Ubiquitin-dependent regulation of COPII
coat size and function
Lingyan Jin1*, Kanika Bajaj Pahuja1,2*, Katherine E. Wickliffe1, Amita Gorur1,2, Christine Baumga ¨rtel1, Randy Schekman1,2
& Michael Rape1
Packaging of proteins from the endoplasmic reticulum into COPII vesicles is essential for secretion. In cells, most COPII
vesicles are approximately 60–80nm in diameter, yet some must increase their size to accommodate 300–400nm
procollagen fibres or chylomicrons. Impaired COPII function results in collagen deposition defects, cranio-
lenticulo-sutural dysplasia, or chylomicron retention disease, but mechanisms to enlarge COPII coats have remained
elusive. Here, we identified the ubiquitin ligase CUL3–KLHL12 as a regulator of COPII coat formation. CUL3–KLHL12
catalyses the monoubiquitylation of the COPII-component SEC31 and drives the assembly of large COPII coats. As a
result, ubiquitylation by CUL3–KLHL12 is essential for collagen export, yet less important for the transport of small
cargo. We conclude that monoubiquitylation controls the size and function of a vesicle coat.
The extracellular matrix provides a scaffold for cell attachment and
binding sites for membrane receptors, such as integrins, making it
essential for the development of all metazoans1,2. When engaged with
the extracellular matrix, integrins trigger signalling cascades that
regulate cell morphology and division, yet in the absence of a func-
tional extracellular matrix, integrins are removed from the plasma
membrane by endocytosis3. The proper interplay between integrins
and the extracellular matrix is particularly important during early
development4, as stem cells depend on integrin-dependent signalling
for division and survival5.
The establishment of the extracellular matrix requires secretion of
several proteins, including its major constituent collagen. Following
cells depends on COPII vesicles6–9, and mutations in genes encoding
COPII proteins lead to collagen deposition defects, skeletal aberra-
tions and developmental diseases, such as cranio-lenticulo-sutural
COPII vesicles are surrounded by a coat consisting of the SAR1
GTPase, SEC23–SEC24 adaptors, and an outer layer of SEC13–SEC31
structures with a diameter of approximately 60–80nm, which are too
small to accommodate a procollagen fibre with a length of 300–
400nm13–15. Thus, collagen transport in cells must involve factors that
known as MIA3) and its partner cTAGE5 interact with collagen and
SEC23–SEC24, thereby recruiting collagen to nascent COPII coats16,17.
The deletion of Tango1 in mice resulted in collagen deposition defects
similar to those caused by loss of COPII18, and mutations in human
TANGO1 are associated with premature myocardial infarction19.
However, TANGO1 is not known to regulate the size of COPII coats
and mechanisms that permit the COPII coat to accommodate a large
cargo remain poorly understood.
By analysing mouse embryonic stem (ES) cell division, we have
identified CUL3–KLHL12 as a regulator of COPII coat formation.
COPII coats. As a result, ubiquitylation by CUL3–KLHL12 is essential
ES cell division. We conclude that monoubiquitylation determines the
size and function of a vesicle coat.
CUL3 regulates mouse ES cell morphology
To provide insight into stem cell-specific division networks, we
depleted ubiquitylation enzymes from mouse ES cells and scored
for effects on proliferation and morphology. We found that loss of
the ubiquitin ligase CUL3 caused mouse ES cells to form tightly
as seen by confocal microscopy analysis of actin and vinculin local-
ization (Fig. 1a). A similar phenotype was observed upon depletion
of UBA3, a component of the NEDD8 pathway that activates CUL3
(Supplementary Fig. 1a). CUL3-depleted mouse ES cells were
delayed in proliferation (Supplementary Fig. 1b, d), yet retained their
the absence of differentiation markers in expression analyses (Sup-
of CUL3 had weaker consequences in fibroblasts (Fig. 1a), although a
previously reported increase in multinucleation was observed (Sup-
plementary Fig. 1g; ref. 20).
Several observations show that the mouse ES cell phenotypes were
caused by specific depletion of CUL3. First, several short interfering
RNAs targeting distinct regions of the Cul3 messenger RNA had the
same effects on mouse ES cells, with a close correlation between
knockdown efficiency and strength of phenotype (Supplementary
Fig. 2a). Second, microarray analysis showed a strong reduction in
Cul3 mRNA upon siRNA treatment, whereas no other gene was sig-
nificantly and reproducibly affected (Supplementary Fig. 2b). Third,
siRNAs that target closely related proteins, such as other cullins, did
The aberrant morphology of CUL3-depleted mouse ES cells was
reminiscent of increased RhoA GTPase activity, which triggers actin
ES cells from compaction (Supplementary Fig. 3a). Among several
possibilities, higher RhoA activity in the absence of CUL3 could result
from RhoA stabilization or defective integrin signalling. Stabilization
*These authors contributed equally to this work.
