Polycystin-2 is regulated by endoplasmic
Genqing Liang1, Qiang Li1, Yan Tang1, Koichi Kokame2, Tadashi Kikuchi2, Guanqing Wu3
and Xing-Zhen Chen1,?
1Membrane Protein Research Group, Department of Physiology, Faculty of Medicine and Dentistry, University of
Alberta, Edmonton, Alberta, Canada T6G 2H7,2National Cardiovascular Centre Research Institute, Suita, Osaka
565-8565, Japan and3Department of Medicine, Vanderbilt University, Nashville, TN 32232-0275, USA
Received November 23, 2007; Revised and Accepted December 26, 2007
Endoplasmic reticulum(ER)-associated degradation (ERAD) is an essential process for cell homeostasis and
remains not well understood. During ERAD, misfolded proteins are recognized, ubiquitinated on ER and sub-
sequently retro-translocated/dislocated from ER to the 26S proteasome in the cytosol for proteolytic elimin-
ation. Polycystin-2 (PC2), a member of the transient receptor potential superfamily of cation channels, is a Ca
channel mainly located on ER and primary cilium membranes of cells. Mutations in PC2 are associated with
autosomal dominant polycystic kidney disease (ADPKD). The cellular and molecular mechanisms underlying
the PC2-associated pathogenesis remain unclear. Here we show that PC2 degradation is regulated by the
ERAD pathway through the ubiquitin–proteasome system. PC2 interacted with ATPase p97, a well-known
ERAD component extracting substrates from ER, and immobilized it in perinuclear regions. PC2 also inter-
acted with Herp, an ubiquitin-like protein implicated in regulation of ERAD. We found that Herp is required
for and promotes PC2 degradation. ER stress accelerates the retro-translocation of PC2 for cytosolic degra-
dation, at least in part through increasing the Herp expression. Thus, PC2 is a novel ERAD substrate. Herp
also promoted, to varied degrees, the degradation of PC2 truncation mutants, including two pathogenic
mutants R872X and E837X, as long as they interact with Herp. In contrast, Herp did not interact with, and
has no effect on the degradation of, PC2 mutant missing both the N- and C-termini. The ERAD machinery
may thus be important for ADPKD pathogenesis because the regulation of PC2 expression by the ERAD path-
way is altered by mutations in PC2.
The endoplasmic reticulum (ER) plays a crucial role in the
protein folding and quality control (1). Conditions disrupting
the ER homeostasis can cause unfolded protein accumulation,
which triggers ER stress and unfolded protein response (UPR)
(2). In a process termed ER-associated degradation (ERAD),
unfolded proteins are delivered into the cytosol where they
are ultimately destroyed by the 26S ubiquitin–proteasome
system (1,3,4). Several steps are involved in protein degra-
dation by ERAD, including the recognition of a target sub-
strate on the ER, retro-translocation of the substrate to the
cytosol, and transferring of the substrate to the 26S protea-
some for final destruction. Efforts to elucidate the pathway
have identified some factors involved in ERAD in mammals.
E3 ubiquitin ligases, such as HRD1 and gp78, have been
shown to mediate the ubiquitination of ERAD substrates
(5,6). A protein complex formed by AAA ATPase p97 and
its cofactors Ufd1 and Npl4 retro-translocates ubiquitinated
substrates from the ER to the cytosol (7–9). Recently, mem-
brane proteins Derlin-1, VIMP, Herp and signal peptide pepti-
dase were demonstrated to mediate the extraction of some
complex (MHC), CD3-delta and cystic fibrosis transmembrane
conductance regulator (10–14). Identification and characteriz-
ation of new substrates and components of the ERAD pathway
will bring novel insights into the molecular mechanism under-
lying ERAD, which remains largely unclear.
?To whom correspondence should be addressed. Tel: þ1 780 492 2294; Fax: þ1 780 492 8915; Email: firstname.lastname@example.org
# 2008 The Author(s)
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Human Molecular Genetics, 2008, Vol. 17, No. 8
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Polycystin-2 (PC2) with a molecular mass of ?110 kDa is a
non-selective cation channel permeable to Ca. It is mainly
localized on the ER membrane as a Ca release channel
(15,16), on epithelial primary cilia membrane of renal
tubular and embryonic nodal cells as part of flow sensor
(17,18), and may also be present on the plasma membrane
for Ca entry (19). The channel is involved in multiple signal
transduction pathways such as Wnt, cAMP and Ras/MAPK
(20). Mutations in PC2 are associated with autosomal domi-
nant polycystic kidney disease (ADPKD), abnormalities in
vascular structure/function and organ left-right asymmetry
development (20). At the cellular level, ADPKD is associated
with altered cell proliferation, adhesion and differentiation.
