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Figure legends
Figure 1. SEL1L degradation intermediates can be detected in cells treated with proteasome
inhibitor.
(A) Schematic representation of the domain organization of SEL1L protein. FN type II,
fibronectin type II domain; TM, transmembrane.
(B) HEK 293 cells were transfected with wild-type SEL1L, S-SEL1L, or SEL1L-HA, and then
treated with MG132 for 6 h. Cells were extracted in a buffer containing 1% NP-40, and
supernatant was collected. After separation by 10% SDS-PAGE, specific signals were detected
by Western blotting. Blue, black, and gray arrows indicate endogenous SEL1L, full-length SSEL1L, and full-length SEL1L-HA, respectively. Angle brackets denote the SEL1L degradation
intermediates. Asterisks show signals non-specifically detected by the antibodies. Lane numbers
in parentheses indicate the same sample loaded in the left panel.
(C) Same as in A, except that cells were transfected with FolA-S-HA, which was detected with
anti-HA-tag antibody. Arrow indicates full-length FolA-S-HA.
(D) HEK 293 cells were transfected with WT SEL1L and HA-Ubiquitin, and SEL1L protein was
immunoprecipitated with anti-SEL1L antibody. The bracket indicates ubiquitinated SEL1L
protein detected by the anti-HA antibody. ** indicates rabbit IgG contained in the anti-sera used
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for immunoprecipitation.
(E) Pulse-chase analysis of S-SEL1L expressed in HEK 293 cells. Cells were treated with MG132
for 3 h prior to metabolic labeling for 15 min (MG132 +) and chased for the indicated periods.
Cell lysates were immunoprecipitated using anti–S-tag or anti-SEL1L antibodies. The arrow
indicates full-length S-SEL1L, and the blue arrow indicates endogenous SEL1L. Angle brackets
denote degradation intermediates of S-SEL1L. Asterisk indicates a non-specific signal in MG132treated cells. Full-length S-SEL1L was quantified, and results are shown as means ± SD from
three independent experiments. **, P < 0.01; ***, P < 0.001 (two-tailed Student’s t-test, compared
with cells without MG132 treatment).
Figure 2. SEL1L degradation intermediates accumulate in the cytosol.
(A–C) HEK 293 cells expressing SEL1L-HA or S-SEL1L were fractionated into cytosol (CE),
membrane (ME), and nuclear extracts (NE), and separated by 10% SDS-PAGE. Specific signals
were detected by Western blot using anti-SEL1L (A) or anti–S-tag antibodies (B), or antibodies
against ER (calnexin [CNX]), and cytosolic proteins (actin, HSC70/HSP70) (C). Blue brackets
and arrows indicate endogenous SEL1L and full-length S-SEL1L, respectively. Angle brackets
denote SEL1L degradation intermediates. Lane numbers in parenthesis in (B) indicate the same
samples loaded in (A) and (C).
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(D, E) HEK 293 cells were homogenized by passing them through a 30-G needle in an isotonic
buffer containing 250 mM sucrose, and then cytosol (S: supernatant) and microsome (P: pellet)
fractions were separated by ultracentrifugation at 100,000 × g for 1 h. Specific signals were
detected by Western blotting using anti-SEL1L (D) or anti–S-tag antibodies (E). Blue brackets,
arrows and angle brackets are the same as in (A). Lane numbers in parenthesis in (E) indicate the
same samples loaded in (D).
Figure 3. SEL1L degradation intermediates are reduced and deglycosylated.
(A) Extracts of HEK 293 cells expressing the indicated plasmids were separated by 10% SDSPAGE under reducing (DTT +) or non-reducing (DTT -) conditions, and specific signals were
detected by Western blot analysis using anti-SEL1L antibody. Blue, gray, and black arrows
indicate endogenous SEL1L, full-length SEL1L-HA, and full-length S-SEL1L, respectively.
Angle brackets denote SEL1L degradation intermediates.
(B) Western blot analysis of SEL1L-HA and DssSEL1L-HA expressed in HEK 293 cells. Arrow
and arrowhead indicate full-length SEL1L-HA and DssSEL1L-HA, respectively. Angle brackets
denote SEL1L degradation intermediates. Asterisks indicate non-specific signals detected by antiHA antibody.