1Department of Molecular and Cell Biology, University of California at Berkeley, California 94720, USA.2Howard Hughes Medical Institute, University of California at Berkeley, California 94720, USA.
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of RhoA by co-depletion of all RhoA-specific CUL3 adaptors, the
BACURDs22, did not affect mouse ES cell morphology (data not
shown). By contrast, depletion of components of integrin signalling
pathways phenocopied the loss of CUL3 in mouse ES cells (Sup-
lethalitywithdasatinib,aninhibitorofthe SRC kinasethat actsdown-
stream of integrin activation (Supplementary Fig. 3c); and integrin b1
was absent from the plasma membrane of CUL3-depleted mouse ES
cells (Fig. 1b).
CUL3 could regulate integrin synthesis and trafficking, or it could
exogenous extracellular matrix. Strikingly, under these conditions,
integrin b1 was found at the plasma membrane of CUL3-depleted
mouse ES cells and no cell clustering was observed (Fig. 1b). Thus,
CUL3 controls integrin signalling in mouse ES cells, most likely by
supporting the establishment of a functional extracellular matrix.
KLHL12 is a key CUL3 adaptor in mouse ES cells
siRNA approaches did not yield roles for BTB proteins in ES cells. As
an alternative strategy to isolate CUL3 adaptors, we made use of the
observation that stem cell regulators are highly expressed in ES cells,
but downregulated upon differentiation27. Using affinity purification
and mass spectrometry, we identified 31 BTB proteins that interact
with CUL3 in mouse ES cells (Supplementary Fig. 4a; Supplementary
Table 1). When analysed by quantitative polymerase chain reaction
with reverse transcription (qRT–PCR) and immunoblot, we found
that three adaptors, KLHL12, KBTBD8 and IBTK, were highly
expressed in mouse ES cells, but downregulated upon differentiation
from mouse ES cells that were sensitized for changes in integrin sig-
but no other BTB protein, resulted in mouse ES cell compaction, as
seen with loss of CUL3 (Fig. 2c). Accordingly, endogenous KLHL12
effectively binds CUL3 in mouse ES cells (Supplementary Fig. 4b).
These experiments, therefore, identify KLHL12 as a key substrate-
ligase as an important regulator of mouse ES cell morphology.
CUL3 monoubiquitylates SEC31
To isolate the substrates of CUL3–KLHL12, we constructed 293T cell
lines that allowed for the inducible expression of Flag–KLHL12. By
affinity chromatography and mass spectrometry, we identified the
Control siCul3 Control siCul3
Mouse ES cells
Integrin β1 DNA Actin Merge
Figure 1 | CUL3 regulates mouse ES cell morphology. a, Left, D3 mouse ES
cells were plated on gelatin and transfected with siRNAs targeting Cul3
(siCul3),whichresultedincell clustering (phase microscopy;upperpanel)and
compaction (confocal microscopy: vinculin, green; actin, red; DNA, blue).
Right, Depletion of CUL3 from mouse 3T3 fibroblasts did not cause cell
compaction. Phase images original magnification was 310, fluorescence
images 340. b, CUL3 is required for integrin localization to the mouse ES cell
plasma membrane. D3 mouse ES cells were plated on gelatin (top two rows),
compaction and integrin-targeting to the plasma membrane were analysed by
confocal microscopy (actin, red; integrin b1, green; DNA, blue). Original
EB, 6 days
EB, 9 ddays
EB, 6 days
EB, 9 days
Figure 2 | KLHL12isa substrateadaptor for CUL3in mouseEScells. a,D3
mouseEScellswere subjected todifferentiation,andmRNAlevels ofindicated
is downregulated upon differentiation, as observed by immunoblot of above
cells were sensitized towards altered integrin-signalling with dasatinib and
monitored for compaction by phase (upper panel) or confocal microscopy
(actin, red; vinculin, green; DNA, blue). Original magnification 340.