However, the cellular regulation of PC2 remains largely
unknown. In this study, we examined how PC2 and its
mutants interact with components of ERAD, in particular,
Herp, and how their degradation is regulated by the ERAD
PC2 is poly-ubiquitinated
To test whether PC2 is a potential substrate of ERAD, we first
examined the possible in vivo ubiquitination of the ER Ca
channel protein. For this end, mouse inner medullary collect-
ing duct (IMCD) cells were treated with proteasome inhibitor
MG-132. Immunoprecipitates of the cell extracts with an
anti-PC2 antibody (21,22) were immunoblotted by an anti-
ubiquitin antibody. Indeed, ubiquitinated PC2 could be
precipitated and the amount of the ubiquitinated fraction was
significantly increasedin cells
(Fig. 1A, left panel). Reciprocally, MG-132-induced accumu-
lation of ubiquitinated PC2 was also observed in the immuno-
precipitate obtained using the anti-ubiquitin antibody (Fig. 1A,
right panel). These data indicate that PC2 is ubiquitinated in
vivo and that the ubiquitinated PC2 proteins accumulate
when proteasome-dependent degradation is inhibited. We
also examined ubiquitination of the over-expressed PC2 in
HeLa cells and obtained similar results (Fig. 1B). We then per-
formed similar experiments using HeLa cells transiently
expressing GFP-ubiquitin or GFP to examine the effect of
ubiquitin on PC2. We found that, similar to the effect of
MG-132, over-expression of ubiquitin increased the pro-
portion of ubiquitinated PC2 and also leads to a decreased
PC2 level (Fig. 1C), suggesting that PC2 degradation is
promoted by ubiquitin. Thus, our data indicate that PC2
is ubiquitinated, consistent with a recent report that PC2 is
likely ubiquitinated (23).
PC2 degradation is proteasome-dependent
We next explored further documentations for the involvement
of the 26S proteasome in PC2 degradation. For this we inhib-
ited protein synthesis by cycloheximide (CHX) in native HeLa
cells and Madin-Darby canine kidney (MDCK) stably expres-
sing GFP-tagged human PC2. Eight hours after treatment with
CHX, PC2 protein was decreased by 62+8% (N ¼ 3)
(Fig. 2A) and 68+7% (N ¼ 3), respectively (Fig. 2B). In
cells incubated with proteasome inhibitor lactacystin or
MG-132, the degradation of PC2 was suppressed. Tunicamycin
(Tm) is a known ER stress inducer that enhances the degra-
dation of ERAD substrates in both yeast and mammals
(24–26). We found that the endogenous PC2 level is indeed
reduced in IMCD and HeLa cells incubated with Tm, but
not in cells incubated with DMSO (as a vehicle control)
(Fig. 2C and D). Because both the mRNA level and protein
synthesis of PC2 were not significantly affected by Tm, as
assessed by RT–PCR and
tively (data not shown), this result indicates that Tm promotes
PC2 degradation. In this experiment, the expression of immu-
noglobulin heavy-chain binding protein (BiP, also called
GRP78), an ER chaperone protein activated during the ER
stress, was used as a marker of the Tm-induced ER stress.
