THE JOURNAL OF BIOLOGICAL CHEMISTRY
19419 –19425, 2005
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
Degradation of Trafficking-defective Long QT Syndrome Type II
Mutant Channels by the Ubiquitin-Proteasome Pathway*
Published, JBC Papers in Press, March 10, 2005, DOI 10.1074/jbc.M502327200
Qiuming Gong‡, David R. Keeney‡, Maurizio Molinari§, and Zhengfeng Zhou‡¶
From the ‡Division of Molecular Medicine, Department of Medicine, Oregon Health and Science University, Portland,
Oregon 97239 and §Institute for Research in Biomedicine, CH-6500 Bellinzona, Switzerland
Mutations in the human ether-a-go-go-related gene
(hERG) cause chromosome 7-linked long QT syndrome
type II (LQT2). We have shown previously that LQT2
mutations lead to endoplasmic reticulum (ER) retention
and rapid degradation of mutant hERG proteins. In this
study we examined the role of the ubiquitin-proteasome
pathway in the degradation of the LQT2 mutation
Y611H. We showed that proteasome inhibitors N-acetyl-
L-leucyl-L-leucyl-L-norleucinal and lactacystin but not
lysosome inhibitor leupeptin inhibited the degradation
of Y611H mutant channels. In addition, ER mannosidase
I inhibitor kifunensine and down-regulation of EDEM
(ER degradation-enhancing ␣-mannosidase-like pro-
tein) also suppressed the degradation of Y611H mutant
channels. Proteasome inhibition but not mannosidase
inhibition led to the accumulation of full-length hERG
protein in the cytosol. The hERG protein accumulated
in the cytosol was deglycosylated. Proteasome inhibi-
tion also resulted in the accumulation of polyubiquiti-
nated hERG channels. These results suggest that the
degradation of LQT2 mutant channels is mediated by
the cytosolic proteasome in a process that involves man-
nose trimming, polyubiquitination, and deglycosylation
of mutant channels.
Long QT syndrome is a cardiac disorder characterized by
prolongation of QT intervals on the electrocardiogram and a
high risk of sudden death due to ventricular arrhythmias. The
chromosome 7-linked form of the inherited long QT syndrome
(long QT syndrome type II (LQT2))1 is caused by mutations of
human ether-a-go-go-related gene (hERG) (1). hERG encodes
the pore-forming subunit of the rapidly activating-delayed rec-
tifier K⫹ channel (IKr) in the heart (2, 3). Studies of LQT2
mutant channels expressed heterologously in oocytes or mam-
malian cells have shown that LQT2 mutations cause hERG
channel dysfunction by multiple mechanisms including defec-
tive protein trafficking, abnormal gating or permeation, and
* This work was supported by Grant HL68854 (to Z. Z.) from the
National Institutes of Health and an award to Oregon Health and
Science University under the Howard Hughes Medical Institute Bio-
medical Research Support Program for Medical Research. The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Div. of Molecular
Medicine, NRC3, Oregon Health and Science University, 3181 S.W.
Sam Jackson Park Rd., Portland, OR 97239. Tel.: 503-494-2713; Fax:
503-494-7368; E-mail: zhouzh@ohsu.edu.
1
The abbreviations used are: LQT2, long QT syndrome type II;
hERG, human ether-a-go-go-related gene; ER, endoplasmic reticulum;
ERAD, ER-associated degradation; ALLN, N-acetyl-L-leucyl-L-leucyl-L-
norleucinal; PNGase, peptide:N-glycanase; GFP, green fluorescence
protein; siRNA, small interfering RNA; siGFP, GFP-directed siRNA;
siEDEM, EDEM-directed siRNA.
This paper is available on line at http://www.jbc.org
Vol. 280, No. 19, Issue of May 13, pp.
Printed in U.S.A.
Received for publication, March 2, 2005
dominant negative suppression of wild type hERG channel
function (4).
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We have previously shown that the LQT2 mutation Y611H is
trafficking-defective (5). This mutant expresses only the imma-
ture form of hERG channel protein, which fails to reach the
plasma membrane. The Y611H mutant protein is retained in
the endoplasmic reticulum (ER) and rapidly degraded. It is well
recognized that newly synthesized proteins in the ER are under
stringent surveillance by a quality control system (6 – 8). The
ER quality control system ensures that only properly folded
and assembled proteins are exported from the ER to the Golgi.