(C) HEK 293 cells expressing SEL1L-HA were treated with the PNGase inhibitor Z-VAD-FMK,
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and cell lysates were analyzed by Western blot. Red angle brackets indicate SEL1L-HA
degradation intermediates, whose migration was delayed by Z-VAD-FMK treatment.
(D) Two-dimensional isoelectric focusing (IEF) electrophoresis of SEL1L and DssSEL1L-HA.
Cells extracted in a buffer containing 9.5 M urea were separated by IEF containing Ampholine
(pH 3–10 and 4–7) in a tube gel, and then by 10% SDS-PAGE. After blotting onto PVDF
membranes, specific signals were detected by Western blotting using anti-HA antibody (left
panel). Double-headed arrows indicate full-length SEL1L-HA. Signals exhibiting different
mobility in IEF are indicated by arrows (red in SEL1L-HA and black in DssSEL1L-HA). The
same membranes were blotted sequentially with anti-actin (1), anti-BiP (2), and anti-calnexin (3,
3’) antibodies to confirm comparable IEF separation on both membranes (right panel). Asterisks
indicate non-specific signals detected by the antibodies.
(E) Same as in E, except that p97/VCP inhibitor MNS-873 was used.
Figure 4. Accumulation of SEL1L degradation intermediates is inhibited by co-expression of OS9 and XTP3-B
(A–C) HEK 293 cells co-transfected with S-SEL1L and NHK-QQQ, OS-9v2-HA, or XTP3-BHA were treated with proteasome inhibitor (MG132 +). Cell lysates were separated by 10% SDSPAGE, and specific signals were detected by Western blotting with anti-SEL1L (A, upper panel),
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anti-S-tag (A, lower panel), or indicated antibodies (B). Blue and black arrows indicate
endogenous SEL1L and full-length S-SEL1L, respectively. Angle brackets denote SEL1L
degradation intermediates.
(C) HEK 293 cells were co-transfected with WT SEL1L and the indicated ER proteins, and cell
lysates were subjected to Western blotting with the indicated specific antibodies. MG132 was
added from 6 h prior to harvest. Bracket indicates endogenous SEL1L and transfected full-length
SEL1L, and angle brackets indicate SEL1L degradation intermediates.
(D–G) Western blot analysis of HEK 293 cells transfected with the indicated siRNAs for 48 h
(D). Levels of endogenous SEL1L (E), OS-9 (F, including both OS-9v1 and v2), and XTP3-B (G)
in D were quantified relative to cells treated with negative control siRNA (left-most lane). Results
are shown as means ± SD from three independent experiments. ns, P > 0.05; **, P < 0.01; ***, P
< 0.001 (two-tailed Student’s t-test, compared with cells transfected with negative control siRNA).
(H) Pulse-chase analysis of NHK-QQQ degradation. HEK 293 cells co-transfected with NHKQQQ, SEL1L, and OS-9v2-HA were metabolically labeled for 15 min and chased for the
indicated period. NHK-QQQ was immunoprecipitated and separated by 10% SDS-PAGE.
Radioisotope incorporation into NHK-QQQ is quantified in the graph. Error bars indicate means
± SD from three independent experiments. *, P < 0.05; ***, P < 0.001 (two-tailed Student’s t-
test, compared with cells co-transfected with SEL1L).
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(I) Pulse-chase analysis of S-SEL1L degradation. Same as in A, except cells were transfected
with S-SEL1L and OS-9v2-HA and immunoprecipitated using anti–S-tag or anti-HA antibodies.
Radioactive incorporation to S-SEL1L was quantified in the graph. Error bars indicate means ±
SD from three independent experiments. *, P < 0.05; **, P < 0.01 (two-tailed Student’s t-test).
Figure 5. Degradation intermediates of endogenous SEL1L are detected in HEK 293 cells
exposed to ER stress
(A) Western blot analysis of endogenous SEL1L upon treatement with thapsigargin (Tg) or
tunicamycin (Tm). For clarity, the obtained images are shown in grayscale (top) or using the “fire”
lookup table of ImageJ. Blue brackets indicate full-length endogenous SEL1L. Angle brackets
denote SEL1L degradation intermediates. Double angle brackets indicate deglycosylated SEL1L.