We also examined the effect of Tm on GFP-tagged human
PC2 stably expressed in MDCK cells and found similar
results (Fig. 2E). The effect of Tm on PC2 degradation was
confirmed by another ER stress inducer dithiothreitol (DTT)
(data not shown). As expected, the Tm-promoted degradation
was inhibited by proteasome inhibitors MG-132 and lactacys-
tin (Fig. 2C–E). Together with the accelerated PC2 degra-
dation promoted by ubiquitin expression (Fig. 1C), our data
indicate that PC2 turnover is regulated by the ubiquitin–
35S-methionine labeling, respec-
PC2 interacts with components of the ERAD pathway
Given the ER membrane localization of PC2 we reasoned that
ERAD components might be involved in the PC2 degradation
through the 26S ubiquitin–proteasome system. Numerous
(p97-Ufd1-Npl4) plays a key role in the transport of ERAD
substrates from the ER to the cytosol (7–9,27,28). To test a
potential association of PC2 with p97, we immunoprecipitated
the extracts of IMCD cells using the anti-PC2 antibody. We
found that p97 was indeed co-precipitated with PC2
(Fig. 3A, left panel, lanes 2 versus 1) and that MG-132 sub-
stantially increased the amount of the precipitated p97
(Fig. 3A, left panel, lanes 3 versus 2), which should be due
at least in part to an increased amount of ubiquitinated PC2
(Fig. 1). This result is paralleled by a previous report that pro-
teasome inhibitors enhance the level of the association of sub-
strate MHC with the ERAD component Derlin-1 that mediates
substrate retro-translocation (10). In negative control experi-
ments we found that ER membrane proteins ATF6a (29)
and Sec61a, a subunit of the Sec61 translocon complex
(30), are not co-precipitated with PC2 (Fig. 3A). Further,
p97 was not immunoprecipitated by the PC2 antibody using
PC2-knockout mouse embryos (31) or HeLa cells in which
PC2 expression was substantially reduced (to 20%) by small
interference RNA (siRNA) (Fig. 3B). The data on the PC2–
p97 specific interaction in vivo thus support that PC2 is an
Immunofluorescence microscopy was performed using
MDCK cells stably expressing GFP-PC2 to examine the
co-localization of PC2 and p97. GFP-PC2 was mainly distri-
buted to perinuclear regions typical of ER localization,
whereas GFPalone had
distribution (Fig. 3C, panels 2 versus 6). Interestingly, in
adifferent pattern of
1110Human Molecular Genetics, 2008, Vol. 17, No. 8
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Figure 1. PC2 is poly-ubiquitinated in vivo. (A) Ubiquitination of polycystin-2 (PC2). Inner medullary collecting duct (IMCD) cells were treated with 10 mM
MG-132 for 4 h, followed by protein preparation and immunoprecipitation (IP) with an anti PC2 antibody (1A11) or ubiquitin (Ub) antibody. Precipitated PC2
and an input (10%) of poly-ubiquitinated PC2 were detected by immunoblotting with the PC2 or ubiquitin antibody. (B) Ubiquitination of GFP-PC2. Extracts of
HeLa cells transfected with pEGFP-PC2 or pEGFP (5 mg in 100-mm plates) for 40 h, followed by the MG-132 (10 mM) treatment for 4 h, were subjected to IP
with a GFP (gaot) antibody and immunoblotting with the ubiquitin or a GFP (rabbit) antibody. Cell extracts (Input, 30%) was used for immunoblotting with the
ubiquitin or GFP (goat) antibody. (C) Effect of over-expression of ubiquitin on PC2 ubiquitination. Extracts of HeLa cells transfected with pEGFP-ubiquitin or
vector pEGFP (5 mg in 100-mm plates) for 40 h, followed by the MG-132 (10 mM) treatment for 4 h, were subjected to IP with the PC2 antibody and immuno-
blotting using the ubiquitin or PC2 antibody. Cell extracts (Input, 10%) were utilized for detecting cellular ubiquitination, GFP-ubiquitin and PC2 expression by
immunoblotting with the ubiquitin, GFP (goat) and PC2 antibodies, respectively. b-actin was used as a loading control.
Human Molecular Genetics, 2008, Vol. 17, No. 81111
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cells, the p97 distribution pattern was similar to that of
PC2 (Fig. 3C). These data together with the complexing
between p97 and PC2 (Fig. 3A and B) indicate that the
PC2–p97 interaction may have immobilized p97 in the peri-
nuclear regions, while leaving its steady state level unaffected
Since ERAD substrates are eventually destroyed by the 26S
proteasome after their retro-translocation from the ER to the
cytosol, we also examined potential association of PC2 with
the proteasome. We found that S12, a subunit of the 19S pro-
teasome believed to recognize ubiquitinated substrates for
degradation by the 20S proteasome (32), is in the same
complex as PC2, and like p97, its complexing with PC2 sub-
stantially augmented in the presence of MG-132 (Fig. 3A, left
panel, lanes 3 versus 2). Similar to p97, S12 was not
embryos or PC2-knockdown cells (Fig. 3B). The data indicate
that poly-ubiquitinated PC2 is indeed retro-translocated from
the ER to the cytosol where it is recognized by the 19S protea-
some for degradation.