Misfolded and unassembled proteins are retained in the ER
and eventually degraded by a process termed ER-associated
degradation (ERAD). According to current models, ERAD sub-
strates undergo retrotranslocation or dislocation from the ER
to the cytosol, in which they are degraded by the proteasome
(8). In most cases, the degradation by the proteasome is pre-
ceded by polyubiquitination. In addition, mannose trimming by
ER mannosidase I and deglycosylation by cytosolic peptide:N-
glycanase (PNGase) are important processes in proteasomal
degradation of glycoproteins (8). Recently EDEM (ER degrada-
tion-enhancing ␣-mannosidase-like protein) has been shown to
play a crucial role in the degradation of misfolded glycoproteins
(9, 10).
The importance of ERAD is underscored by the fact that
proteasomal degradation of misfolded proteins has been shown
in an increasing number of human diseases. The role of ERAD
in the degradation of misfolded proteins has been studied ex-
tensively in cystic fibrosis and ␣1-antitrypsin deficiency (11,
12). Defective trafficking of mutant channels to the plasma
membrane has been shown as the most common mechanism of
hERG channel dysfunction in LQT2. More than 15 LQT2 mu-
tations have been reported to cause defective trafficking of
mutant channels (13, 14). These LQT2 mutations lead to mis-
folding, ER retention, and the degradation of mutant hERG
channels (5, 13, 14). Although degradation of mutant hERG
channels by the proteasome has been suggested (15, 16), little
is known about how mutant hERG channels are targeted to the
cytosolic proteasome for degradation. In this study, we ana-
lyzed the role of the ubiquitin-proteasome pathway in the deg-
radation of the LQT2 mutant Y611H. Our results suggest that
the mutant hERG protein is targeted to the proteasome for
degradation by dislocation from the ER membrane to the cy-
tosol. The proteasome-dependent degradation involves man-
nose trimming, ubiquitination, and deglycosylation of
mutant channels.
EXPERIMENTAL PROCEDURES
Reagents and Cell Transfection—The proteasome inhibitors lactacys-
tin and N-acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN) were purchased
from Kamiya Biomedical Co. (Seattle, WA) and Calbiochem, respec-
tively. The ER mannosidase I inhibitor kifunensine was purchased from
Toronto Research Chemicals, Inc. (North York, Ontario, Canada). Anti-
19419
19420 Proteasomal Degradation of Mutant hERG Channels
hERG antibody was raised against the hERG-thioredoxon fusion pro-
tein as described previously (5). Anti-ubiquitin monoclonal antibody
was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-
calnexin antibody was purchased from Stressgen (Victoria, British Co-
lumbia, Canada). Expre35S35S protein labeling mix was purchased from
PerkinElmer Life Sciences. HEK293 cells stably expressing wild type
hERG or the Y611H mutant were cultured as described previously (5).
For down-regulation of EDEM, HEK293 cells were cotransfected with 6
␮g of pcDNA3-Y611H and 10 ␮g of pcDNA3-siEDEM or pcDNA3-siGFP
utilizing Lipofectamine 2000 reagent (Invitrogen) (9).
Western Blot Analysis—Western blot procedures were described pre-
viously (5). Briefly, cells were lysed in lysis buffer containing 50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and
proteinase inhibitors. After centrifugation at 14,000 ⫻ g for 10 min at
4 °C, cell lysates were subjected to 7.5% SDS-PAGE and electrophoreti-
cally transferred onto nitrocellulose membranes. hERG proteins were
detected with anti-hERG antibody (1:10000 dilution) and visualized
with a horseradish peroxidase-conjugated second antibody and ECL
detection kit. Deglycosylation of hERG proteins by PNGase was per-
formed as described previously (17).
Metabolic Labeling and Immunoprecipitation—Cells in 35-mm
plates were pulse-labeled for 1 h in methionine- and cysteine-free Dul-
becco’s modified Eagle’s medium containing 110 ␮Ci/ml [35S]methi-
onine/cysteine and chased in Dulbecco’s modified Eagle’s medium with
2 mM unlabeled methionine and cysteine for time intervals up to 24 h.
For drug treatment, 50 ␮M ALLN, 20 ␮M lactacystin, 100 ␮M leupeptin,
or 100 ␮M kifunensine was added 1 h before pulse labeling and was
continuously included in the culture medium during the pulse and the
chase periods. At the end of chase period, cells were lysed in 600 ␮l of
immunoprecipitation buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1%
SDS, 1 mg/ml bovine serum albumin, and protease inhibitors (100 ␮M
phenylmethylsulfonyl fluoride, 1 ␮g/ml pepstatin A, 1 ␮g/ml leupeptin,
4 ␮l/ml aprotinin). After centrifugation at 14,000 ⫻ g for 10 min at 4 °C,
cell lysates were precleared by incubation with protein A-agarose beads
(Pierce). hERG antiserum (1:100 dilution) was then added, and the
mixture was incubated at 4 °C overnight. The antigen-antibody com-
plexes were isolated with protein A-agarose beads, subjected to 7.5%
SDS-PAGE, and visualized with autoradiography. For quantitative
analysis, the dried gels were exposed to a K-screen and quantified by
phosphorimaging analysis using Quantity One soft ware (Bio-Rad, Mo-
lecular Imager FX).