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COPII proteins SEC13 and SEC31 as specific binding partners of
KLHL12 (Fig. 3a and Supplementary Table 2). Immunoblotting con-
firmed retention of endogenous SEC13 and SEC31 in KLHL12 purifi-
cations, but not in precipitates of other BTB proteins (Supplementary
Fig. 5a). As seen in pull-down assays, KLHL12 directly bound SEC31,
but not SEC13 (Supplementary Fig. 5c, d), and this interaction was
mediated by the amino terminus of SEC31 (Supplementary Fig. 6a)
and the Kelch domain of KLHL12 (Supplementary Fig. 6b). In cells,
approximately 30% of endogenous KLHL12 was associated with
SEC13–SEC31 (Fig. 3b and Supplementary Fig. 5b). Consistent with
in punctae, which are likely to represent endoplasmic reticulum exit
COPII resulted in mouse ES cell compaction (Fig. 3d), indicating that
CUL3–KLHL12 and the COPII coat act in the same pathway.
In vitro, CUL3–KLHL12 catalysed the monoubiquitylation of
SEC31 (Fig. 3e), which was not observed if a KLHL12 mutant with
monoubiquitylated in cells, which was strongly increased upon
expression of KLHL12 (Fig. 3g). KLHL12 mutants unable to bind
SEC31 abolished its monoubiquitylation (Fig. 3h), which is likely to
be due to dimerization with and inactivation of endogenous KLHL12
(Fig. 3a and Supplementary Fig. 6c). SEC31 monoubiquitylation was
also strongly diminished upon expression of dominant-negative
CUL3 (Fig. 3g) or depletion of CUL3–KLHL12 by siRNA (Fig. 3i).
As seen upon expression of lysine-free ubiquitin, SEC31 was mono-
ubiquitylated at one preferred and an alternative, less prominently
used lysine (Fig. 3g), consistent with proteomic analyses that iden-
tified Lys647 and Lys1217 in SEC31A as ubiquitylation sites29,30.
However, neither mutation of these residues nor any other of the 65
lysine residues of SEC31 blocked ubiquitylation by CUL3–KLHL12
(data not shown), revealing flexibility in the actual modification site.
Co-expression of KLHL12 and CUL3 triggered SEC31 multiubi-
quitylation and degradation (Figs 3g, 4e and Supplementary Fig. 6d),
whichwasnotobservedwithlysine-free ubiquitin (Fig.3g).However,
whereas SEC31 was monoubiquitylated by endogenous CUL3–
KLHL12, its multiubiquitylation was only seen when CUL3 and
some inhibition did not change SEC31 levels in untransfected cells
(Fig. 3i and Supplementary Fig. 6e), and blockade of ubiquitin chain
formation or proteasome inhibition did not impair CUL3–KLHL12
function (see Fig. 5). Thus, multiubiquitylation of SEC31 is unlikely a
key outcome of CUL3–KLHL12 activity in mouse ES cells. Instead, it
seems that CUL3–KLHL12 acts by catalysing monoubiquitylation,
with the COPII protein SEC31 as a major substrate.
CUL3 regulates the size of COPII coats
To identify a role for monoubiquitylation by CUL3–KLHL12, we
induced KLHL12 expression in cells and followed the fate of SEC31
by microscopy. Shortly after KLHL12 induction, the majority of
(Fig. 4a, b). As seen by high-resolution confocal imaging, the large
structures were hollow and spherical with a diameter of 200–500nm,
and they were decorated with the proteins of the COPII coat and
with KLHL12 (Fig. 4c).Accordingly,thin-section electronmicroscopy
lum origin, in cells transfected with KLHL12 (Fig. 4d). Immunogold-
labelling electron microscopy showed comparable structures of
100 4 41rel. SEC31-ubi
Ni-NTA pull-down Input
Figure 3 | CUL3–KLHL12 monoubiquitylates SEC31.