(B) Western blot analysis of endogenous SEL1L in HEK 293 cells transfected with indicated
siRNAs. Thirty hours after transfection of siRNAs, cells were treated with MG132 for 6 h
(MG132 +). The same image is shown using the “fire” lookup table of ImageJ. The asterisk
indicates a non-specific signal detected by anti–OS-9 antibody.
(C) Immunoprecipitation analysis of S-SEL1L and HRD1-myc co-expressed in HEK 293 cells.
Cells were lysed in a buffer containing 1% NP-40 (NP-40) or 3% digitonin (Dig), and
immunoprecipitated using anti-S-tag or anti-c-myc antibodies. ** indicates mouse IgG used for
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immunoprecipitation.
(D) Alkaline extraction of SEL1L-HA, S-SEL1L and HRD1-myc expressed in HEK 293 cells.
The microsomal fraction was prepared as in Fig. 2C and D from cells co-expressing SEL1L-HA
and HRD1-myc (left panel) or S-SEL1L and HRD1-myc (right panel). After incubation in an
alkaline solution containing 0.1 M Na2CO3 or in a buffer containing 1% NP-40, a supernatant
(Sup) and pellet (Ppt) were fractionated by ultracentrifugation at 100,000 × g.
(E) Pulse-chase analysis of S-SEL1L co-expressed with or without HRD1-myc in HEK 293 cells.
Cells were pulse-labeled for 15 min and chased for the indicated periods. Cell lysates were
immunoprecipitated using anti–S-tag or anti-SEL1L antibodies. The bracket indicates full-length
S-SEL1L. The blue arrow indicates endogenous SEL1L. Full-length S-SEL1L was quantified.
Results are shown as means ± SD from three independent experiments. *, P < 0.05; **, P < 0.01;
***, P < 0.001 (two-tailed Student’s t-test, compared with cells without co-expression of HRD1myc).
(F) Cycloheximide-chase analysis of NHK degradation. HEK 293 cells co-transfected with NHK,
HRD1-myc, and S-SEL1L were chased for the indicated period after addition of cycloheximide.
The cell lysate was separated by 10% SDS-PAGE and analyzed by Western blotting. The signal
intensity of NHK is quantified in the graph. Error bars indicate means ± SD from three
independent experiments. *, P < 0.05; **, P < 0.01 (two-tailed Student’s t-test, compared with
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cells co-transfected with HRD1-myc).
Figure 6. Deletion of SEL1L proline-rich region promotes SEL1L degradation.
(A) Western blot analysis of HEK 293, HeLa, HepG2 (HG2), RD, and Capan2 cells (upper panel),
and Coomassie Brilliant Blue (CBB) staining of the same membrane (lower panel). Twenty
micrograms of each cell lysate was separated by SDS-PAGE and blotted with anti-SEL1L
antibody.
(B) Cells were transfected with SEL1L-HA and treated with MG12 for 6 h (MG132 +). Arrows
indicate full-length SEL1L-HA, and angle brackets denote SEL1L-HA degradation intermediates.
Asterisks indicate non-specific signal detected by anti-HA antibody.
(C) Western blot analysis of SEL1L-HA and SEL1L-DPR-HA expressed in HEK 293 cells. Red
arrow and angle brackets denote full-length SEL1L-DPR-HA and degradation intermediates,
respectively.
(D) Pulse-chase analysis of NHK-QQQ and SEL1L-DPR-HA degradation. HEK 293 cells cotransfected with NHK-QQQ and SEL1L-HA or SEL1L-DPR-HA were metabolically labeled for
15 min and chased for the indicated period. Radioisotope incorporation into NHK-QQQ and
SEL1L-HA or SEL1L-DPR-HA is quantified in the graph. Error bars indicate means ± SD from
three independent experiments. *, P < 0.05; **, P < 0.01 (two-tailed Student’s t-test).
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Figure 7. Htt-polyQ76-GFP aggregates form in HEK293 cells.