Detection of Ubiquitinated hERG Proteins—Cells were lysed in 600
␮l of immunoprecipitation buffer. Cell lysates containing an equal
amount of protein were immunoprecipitated with anti-hERG antibody
at 1:100 dilution as described above. The immunoprecipitates were
subjected to 5.5% SDS-PAGE and electrophoretically transferred onto
nitrocellulose membranes. The membranes were probed with mono-
clonal anti-ubiquitin antibody at 1:500 dilution. The membranes were
also probed with anti-hERG antibody after stripping of the bound
anti-ubiquitin antibodies.
Subcellular Fractionation—Cells were homogenized in 600 ␮l of ho-
mogenization buffer containing 10 mM Tris-HCl. pH 7.4, 1 mM EDTA,
and proteinase inhibitors with a Dounce homogenizer. Cells metaboli-
cally labeled by [35S]methionine/cysteine were homogenized in 600 ␮l of
homogenization buffer by freezing and thawing the cell suspension and
passing it 10 times through a 25-gauge needle as described previously
(18). Homogenates were centrifuged at 1000 ⫻ g for 10 min to remove
unbroken cells and nuclei. The postnuclear supernatant was then cen-
trifuged at 10,000 ⫻ g for 30 min. The supernatant from the 10,000 ⫻
g centrifugation was further centrifuged at 100,000 ⫻ g for 60 min.
Membrane pellets from 10,000 ⫻ g and 100,000 ⫻ g spins were washed
with homogenization buffer and solubilized in a buffer containing 50
mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1%
sodium deoxycholate, 0.1% SDS, 1 mg/ml bovine serum albumin, and
protease inhibitors. The supernatant from the 100,000 ⫻ g centrifuga-
tion (cytosolic fraction) was adjusted to a final concentration of 150 mM
NaCl, 1% Triton X-100, and 1 mg/ml bovine serum albumin. Solubilized
membranes and cytosolic fractions were analyzed by immunoprecipita-
tion and immunoblot as described above. Equal fractions from each
pellet and supernatant were analyzed.
RNase Protection Assay—Twenty-four h after transfection with
siRNAs, the cells were harvested and total RNA isolated using the
RNeasy method (Qiagen, Valencia, CA). RNase protection probes were
made by subcloning a 452-nucleotide cDNA fragment corresponding to
1386 –1838 bp of EDEM and a 290-nucleotide cDNA fragment corre-
sponding to 466 –756 bp of actin into pCRII vector (Invitrogen). The
antisense RNA probes were transcribed using a MAXIscript in vitro
FIG. 1. Effects of ALLN, lactacystin, and leupeptin on protein
levels of wild type hERG and the Y611H mutant. HEK293 cells
stably transfected with wild type hERG or Y611H were treated with (⫹)
or without (⫺) 50 ␮M ALLN, 20 ␮M lactacystin (LACT), or 100 ␮M
leupeptin (LEUP) for 24 h. The cell lysates were subjected to SDS-
PAGE and immunoblotted with anti-hERG antibody.
transcription kit (Ambion, Austin, TX) and biotin-16-UTP (Roche Ap-
plied Science). Thirty ␮g of total RNA or yeast tRNA were analyzed
with EDEM riboprobe, and 10 ␮g of total RNA or yeast tRNA were
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analyzed by actin riboprobe using RPAII and BrightStart BioDetect
kits (Ambion). Yeast tRNA was used as a control for the complete
digestion of the probes by RNase.
RESULTS
The involvement of the proteasome in the degradation of
hERG channel was initially assessed by examining the effect of
proteasome inhibitors ALLN and lactacystin on the steady
state protein levels of wild type hERG and the LQT2 mutant
Y611H. Fig. 1 shows Western blot analysis of hERG channel in
HEK293 cells stably transfected with wild type hERG or
Y611H. Wild type hERG expressed two protein bands, an upper
band of 155 kDa and a lower band of 135 kDa, whereas the
Y611H mutant expressed only the 135-kDa lower band. We
have previously shown that the upper band is the fully glyco-
sylated, mature form of the channel protein located in the
plasma membrane, and the lower band is a core-glycosylated,
immature form of the channel protein located in the ER (5).