cells, butnotin GFP-expressing
Herp associates with PC2 and regulates its degradation
Herp (or Mif1) is a single-transmembrane ER membrane
protein with its N- (amino acids 1–285) and C-termini
(amino acids 308–391) putatively localized to the cytosol,
and possesses an ubiquitin-like (UBL) domain (amino acids
14–85) on the N-terminus (33,34). Since Herp is highly
up-regulated during UPR and known to be implicated in
ERAD (12,33,35,36), we examined whether Herp is involved
in the PC2 degradation. For this HeLa cells were transfected
with vector pcDNA3.1 or pcDNA3.1-mHerpf in which the
Herp N- and C-termini are tagged with Myc and FLAG,
respectively (33,37). It was reported that the full-length
mHerpf is ?61 kDa and resides on the ER (33). In cells over-
expressing Herp PC2 degradation accelerated (Figs 4A and
5A, lanes 3 versus 1). Multiple cleaved N-terminal fragments,
at 50, 37, 30 and 15 kDa, were present in the cells (as detected
by a Myc antibody), of which the 50-kDa fragment has com-
parable high abundance as the full-length Herp (Fig. 4A, blot
Myc, lanes 3 and 4). Fractionation analyses showed that the
50-kDa proteolytic fragment of Herp associates with the ER
Figure 2. Polycystin-2 (PC2) degradation is regulated by proteasome system. (A) and (B) Effects of proteasome inhibitors on PC2 degradation. HeLa cells (A)
and stable Madin-Darby canine kidney (MDCK) cells expressing GFP-PC2 (B) were treated by 50 mg/ml cycloheximide (CHX) (Sigma-Aldrich Canada) along
with 10 mM proteasome inhibitor lactacystin or MG-132 (Sigma-Aldrich Canada) for 8 h, followed by immunoblotting of the resulting cell extracts with the PC2
antibody. (C), (D) and (E) Effect of proteasome inhibitors on the Tm-induced degradation of PC2. Inner medullary collecting duct (IMCD) (C), HeLa (D) and
stable MDCK cells expressing GFP-PC2 (E) were treated by 2 mg/ml Tm with or without 10 mM MG-132 or lactacystin for 8 h, followed by immunoblotting of
cell extracts with antibodies, as indicated. Arrow in (E) suggests the unglycosylated form of GFP-PC2. The expressions of BiP were used as an indication of
unfolded protein response (UPR).
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membrane (Fig. 4B). As a method control, IP3R-1, a subunit
of the ER IP3R Ca channel complex (38), was purified from
the membrane fraction. In contrast, only the full-length Herp
was detected with an antibody against the C-terminal FLAG
(Fig. 4A, blot FLAG, lanes 3 and 4), indicating that removal
of N-terminal fragments makes the rest of the protein
(tagged with FLAG) unstable. Our data suggest that a frag-
ment of ?15 kDa at the Herp N-terminal end, that contains
the UBL domain (amino acids 14–85), is important for the
protein stability. Next, we tested possible association of PC2
with Herp in these HeLa cells. Indeed, both the full-length
and the 50-kDa fragment, but not smaller fragments
Figure 3. Polycystin-2 (PC2) interacts with p97 and the 26S proteasome. (A) Immunoprecipitates by PC2 were prepared similarly as in Figure 1A and used for
immunoblotting with a p97, S12 or ATF6a antibody. Cell extracts (Input, 10%) were utilized for detecting p97, S12, Sec61a and ATF6a by immunoblotting. (B)
Immunoprecipitates using a PC2 antibody were obtained from extracts of E13.5 embryos of PC2 knockout (2/2) or wild type (þ/þ) mice, and of HeLa cells
with PC2 or control (Ctrl) small interference RNA (siRNA). These precipitates were then used for immunoblotting with a p97 or S12 antibody. Mouse tissue and
HeLa cell extracts (Input, 10%) were utilized to detect p97 and S12 by immunoblotting. (C) Co-localization between PC2 and p97 determined by immunofluor-
escence. Madin-Darby canine kidney (MDCK) cells stably expressing GFP-PC2 or GFP were stained with the p97 antibody (panels 3 and 7). PC2 was monitored
by GFP (panels 2 and 6). Cells were also stained with 4,6-diamidino-2-phenylindole (DAPI) (panels 1 and 5). (D) Effect of over-expressed PC2 on the expression
of the endogenous p97 in MDCK cells. Proteins extracted from MDCK cells stably expressing GFP-PC2 or GFP were subjected to immunoblotting with the p97
antibody. Lysis buffer was used as a negative control.