a, Immunoprecipitates of Flag–KLHL12 or Flag–Klhl9 were analysed by silver
staining and mass spectrometry. Asterisk, non-specific band; double asterisk,
colocalizes with COPII, as seen by confocal microscopy (KLHL12, green;
SEC13, red; DNA, blue). Original magnification 360. d, D3 mouse ES cells
(top) or confocal microscopy (actin, red; vinculin, green; DNA, blue). Original
magnification 340. e, CUL3–KLHL12 monoubiquitylates SEC31. CUL3–
NEDD8–RBX1 was incubated with KLHL12, SEC13/31 and ubiquitin (ubi) or
His-ubiquitin (His-ubi). f, In vitro ubiquitylation of SEC31by CUL3–KLHL12
or CUL3–KLHL12(FG289AA) (FG289AA) was performed as above. g, SEC31
ismonoubiquitylatedinvivo. Upperpanels, ubiquitinconjugateswere purified
under denaturing conditions from MG132-treated 293T cells expressing His–
ubiquitin, haemagglutinin–SEC31, KLHL12, CUL3 or dominant-negative
CUL3(dnCUL3), andanalysedbyanti-SEC31 Western blot.Lowerpanels,the
same experiment was performed with lysine-free His–ubiquitin, which only
allowed SEC31-monoubiquitylation on at least two sites (SEC31-ubi and
SEC31-ubi*). SEC31-ubi-n denotes multiubiquitylated SEC31. h, Ubiquitin
conjugates were purified from 293T cells expressing KLHL12 or SEC31-
binding deficient KLHL12 mutants. i, CUL3 is essential for SEC31
ubiquitylation in vivo. 293T cells were transfected with His–ubiquitin and
siRNAs, and ubiquitin conjugates were analysed for SEC31 by Western blot.
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which is absent from procollagen transport vesicles31; endoplasmic
reticulum membrane markers that do not accumulate at endoplasmic
reticulum exit sites32; nor endosomal or autophagosomal markers
(Supplementary Fig. 7a–c). Importantly, SEC31-binding deficient
mutants, including KLHL12(FG289AA), neither colocalized with
SEC31 nor induced formation of large structures (Fig. 4e and Sup-
structures by KLHL12 (Fig. 4b). Thus, binding of KLHL12 to SEC31
triggers formation of large COPII-containing structures.
When KLHL12 was expressed with a CUL3 mutant that blocks
SEC31 ubiquitylation (CUL3(1–250)), COPII structures were not
enlarged (Fig. 4e). In addition, depletion of CUL3 by siRNAs, which
also abolishes SEC31 monoubiquitylation, prevented formation of
large COPII structures by KLHL12 (Fig. 4a, c). By contrast, if
KLHL12 was expressed with lysine-free ubiquitin to allow mono-,
but not multiubiquitylation, large COPII structures were readily
detected (Fig. 4c, e), and these structures were enriched for ubiquitin,
consistent with monoubiquitylation being non-proteolytic (Sup-
plementary Fig. 7e). Thus, monoubiquitylation by CUL3–KLHL12
promotes formation of large COPII structures, which probably rep-
resent a mixture of nascent coats at endoplasmic reticulum exit sites
and budded coats on large COPII vesicles or tubules.
CUL3 is required for collagen export
extracellular matrix, which requires collagen secretion. Thus, the
CUL3–KLHL12-dependent increase in COPII size might function to
promote collagen export from the endoplasmic reticulum. To test this
accumulate collagen in the endoplasmic reticulum due to inefficient
export.Strikingly, KLHL12, butnotKLHL12(FG289AA) or unrelated
BTB proteins, triggered depletion of procollagen I from intracellular
endoplasmic reticulum pools (Fig. 5a). As a result, increased collagen
not KLHL12(FG289AA) (Fig. 5b). When secretion was inhibited with
brefeldin A, or if collagen folding in the endoplasmic reticulum was
impaired by removal of ascorbate from the medium, procollagen
remained within KLHL12-expressing cells (Fig. 5a). Time-resolved
from IMR90 cells (Fig. 5c). Shortly after inducing secretion, KLHL12
and collagen were detected at overlapping locations (Supplementary
Fig. 7f), all of which indicates that CUL3–KLHL12 facilitates collagen
traffic from the endoplasmic reticulum.
Blockade of SEC31 ubiquitylation by dominant-negative CUL3
interfered with the KLHL12-dependent export of collagen from
IMR90 cells (Supplementary Fig. 8a). Similarly, depletion of CUL3–
KLHL12 from engineered HT1080 fibrosarcoma cells severely
3 h5 h24 h
SEC31 SEC13 SEC24C
KLHL12 ± CUL3/
Figure 4 | CUL3–KLHL12-dependent
monoubiquitylation enlarges COPII-structures.
a, Localization of doxycycline (dox)-induced Flag–
KLHL12 (dox::Klhl12, green) and SEC31 (red) in
293T cells, monitored by confocal microscopy.
Scale bar, 3mm. b, KLHL12-expressing HeLa cells
were analysed for KLHL12 (green) and SEC31,
SEC13 or SEC24C (red) by confocal microscopy.