Exposure to 50 ␮M ALLN or 20 ␮M lactacystin for 24 h signif-
icantly increased protein levels of the immature form without
an apparent increase in the mature form of wild type hERG or
Y611H. The lysosome inhibitor leupeptin had no effect on pro-
tein levels of wild type hERG or Y611H. These results strongly
suggest that the proteasome is involved in the degradation of
the immature form of hERG channels.
To further examine the role of the proteasome in the degra-
dation of hERG channels, we performed pulse-chase analysis.
In these experiments, cells expressing wild type hERG or
Y611H were metabolically labeled with [35S]methionine and
[35S]cysteine for 1 h and chased with unlabeled methionine and
cysteine for up to 24 h under control conditions or in the
presence of 50 ␮M ALLN (Fig. 2A). Under control conditions,
wild type hERG was initially synthesized as the 135-kDa im-
mature form, which was gradually converted to the 155-kDa
mature form. The kinetics of hERG channel maturation and
degradation were analyzed by phosphorimaging quantification.
As shown in Fig. 2B, the conversion of wild type hERG from the
immature to the mature form became obvious at 2 h of chase
and reached a maximum at 8 h. About 60% of the immature
form was converted to the mature form. In the presence of
ALLN, there was a significant increase in the amount of la-
beled immature form of wild type hERG. The increase in the
immature form, however, did not result in an increase in the
mature form during the chase. In fact, ALLN significantly
reduced the efficiency of hERG channel maturation, as only
about 20% of the immature form was converted to the mature
form in the presence of ALLN.
In pulse-chase experiments, the Y611H mutant was initially
synthesized as the 135-kDa immature form and was not con-
verted to the mature form during the chase (Fig. 2C). Rather,
the mutant protein underwent progressive degradation with
complete disappearance of the 135-kDa band by 24 h. Kinetic
 Proteasomal Degradation of Mutant hERG Channels
FIG. 2. Pulse-chase analysis of wild type hERG and Y611H
mutant channels. HEK293 cells stably transfected with wild type
hERG (A) or Y611H (C) were pulse-labeled for 1 h and chased for the
indicated times in the absence or presence of 50 ␮M ALLN. The cells
were lysed and hERG was analyzed by immunoprecipitation with anti-
hERG antibody and SDS-PAGE. The amount of mature and immature
forms of hERG was quantified by phosphorimaging analysis and plotted
as percentage of the value at time point 0 (B and D). The data shown are
means from two (wild type, B) or four (Y611H, D) independent
experiments.
analysis shown in Fig. 2D revealed that the immature form of
Y611H was degraded with an estimated half-life of 4.5 h. ALLN
inhibited the degradation of the Y611H mutant channel,
thereby prolonging its half-life to 12 h (Fig. 2, C and D). How-
ever, inhibition of the degradation did not cause conversion of
the immature to the mature form. We also performed pulse-
chase experiments to study the effects of the specific protea-
some inhibitor lactacystin and the lysosome inhibitor leupeptin
on the degradation of the Y611H mutant channel. In these
experiments, cells expressing Y611H were labeled for 1 h and
chased for 8 h under control conditions or in the presence of 10
␮M lactacystin or 100 ␮M leupeptin. As shown in Fig. 3, lacta-
cystin inhibited the degradation of the Y611H mutant. After
8 h of chase, 26% of the 35S-labeled hERG protein remained
under control conditions, whereas in the presence of lactacystin
68% of the 35S-labeled hERG protein remained. In contrast,
leupeptin had no effect on the degradation of the Y611H mu-
tant. After 8 h of chase, 17 and 21% of the 35S-labeled hERG
protein remained under control conditions and in the presence
19421
FIG. 3. Effects of lactacystin and leupeptin on the degradation
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of the Y611H mutant channel. Cells expressing Y611H mutant were
pulse-labeled for 1 h and chased for 8 h in the absence or presence of 20
␮M lactacystin (LACT) (A), or 100 ␮M leupeptin (LEUP) (B). hERG
channels were immunoprecipitated with anti-hERG antibody and sub-
jected to electrophoresis on SDS-polyacrylamide gel for analysis. Quan-
tified data are plotted as percentage of the value at time point 0, and the
data shown are means from two independent experiments.
of leupeptin, respectively. Thus, these pulse-chase results are
consistent with our Western blot data and suggest that the
cytosolic proteasome is involved in the degradation of the
Y611H mutant channel.