Human Molecular Genetics, 2008, Vol. 17, No. 8 1113
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Figure 4. Herp interacts with polycystin-2 (PC2) on the endoplasmic reticulum (ER) membrane. (A) Cleavage of Herp. Extracts of HeLa cells transfected with
pcDNA3.1-mHerpf or vector pcDNA3.1 were subjected to immunoblotting with an FLAG or Myc (rabbit) antibody to detect the full-length Herp (blots FLAG
and Myc) and cleaved Herp (blot Myc). The bands with the largest molecular weight indicated by an arrow may correspond to a non-specific signal as they are
present in control cells as well (also see panel B). (B) Solubility of cleaved fragments of Herp. Membrane and cytosolic fractions were separated from the lysates
of HeLa cells transiently transfected with pcDNA3.1-mHerpf or the pcDNA3.1 vector (5 mg in 100-mm dishes) for 40 h. Full-length and cleaved fragments of
Herp were detected by immunoblotting with the Myc (rabbit), IP3R-1 or b-actin antibody. (C) Complexing between Herp and PC2 determined by
co-immunoprecipitation (IP). IP with the PC2 antibody using cell extracts in (A) was performed as in Figure 1A. Immunoprecipitates were blotted with the
FLAG, Myc (rabbit), or PC2 antibody. (D) Confirmation of the specific interaction of PC2 with Herp. Immunoprecipitates from the extracts used in (A)
with the Myc (rabbit) antibody were immunoblotted with the PC2 or Myc (mouse) antibody.
1114 Human Molecular Genetics, 2008, Vol. 17, No. 8
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(perhaps due to their lower abundance), of Herp were found to
interact with PC2 (Fig. 4C, blots FLAG and Myc, lanes 3 and
4). Reciprocally, PC2 was co-immunoprecipitated with Herp
and the level of the Herp-associated PC2 seemed to increase
by MG-132 (Fig. 4D, blot PC2, lanes 4 versus 3), presumably
by delaying the retro-translocation of PC2 from the ER. Of
note, a band corresponding to a size slightly smaller than
that of the full-length PC2 was present in the precipitates of
Figure 5. Herp promotes polycystin-2 (PC2) degradation. (A) Effect of Herp over-expression on PC2 degradation. HeLa cells were transfected with
pcDNA3.1-mHerpf or pcDNA3.1 vector (1 mg in 35-mm dishes) for 40 h before immunoblotting. Treatment with Tm (8 h) was used as a positive control.
(B) Effect of Herp knockdown on PC2 degradation. HeLa cells were transfected with Herp small interference RNA (siRNA) or control siRNA (Ctrl) (20 ml
of 20 mM in 35-mm dishes) for 40 h before immunoblotting. Treatment with Tm (8 h) was used to induce PC2 degradation. (C) Effects of Herp on the degra-
dation of PC2 mutants. Extracts of HeLa cells co-transfected with plasmid GFP-PC2 or a PC2 mutant, and with pcDNA3.1-mHerpf (þ) [or vector pcDNA3.1
(2), as a control] were subjected to immunoblotting with a GFP, FLAG or b-actin antibody. (D) Interaction of PC2 mutants with Herp. Extracts of HeLa cells
co-transfected with pcDNA3.1-mHerpf and with pEGFP, pEGPF-PC2 or a PC2 mutant plasmid for 48 h were subjected to immunoprecipitation (IP) with the
GFP antibody and immunoblotting with the FLAG or GFP antibody. Cell extracts (Input, 10%) were utilized for detecting transfection efficiency.
Human Molecular Genetics, 2008, Vol. 17, No. 8 1115
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cells over-expressing Herp (Fig. 4C, blot PC2, lane 3, arrow).