(A) HEK 293 cells were transfected with Htt-polyQ76-GFP and SEL1L-HA or DssSEL1L-HA.
Twenty-four hours after transfection, nuclei were stained with Hoechst 33342 and analyzed by
fluorescence microscopy. MG132 was added 6 h prior to microscopic observation (MG132 +).
Scale bars, 50 µm.
(B) Cells in A containing Htt-polyQ76-GFP aggregates were counted, and the percentage of GFPpositive cells (~300–500 cells) with aggregates was calculated. Error bars indicate means ± SD
from three independent experiments. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (twotailed Student’s t-test, compared with mock-transfected cells (MG132 -), or between the samples
indicated by the bracket).
(C) Immunocytochemistry of HeLa cells transfected with SEL1L-HA or DssSEL1L-HA, treated
with or without MG132. Cells were fixed and stained with anti-HA and anti-calreticulin (CRT,
ER marker, left panel) or anti-HA and anti-HSP70/HSC70 (cytosolic marker) antibodies (right
panel). SEL1L-HA and DssSEL1L-HA were visualized with Alexa Fluor 594, CRT and
HSP70/HSC70 were stained with Alexa Fluor 488, and images were acquired by confocal
microscopy. Scale bars, 10 µm.
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Figure 8. Immunostaining of polyQ inclusions.
HEK 293 cells (A, B, C, E, and F) and HeLa cells (D) were transfected with Htt-polyQ76-GFP
and SEL1L-HA (A, B and D), Htt-polyQ76-GFP and DssSEL1L-HA (C, D), Htt-polyQ23 and
SEL1L-HA (E), or Htt-polyQ23 and DssSEL1L-HA (F). Twenty-four hours after transfection,
cells were fixed and stained with anti-GFP and anti-HA antibodies. GFP was visualized with
Alexa Fluor 488, HA was stained with Alexa Fluor 596, and images were analyzed by confocal
microscopy. MG132 was added 6 h prior to microscopic observation (MG132 +). Nuclei were
counterstained with DAPI. Scale bars, 10 µm (upper panel) and 5 µm (magnified).
Figure 9. SEL1L SLR expressed in the cytosol promotes polyQ aggregation.
(A, B) Htt-polyQ76-GFP aggregation formation analyzed by fluorescence microscopy. Same as
in Fig. 7A and B, except that cells were co-transfected with SEL1L-R5-9-HA.
(C) Immunostaining of Htt-polyQ76-GFP. Same as in Fig. 8B, except that cells were cotransfected with SEL1L-R5-9-HA.
(D, E) Filter-trap assays of Htt-polyQ76-GFP co-transfected with SEL1L-HA, Htt-polyQ23-GFP
(D), and Htt-polyQ76-GFP co-transfected with mock, DssSEL1L-HA, or SEL1L-R5-9-HA (E). Cell
lysates were serially diluted, and the indicated amounts of protein were applied to the filter.
(F) Model of HRD1–SEL1L complex formation. SEL1L and HRD1 form a stable complex in the
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ER membrane, but SEL1L expressed in excess is rapidly degraded by ERAD (left). OS-9 and
XTP3-B associate with and stabilize SEL1L until the HRD1–SEL1L complex is formed.
Alternatively, SEL1L–lectin complex surveils the ER for misfolded proteins and dynamically
forms a HRD1-containing membrane complex that is competent for retrotranslocation (right).
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Figure 1
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Figure 3
A Self-archived copy in
Kyoto University Research Information Repository
https://repository.kulib.kyoto-u.ac.jp
Figure 4
A Self-archived copy in
Kyoto University Research Information Repository
https://repository.kulib.kyoto-u.ac.jp
Figure 5
A Self-archived copy in
Kyoto University Research Information Repository
https://repository.kulib.kyoto-u.ac.jp
Figure 6
A Self-archived copy in
Kyoto University Research Information Repository
https://repository.kulib.kyoto-u.ac.jp
Figure 7
A Self-archived copy in
Kyoto University Research Information Repository
https://repository.kulib.kyoto-u.ac.jp
Figure 8
A Self-archived copy in
Kyoto University Research Information Repository
https://repository.kulib.kyoto-u.ac.jp
Figure 9
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