Degradation of ERAD substrate proteins by the cytosolic
proteasome implies that they must be dislocated from the ER to
the cytosol. It has been reported that some membrane proteins
can be dislocated from the ER to the cytosol in an intact form
when proteasome activity is inhibited (18 –20). To test whether
hERG channel proteins undergo similar dislocation, we per-
formed subcellular fractionation experiments (Fig. 4A). After
removal of unbroken cells and nuclei by centrifugation at
1000 ⫻ g, postnuclear supernatant was separated into three
fractions: 10,000 ⫻ g pellet (crude membranes), 100,000 ⫻ g
pellet (residual microsome membranes), and 100,000 ⫻ g su-
pernatant (cytosolic fraction) (18). Western blot analysis
showed that in the absence of ALLN, both wild type hERG and
Y611H mutant proteins were recovered only from the 10,000 ⫻
g pellet membrane fraction. In the presence of ALLN, they were
also present in the 100,000 ⫻ g supernatant cytosolic fraction.
As a control, the ER membrane protein calnexin was shown to
be present only in the 10,000 ⫻ g pellet membrane fraction.
Thus, these results suggest that an intact form of hERG chan-
nel can be dislocated from the ER to the cytosol. Because ERAD
substrate proteins dislocated to the cytosol have been reported
to undergo deglycosylation prior to degradation by the protea-
some (18, 21–23), we examined whether the hERG protein
recovered from the cytosolic fraction represents the deglycosy-
lated form. As shown in Fig. 4B, the Y611H mutant protein
recovered from the cytosolic fraction had a molecular mass of
132 kDa and was resistant to PNGase treatment. In contrast,
the mutant protein recovered from membrane fraction had a
molecular mass of 135 kDa and was sensitive to PNGase, with
its molecular mass being reduced to 132 kDa. These results
suggest that Y611H mutant protein recovered from the cytoso-
lic fraction represents the deglycosylated form.
To demonstrate that the presence of hERG channels in the
cytosol is due to dislocation of those retained in the ER, rather
than the failure of incorporation of newly synthesized hERG
channels into the ER, we performed pulse-chase experiments
followed by cell fractionation. Cells expressing the Y611H mu-
tant were labeled for 1 h and chased for up to 24 h in the
presence of 50 ␮M ALLN. hERG proteins were immunoprecipi-
tated from the 10,000 ⫻ g pellet membrane fraction and the
100,000 ⫻ g supernatant cytosolic fraction. As shown in Fig. 5,
19422 Proteasomal Degradation of Mutant hERG Channels
FIG. 4. A, accumulation of hERG channels in the cytosol. HEK293
cells stably transfected with wild type hERG (top and bottom panels) or
Y611H (middle panel) were treated with (⫹) or without (⫺) 50 ␮M
ALLN for 24 h. The cells were homogenized and subjected to subcellular
fractionation as described under “Experimental Procedures.” Samples
from different fractions were separated on SDS-polyacrylamide gel and
immunoblotted with anti-hERG antibody (top and middle panels) or
anti-calnexin antibody (bottom panel). B, effect of PNGase on hERG
channels. Cells expressing the Y611H mutant were treated with 50 ␮M
ALLN for 24 h. The cells were then homogenized and subjected to
subcellular fractionation. Samples from 10,000 ⫻ g pellet (membrane
fraction) and 100,000 ⫻ g supernatant (cytosolic fraction) were treated
with (⫹) or without (⫺) PNGase and analyzed by immunoblotting with
anti-hERG antibody.
FIG. 5. Pulse-chase analysis of the Y611H mutant channel in
the membrane and cytosolic fractions. Cells expressing the Y611H
mutant were pulse-labeled for 1 h and chased for the indicated times in
the presence of 50 ␮M ALLN. The cells were homogenized and fraction-
ated by centrifugation. Samples from 10,000 ⫻ g pellet (membrane
fraction) and 100,000 ⫻ g supernatant (cytosolic fraction) were ana-
lyzed by immunoprecipitation with anti-hERG antibody and SDS-
PAGE. Quantified data are plotted as percentage of the value of mem-
brane fraction at time point 0, and the data shown are means from two
independent experiments.
nearly all 35S-labeled hERG protein was present in the mem-
brane fraction at the end of the pulse labeling. The amount of
hERG protein in the membrane fraction gradually decreased
during the chase, whereas the amount of hERG protein in the
cytosolic fraction gradually increased, reaching a maximum at
8 h of chase. These pulse-chase experiments strongly suggest
FIG. 6. Ubiquitination of wild type hERG and Y611H mutant
channels. A, untransfected HEK293 cells and wild type hERG- or
Y611H-transfected cells were treated with (⫹) or without (⫺) 50 ␮M
ALLN for 24 h. The cell lysates were analyzed by immunoprecipitation
Downloaded from http://www.jbc.org/ at FUDAN UNIVERSITY SCHOOL OF PHARMACY on May 24, 2018
(IP) with anti-hERG antibody followed by serial immunoblotting (IB)
with anti-ubiquitin (upper panel) and anti-hERG (lower panel) antibod-
ies. B, ALLN-treated cells were homogenized and fractionated. Samples
from membrane (M) and cytosolic (C) fractions were analyzed by im-
munoprecipitation with anti-hERG antibody followed by serial immu-
noblotting with anti-ubiquitin (upper panel) and anti-hERG (lower
panel) antibodies.