Because this band was undetectable when cells were treated
with MG-132 (Fig. 4C, blot PC2, lanes 4 versus 3) it may rep-
resent an intermediate form of PC2 during its degradation by
the proteasome. Thus our data show that the UBL protein
Herp interacts with PC2 and promotes its degradation
through the ERAD pathway.
Note that, although an increase in the Herp expression, like
the treatment with an ER stress inducer, accelerates PC2
degradation, it does not induce ER stress to the cells, as
assessed by the BiP expression (Fig. 5A). We next tested the
effect of Herp knockdown, by siRNA, on PC2 degradation.
A robust reduction in the Herp expression was obtained in
HeLa cells with Herp siRNA (Fig. 5B, lanes 3 versus 1).
The amount of PC2 augmented when Herp expression was
inhibited by siRNA in both normal cells and those under ER
stress triggered by Tm (lanes 3 versus 1 and 4 versus 2).
Thus, the rate of PC2 degradation correlates with the Herp
level. To further document this correlation, instead of directly
altering the Herp level by over-expression or siRNA, we made
use of ER stress inducers, which are known to increase Herp
expression by inducing ER stress (33). Indeed, the Herp
level increased and the PC2 level decreased by Tm (Fig. 5A
and B, lanes 2 versus 1) and DTT (data not shown) in HeLa
cells. Similar results were also obtained in cells with Herp
knockdown (Fig. 5B, lanes 4 versus 3). Taken together, our
data obtained under various conditions demonstrate that
Herp regulates PC2 degradation, presumably via their physical
interaction and its promotion of retro-translocation.
Next, we tested whether the degradation of pathogenic
mutants of PC2 is mediated by Herp, using HeLa cells
co-transfected with a plasmid encoding pEGFP, pEGFP-PC2
ora mutantPC2,and with
pcDNA3.1 vector, as a control). Similar to PC2, the degra-
dation of PC2 pathogenic mutants R872X and E837X, and
PC2 mutants lacking the intracellular C-terminus (PC2DC,
amino acids 1–688 or S689X) or N-terminus (PC2DN,
amino acids 209–968) was promoted by Herp (Fig. 5C).
However, Herp exhibited no effect on the turnover of PC2
mutant lacking both N- and C-termini (PC2DNC, amino
acids 209-688) (Fig. 5C). Because PC2DC and PC2DN, but
not PC2DNC, interact with Herp (Fig. 5D), our data together
suggest that the Herp–PC2 physical interaction is critical for
PC2 degradation by the ERAD pathway.
The present study shows that PC2, an ER Ca release channel,
is a novel ERAD substrate, using several pieces of data,
including ubiquitination of PC2, degradation inhibition by pro-
teasome inhibitors, interaction with the ERAD components
(p97 and Herp) and the 26S proteasome. PC2 is ubiquitinated
under the physiological condition, and PC2 ubiquitination, as
well as the amounts of the PC2–p97 and PC2-S12 complexes,
significantly augments when PC2 retro-translocation and
degradation are blocked by proteasome inhibitors, indicating
that these forms of PC2 are located at the upstream of the pro-
teasome. ATPase p97, which provides a driving force for
retro-translocation of ERAD substrates from the ER, aggre-
gates to perinuclear regions in the presence of over-expressed
PC2, presumably due to and in favor of its physical binding
with PC2. ER stress inducers increase the expression of
Herp, which directly promotes PC2 degradation by increasing
the retro-translocation of PC2 from the ER to the proteasome
complex in the cytosol. Whether other changes induced by ER
stress inducers also contribute to the increased PC2 degra-
dation remains to be determined.
Our data demonstrate that Herp regulates PC2 degradation.
Herp, a known ER stress response protein (12,33,35,36), is
implicated in ERAD. However, it has so far been unclear as
to how this UBL protein regulates the ERAD pathway. Our
data show that Herp is in the same complex as the ERAD
substrate PC2. Direct changes in the Herp level, by over-
expression or siRNA knockdown, positively correlate with
the rate of PC2 degradation. This correlation is further sup-
ported by the effect of indirect changes in the Herp level
induced by ER stress inducers with or without simultaneous
Herp siRNA. Thus, the amount of Herp is critical in determin-
ing the cellular level of PC2, which may account, at least in
part, for the Herp-induced reduction in the ER Ca release in
ER-stressed cells (37).
