 Download full-text
used to calculate the cAMP level/well. The cAMP response was deter-
mined by dividing the cAMP level in CT- or forskolin -treated cells by the
cAMP level in unstimulated cells. Forskolin induced a sevenfold higher
cAMP response in ERp72− cells than in wild-type cells. Results are reported
as a percentage of the wild-type CT-induced cAMP response normalized to
the forskolin-induced cAMP response.
Trypsin sensitivity assay
Purifi ed CTA was incubated with 1 mM glutathione, 2 μg/ml BSA, 2 μg/ml
PDI, or 0.2 μg/ml ERp72 for 30 min at 37°C. 100 or 300 μg/ml trypsin
was added to the samples for 30 min at 4°C. Samples were analyzed by
nonreducing SDS-PAGE followed by immunoblot analysis.
Accumulation of polyubiquitinated proteins
Cells were incubated with or without 20 μM MG132 for 30 min and lysed
in a buffer containing 1% Triton-X, 10 mM NEM, 30 mM Hepes, pH 7.4,
150 mM NaCl, 5 mM EDTA, and protease inhibitors. Cleared lysates were
analyzed by 4–20% SDS-PAGE followed by immunoblot analysis. To
monitor ubiquitin-dependent degradation of IκBα, cells were treated with
10 ng/ml TNFα for 15 min, and the cell lysate was analyzed for the pres-
ence of IκBα.
The cogTg degradation rate was analyzed as previously described
(Tokunaga et al., 2000).
We thank Tom Rapoport for critical review of the manuscript.
B. Tsai is a Biological Scholar at the University of Michigan.
Submitted: 8 February 2006
Accepted: 11 May 2006
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