that newly synthesized hERG channels are incorporated into
the ER membrane and then dislocated from the ER into the
cytosol for proteasomal degradation. The results also show that
despite the presence of hERG protein in the cytosolic fraction,
the majority of hERG protein was still present in the mem-
brane fraction during the chase, indicating that the rate of
dislocation from the membrane to the cytosol is reduced in the
presence of proteasome inhibitors.
Many ERAD substrate proteins are conjugated with poly-
ubiquitin chains that serve as a signal for proteasomal degra-
dation. To demonstrate that the hERG channel is modified by
polyubiquitin chains, cell lysates were immunoprecipitated
with anti-hERG antibody and then immunoblotted with anti-
ubiquitin antibody. In these experiments, untransfected HEK
cells and cells expressing wild type hERG or Y611H were
treated with or without ALLN for 24 h. As shown in Fig. 6A,
treatment of cells with ALLN resulted in the accumulation of
high molecular weight ubiquitinated hERG channels in wild
type hERG and in Y611H-transfected cells but not in untrans-
fected cells. Ubiquitinated hERG channels were barely detect-
able in untreated cells, suggesting that ubiquitinated hERG
channel protein is normally degraded by the proteasome in the
absence of proteasome inhibitor. To determine the subcellular
localization of the ubiquitinated hERG channels, cell fraction-
ation experiments were carried out in which hERG channels
were immunoprecipitated from membrane and cytosolic frac-
tions of ALLN-treated cells and then immunoblotted with anti-
ubiquitin antibody. As shown in Fig. 6B, ubiquitinated hERG
channels were present in both membrane and cytosolic frac-
tions. This result suggests that the ubiquitination of hERG
channels starts before they are released into the cytosol.
The hERG channel protein is modified by N-linked glycosyl-
ation (17, 24). Several studies have shown that the trimming of
mannose by ER mannosidase I is required for proteasomal
degradation of glycoproteins (25–29). We examined the role of
mannose trimming in the degradation of the Y611H mutant
channel by using the ER mannosidase I inhibitor kifunensine.
As shown in Fig. 7, cells expressing the Y611H mutant were
metabolically labeled with [35S]methionine and [35S]cysteine
for 1 h and chased with unlabeled methionine and cysteine for
up to 24 h under control conditions or in the presence of 100 ␮M
kifunensine (Fig. 7A). Similar to proteasome inhibitors, kifu-
nensine inhibited the degradation of the Y611H mutant chan-
nel. In cell fractionation experiments, it was found that inhi-
bition of degradation by kifunensine did not result in the
 Proteasomal Degradation of Mutant hERG Channels
FIG. 7. Effects of kifunensine on the degradation of the Y611H
mutant. Cells expressing the Y611H mutant were pulse-labeled for 1 h
and chased for the indicated times in the absence or presence of 100 ␮M
kifunensine. A, the cell lysates were subjected to immunoprecipitation
with anti-hERG antibody and SDS-PAGE. B, samples from membrane
and cytosolic fractions were analyzed by immunoprecipitation with
anti-hERG antibody and SDS-PAGE.
accumulation of hERG channel protein in the cytosol (Fig. 7B).
These results suggest that mannose trimming is an important
process in the degradation of the Y611H mutant channel.
Recently, it has been shown that EDEM plays an important
role in proteasomal degradation of misfolded glycoproteins (9,
10). EDEM is an ER type II membrane protein homologous to
␣-ER mannosidase but lacking mannosidase activity. To test
whether EDEM is involved in the degradation of mutant hERG
channels, we used RNA-mediated interference to reduce the
intracellular concentration of EDEM. In these experiments, we
cotransfected the Y611H mutant with an EDEM-directed
siRNA (siEDEM) or a control GFP-directed siRNA (siGFP) (9)
into HEK293 cells and performed pulse-chase analysis 24 h
after transfection. A RNase protection assay revealed a de-
crease in EDEM mRNA in siEDEM-transfected cells (57 ⫾ 5%)
compared with siGFP control (n ⫽ 3) (Fig. 8A). Pulse-chase
experiments showed that down-regulation of EDEM inhibited
the degradation of the Y611H mutant channel (Fig. 8, B and C).