accompanied by Herp C-terminal cleavage in cells either tran-
siently over-expressing this ER membrane protein or under ER
stress. The 50-kDa fragment of Herp corresponds likely to a
mutant missing the C-terminus, based on its size and attach-
ment to the membrane (Fig. 4), indicating that the PC2–
Herp complexing is via the N-terminus or the transmembrane
domain of Herp. Of note, the difference in the detected frag-
ments between the over-expressed, tagged Herp (Fig. 4A)
and the increased Herp by Tm (Fig. 5B) should be due to
different antibodies used. It will be interesting to determine
whether Herp cleavage plays a role in its regulation of PC2
degradation; for example, whether its C-terminus may have
an inhibitory effect on its activity with respect to PC2
degradation. The observation of Herp cleavage under these
conditions is consistent with our previous study (35). Also,
it was reported that ATF6 and Luman, two ER membrane-
localized transcription factors mediating UPR, are activated
through cleavage upon UPR or when they are transiently
expressed in cells (35,39,40).
We propose a six-state model to illustrate the proteasome-
dependent PC2 degradation (Fig. 6) in which the involvements
or roles of ubiquitin, Herp, p97, the 26S proteasome complex,
ER stress inducers and proteasome inhibitors, are indicated.
On the ER membrane, PC2 interacts with Herp (state 1) and
is ubiquitinated (state 2), for example, by TAZ-regulated
SCFb-TrcpE3 ligase complex (23). Next, ATPase p97 is
recruited onto ubiquitinated PC2 (state 3). An increase in the
Herp level, induced either directly (by transfection) or
indirectly (by ER stress), promotes Herp cleavage and tran-
sition from state 2 to 3. The retro-translocation of PC2 from
the ER membrane to the cytosol (state 4) may be driven by
ATP hydrolysis of p97 and Herp cleavage, and leads to its rec-
ognition by the proteasome complex (state 5) for ultimate
degradation (state 6). Proteasome inhibitors delay PC2 retro-
translocation and increase the population of PC2 in upstream
states, for example, the PC2-proteasome and PC2–p97 com-
inPC2 degradation is
1116 Human Molecular Genetics, 2008, Vol. 17, No. 8
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It is commonly accepted, and as supported by numerous
studies including those using animal or cellular models, that
loss of function of PC2 or polycystin-1 (PC1), a PC2 interact-
ing partner mutated in ?80% of ADPKD patients, results in
cystogenesis. Interestingly, increased PC2 expression in
TAZ-knockout mice and increased PC1 expression in pkd1
transgenic mice are both associated with renal cystogenesis
(23,41). Thus, together it seems that an altered (either
increased or decreased) PC1 or PC2 expression/function
from their normal ranges may lead to disease. Our present
study found that the degradation of pathogenic mutants
R872X and E837X and truncation mutants PC2DC and
PC2DN is all enhanced by Herp though the enhancement
may be of different degrees (Fig. 5C), possibly due to varied
strength of interaction with Herp. On the other hand, the
level of PC2DNC is not affected by Herp and this mutant
has no interaction with Herp. These data together indicate
that the PC2–Herp physical interaction is essential to
the regulation of PC2 expression (and thus, function). Thus,
the ERAD-regulated PC2 turnover may be important for the
pathogenesis of ADPKD. Future studies should examine (i)
whether/how pathogenic point mutations in PC2 affect its
interaction with and degradation by Herp, and (ii) whether/
how mutations in PC2 impact the ERAD machinery, which
then affects cell’s normal properties.
MATERIALS AND MATHODS
Cell culture, DNA constructs and gene transfection
IMCD, MDCK and HeLa cells were cultured in Dulbecco’s
Modified Eagle’s Medium (high glucose; Invitrogen) contain-
ing 10% (v/v) fetal bovine serum, 1% penicillin and
streptomycin at 378C and 5% CO2. Plasmids pEGFP-PC2,
pEGFP-R872X, pEGFP-E837X, pEGFP-PC2DC, pEGFP-
PC2DN, and pEGFP-PC2DNC were constructed based on a
method described previously (21). HeLa cells were grown to
?70% confluency prior to transfection using Lipofectamine
2000 (Invitrogen). MDCK cells stably expressing GFP-PC2
or GFP were selected as previously described (42) and main-
tained using G418 (300 mg/ml).