Down-regulation of EDEM led to a prolongation of the half-life
of the Y611H mutant channel from 3.2 to 9.5 h, similar to that
found in ALLN-treated samples.
DISCUSSION
The present experiments demonstrate that degradation of
LQT2 mutant hERG channels is mediated by the ubiquitin-
proteasome pathway. We show that the misfolded hERG
protein is targeted to the proteasome for degradation by dislo-
cation from the ER membrane to the cytosol. The proteasome-
dependent degradation involves mannose trimming, ubiquiti-
nation, and deglycosylation of mutant channels.
Our pulse-chase experiments revealed important informa-
tion about the kinetics of the biogenesis of the wild type hERG
channel. About 60% of the core-glycosylated immature form of
wild type hERG is processed to the complex-glycosylated ma-
ture form. Compared with other polytopic membrane proteins,
the hERG channel has intermediate maturation efficiency. For
example, the maturation efficiency of the Shaker potassium
channel is near 100%, whereas less than 40% of newly synthe-
sized wild type cystic fibrosis transmembrane conductance and
delta opioid receptor are processed to the mature forms, and
the remaining immature forms are degraded by the ubiquitin-
proteasome pathway (18, 30, 31). Our data also suggest that a
fraction of wild type hERG protein that fails to mature is
degraded by the ubiquitin-proteasome pathway. In the pres-
ence of proteasome inhibitors, there is an increase in the im-
mature form of wild type hERG protein. In addition, protea-
some inhibition results in the accumulation of ubiquitinated
19423
Downloaded from http://www.jbc.org/ at FUDAN UNIVERSITY SCHOOL OF PHARMACY on May 24, 2018
FIG. 8. Effect of down-regulation of EDEM on the degradation
of the Y611H mutant. The Y611H mutant was cotransfected with
siEDEM or siGFP into HEK293 cells. Twenty-four h after transfection,
the intracellular concentration of EDEM mRNA was determined by
RNase protection assay (A), and the cotransfected cells were pulse-
labeled for 1 h and chased for the indicated times (B). The cell lysates
were subjected to immunoprecipitation with anti-hERG antibody and
SDS-PAGE. The amount of hERG protein was quantified by phosphor-
imaging analysis and plotted as percentage of the value at time point 0
(C). The data shown are means from three independent experiments.
wild type hERG channels. In the case of the delta opioid recep-
tor, proteasome inhibition leads to an increase in the cell sur-
face expression of functional receptors (18). In hERG, however,
inhibition of the proteasome does not result in an increase in
the mature form. Similarly, proteasome inhibition increases
the immature but not the mature form of wild type cystic
fibrosis transmembrane conductance (11).
Degradation of ER luminal and membrane proteins by the
cytosolic proteasome requires dislocation of substrates from the
ER to the cytosol. This retrograde transport is believed to occur
through the Sec61p ER translocation complex (32, 33). How-
ever, the mechanism of dislocation and the link between dislo-
cation and proteolysis by the proteasome are controversial. It
has been reported that dislocation and degradation by the
proteasome are tightly coupled events in several ERAD sub-
strates, suggesting that active proteasome is required for the
extraction of ERAD substrates from the ER (23, 34 –36). In this
model, ER proteins are directly dislocated from the ER mem-
brane into the membrane-bound proteasome for degradation.
The 19 S cap ATPases may provide the driving force for the
membrane extraction. However, some ERAD substrates have
been reported to accumulate in the cytosol when proteasome
activity is inhibited (18 –23). This suggests that proteasome
activity is not required for the dislocation of these ERAD sub-
strates from the ER to the cytosol. Recently, it has been shown
that the cdc48/p97䡠Ufd1䡠Npl4 complex is involved in the dislo-
cation of ERAD substrates from the ER to the cytosol (37, 38).
It is proposed that the ATPase activity of cdc48/p97 is involved
in extracting proteins from the ER membrane (39). In our
experiments, a fraction of the hERG channel protein accumu-
19424 Proteasomal Degradation of Mutant hERG Channels
lates in the cytosol following inhibition of the proteasome.
However, the fact that the rate at which the hERG channel
protein accumulates in the cytosol is much slower than the rate
of degradation in the absence of proteasome inhibitors indi-
cates that the efficiency of dislocation is reduced in the pres-
ence of proteasome inhibitors. A similar reduction in disloca-
tion efficiency by proteasome inhibitors has been reported for
other ERAD substrates (18, 20, 23). Thus, although proteasome
activity is not obligatory for the dislocation of hERG channel,
inhibition of the proteasome affects the efficiency of hERG
channel dislocation.
Our present results show that mannose trimming by ER
mannosidase I is required for the degradation of Y611H mu-
tant channels. We have previously shown that the hERG chan-
nel undergoes N-linked glycosylation at residue Asp-598 in the
extracellular domain between transmembrane segments 5 and
6 (24). N-Linked glycosylation begins with the en bloc transfer
of a preassembled oligosaccharide chain, Glu3Man9GluNA2, to
asparagine residues of glycoproteins in the ER (40). The oligo-
saccharide chain is subject to the trimming of glucose and
mannose residues by a number of glycosidases including glu-
cosidases I and II and ER mannosidases I and II. Several
reports have indicated the prerequisite of mannose trimming
by ER mannosidase I in proteasomal degradation of glycopro-
teins (25–29). In our experiments, the ER mannosidase I in-
hibitor kifunensine suppresses the degradation of Y611H mu-
tant channels. Unlike proteasome inhibitor treatment, in
which a fraction of hERG protein accumulates in the cytosol,
kifunensine treatment does not result in the cytosolic accumu-
lation of hERG protein. The results suggest that mannose
trimming contributes to the initial targeting process that is
upstream of the dislocation of the hERG protein in the ubiq-
uitin-proteasome pathway. This process may involve the inter-
action of the mannose-trimmed hERG channel with EDEM
because down-regulation of EDEM by RNA-mediated interfer-
ence suppresses the degradation of Y611H mutant channels.
Our results support the proposal that EDEM functions as a
lectin that recognizes the mannose-trimmed misfolded gly-
coproteins and promotes their degradation by the cytosolic
proteasome (9, 10, 41).
The present experiments show that the hERG channel is mod-
ified by ubiquitination. The requirement of ubiquitination for
proteasomal degradation has been shown in many ERAD sub-
strates (42). Recent studies also suggest that attachment of
polyubiquitin chains to ERAD substrates is required for their
dislocation from the ER to the cytosol (43– 45). In our experi-
ments, ubiquitinated hERG channels were recovered from both
membrane and cytosolic fractions, suggesting that ubiquitination
of hERG channels starts prior to their dislocation from the ER to
the cytosol. Thus, it is possible that ubiquitin modification may
contribute to dislocation as well as proteasomal degradation of
hERG channels. In addition to ubiquitin modification, hERG
channel accumulated in the cytosol was also found to be degly-
cosylated. The presence of deglycosylated intermediates in the
cytosol has been reported for other glycosylated ERAD substrates
(18, 21–23). These intermediates are believed to be the result of
deglycosylation of ERAD substrates by a cytoplasmic PNGase
prior to their degradation by the proteasome (21, 22). Recently,
genes encoding yeast cytoplasmic PNGase PNG1 and its mouse
homologue mPNG1 have been identified (46). Both Png1p and
mPng1p display N-glycanase activity toward intact glycoproteins
and are believed to be responsible for the deglycosylation of
ERAD substrates in the cytosol (47). The cytoplasmic PNGase
has been shown to interact with the proteasome and may play an
important role in proteasome-mediated degradation of misfolded
glycoproteins (48, 49).
Defective trafficking of mutant channels to the plasma mem-
brane has been increasingly recognized as an important mech-
anism of hERG channel dysfunction in LQT2. Trafficking-de-
fective LQT2 mutations lead to misfolding, ER retention, and
degradation of mutant hERG channels. Our present study sug-
gests that the ubiquitin-proteasome pathway plays an impor-
tant role in the ER retention and degradation of LQT2 mutant
channels. Because hERG belongs to a large superfamily of
voltage-gated potassium channels and defective trafficking of
disease-causing mutations has been reported in several volt-
age-gated potassium channels such as Kv1.1 and KvLQT1 (50 –
52), elucidating the mechanisms underlying the degradation of
LQT2 mutant channels will increase our knowledge of the
pathogenesis of a variety of human diseases involving voltage-
gated potassium channels.
Downloaded from http://www.jbc.org/ at FUDAN UNIVERSITY SCHOOL OF PHARMACY on May 24, 2018
Acknowledgments—We thank Drs. Linda S. Musil and
William R. Skach for helpful advice and discussion of the manuscript.
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Degradation of Trafficking-defective Long QT Syndrome Type II Mutant Channels
by the Ubiquitin-Proteasome Pathway
Qiuming Gong, David R. Keeney, Maurizio Molinari and Zhengfeng Zhou
J. Biol. Chem. 2005, 280:19419-19425.
doi: 10.1074/jbc.M502327200 originally published online March 10, 2005

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