Protein extraction, immunoblotting, immunoprecipitation and
microscopy were performed as described (22). Typically, 20
and 200 mg of total cellular protein were used for immunoblot-
ting and immunoprecipitation, respectively. HeLa cells were
pEGFP-PC2, pcDNA3.1 (Invitrogen) or pcDNA3.1-mHerpf
(33) for immunoprecipitation. At 20 h post-transfection, cells
were split into two equal fractions for treatment with a protea-
some inhibitor. To examine the degradation of PC2 mutants
we transfected HeLa cells with pEGFP-PC2, pEGFP-R872X,
pEGFP-E837X, pEGFP-PC2DC, pEGFP-PC2DN, pEGFP-
PC2DNC, or vector pEGFP. At 6 h post-transfection, cells
were splitinto two equal
co-transfection with pcDNA3.1-mHerpf or pcDNA3.1 at 20
h post-transfection. For the interaction of Herp with PC2
mutants, HeLa cells were transfected with pcDNA3.1-mHerpf.
At 6 h post-transfection, cells were split into five equal frac-
tions for subsequent co-transfection with PC2 or its mutant
plasmids 20 h post-transfection. Fluorescent images were cap-
tured on a motorized Olympus IX81 microscopy installed with
a CCD cooling RT SE6 monochrome camera (Diagnostic
Instruments). Final composite images were made using
Image-Pro Plus 5.0 (Media Cybernetics).
Figure 6. A model for polycystin-2 (PC2) degradation through the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. State 1, PC2 in
complex with Herp. State 2, TAZ-dependent PC2 ubiquitination. State 3, binding of p97 to ubiquitinated PC2 and Herp cleavage. State 4, retro-translocated
PC2 (from the ER membrane) in complex with p97 in the cytosol. State 5, PC2 in complex with the proteasome. State 6, destructed PC2.
Human Molecular Genetics, 2008, Vol. 17, No. 81117
by guest on June 5, 2013
Cycloheximide chase assays
In CHX chase experiments, IMCD cells were incubated with
50 mg/ml of CHX in the presence or absence of a proteasome
inhibitor for 0 or 8 h. Cells were harvested for protein prep-
aration and immunoblotting, as described (22).
Rabbit antibodies against p97, Myc or FLAG, and mouse anti-
ubiquitin antibody were purchased from Cell Signaling Tech-
nology. Mouse anti-Myc antibody was from Chemicon.
Anti-S12, -IP3R and -Sec61a antibodies were from Affinity
BioReagents. b-actin antibody was from Sigma-Aldrich
Canada. Goat anti-BiP and rabbit anti-ATF6a were purchased
from Santa Cruz. GFP antibodies were a gift from Dr Luc
Berthiaume (University of Alberta, Canada; also available at
www.eusera.com). Mouse anti-PC2 and rabbit anti-Herp anti-
bodies were described as before (21,22,33,35). Secondary anti-
bodies were from Amersham or Promega.
HeLa cells over-expressing Herp were homogenized in a cold
fractionation buffer (33). The homogenate was subjected to a
serial of centrifugations, at 1000 g for 10 min, 10 000 g for
10 min and 100 000 g for 60 min. The supernatants and
pellets from the 100 000 g centrifugation that contained the
cytosolic and membrane fractions, respectively, were col-
lected. The pellets were dissolved in the CellLyticTMM Cell
Lysis Reagent (Sigma-Aldrich Canada).
Herp and PC2 knockdown by small interference RNA
Herp Stealth siRNA (sense, 50-UCAGAAUGCUGCUCCUCA
AUU and antisense, 50-UUGAGGAGCAGCAUUCUGAUU)
and its specific control siRNA (sense, 50-ACAUAGCCAUGC
GUUACUCUU and antisense, 50-GAGUAACGCAUGGCUA
UGUUU) were used to transfect HeLa cells using Lipofecta-
mine 2000 reagent following the manufacturer’s instructions.
PC2 knockdown was described previously (22). The efficiency
of the siRNA knockdown was assessed by immunoblotting.
Conflict of Interest statement. None declared.
This work was supported by the Canadian Institutes of Health
Research and the Kidney Foundation of Canada (to X.-Z.C.).
Q.L. is a recipient of the Polycystic Kidney Disease Foun-
dation Fellowship. X.-Z.C. is a Senior Scholar of the Alberta
Heritage Foundation for Medical Research.
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