Scale bar, 3mm. c. COPII-structures in HeLa cells
transfected with Flag–KLHL12, lysine-free
ubiquitin (ubi-K0) or Cul3-siRNA, analysed by
confocal microscopy. Scale bar, 500nm. d, Upper
panel, thin-section electron microscopy (EM) of
KLHL12-expressing or control HeLa cells (red
arrow, KLHL12-dependent structures; blue arrow,
small control vesicles). Scale bar, 500nm. Lower
panel, immunogold-EM of KLHL12 in transiently
transfected HeLa (left) or stable 293T cells (right).
Scale bar, 200nm. e, HeLa cells transfected with
Flag–KLHL12, lysine-free ubiquitin, Flag–
KLHL12(FG289AA), Flag–CUL3(1–250) or Flag–
CUL3 were analysed for localization of KLHL12/
CUL3 (green) and SEC31 (red) by confocal
microscopy. Original magnification 340.
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contrast, smaller COPII cargoes, such as fibronectin or EGF receptor,
were properly localized in the absence of CUL3 (Supplementary Fig.
comparable to the effects observed upon loss of SEC13 (Fig. 5e and
If promoting collagen export were the key role of CUL3 in mouse
ES cells, the phenotypes of CUL3 depletion might be mitigated by
cells were plated on purified collagen IV, depletion of CUL3 did not
cause cell clustering, and integrin b1 was detected at the plasma
membrane (Fig. 1b). We conclude that promoting collagen secretion
is a key a function of CUL3, in agreement with its role in driving the
assembly of large COPII coats.
In this study, we have identified CUL3–KLHL12 as an essential regu-
lator of collagen export, which is required for mouse ES cell division.
Deletion of Cul3 in mice results in early embryonic lethality with
completely disorganized extraembryonic tissues33, a phenotype that
can in part be attributed to its role in collagen secretion. Moreover,
disorder Sjogren’s syndrome34, raising the possibility that aberrant
function of CUL3–KLHL12 might be related to disease.
CUL3–KLHL12 monoubiquitylates SEC31 and promotes forma-
tion of large COPII coats that can accommodate unusually shaped
cargo. As a result, CUL3 is essential for the secretion of procollagen
fibres, whereas it is not required for the transport of smaller or more
flexible molecules, such as fibronectin, EGF receptor or integrin b1.
Thus, CUL3–KLHL12 seems to be specifically required for the
COPII-dependent transport of large cargo.
How ubiquitylation affects COPII coat size or structure is not
known. None of the 65 lysine residues of SEC31 was essential for
ubiquitylation by CUL3–KLHL12, showing that CUL3 can target
alternative lysine residues if the primary site is blocked. Despite this
flexibility, CUL3–KLHL12 does not stoichiometrically ubiquitylate
SEC31. Thus, if SEC31 ubiquitylation performs astructural role, then
few ubiquitylated molecules must suffice to produce large COPII
coats, and these vesicles must tolerate considerable variation in the
proteins, modified SEC31 might recruit an effector that delays COPII
budding or promotes coat polymerization. As CUL3–KLHL12 ubi-
quitylates other proteins35, SEC31 may not be its only substrate in the
secretory pathway. Identification of the complete set of CUL3–
KLHL12 substrates and potential effector molecules should reveal
the mechanism underlying the ubiquitin-dependent regulation of
Our findings have the potential to be translated into therapeutic
or Sar1 mutations in chylomicron retention disease10,11. By contrast,
interfering with CUL3 activity may counteract increased collagen
Cells with secreted
collagen I (%)
Cells with ER-resident
collagen I (%)
0 2040 60
Cells with collagen
secreted from ER (%)
Time after ascorbate
Figure 5 | CUL3–KLHL12 promotes collagen export. a, IMR90 cells
transfected with Flag–KLHL12, Flag–KLHL12(FG289AA) or Flag–KEAP1
were analysed by confocal microscopy (BTB, green; collagen-I, red; DNA,
blue). When noted, cells were treated with chloroquine, MG132, brefeldin A
(BFA) or dialysed medium lacking ascorbate. Errors bars, standard deviation
n53.Originalmagnification360.b, Cell lysate (L)orculture medium(M)of
IMR90 cells transfected with Flag–KLHL12 or Flag–KLHL12(FG289AA) was
analysed by immunoblotting. c, Collagen I localization was analysed in IMR90
340. d, HT1080 cells stably expressing collagen I were transfected with
shRNAs against Cul3 and analysed by confocal microscopy (transfection
control green fluorescent protein (GFP), green; protein disulphide isomerase
(PDI), blue; collagen I, red). Error bars, standard deviation n53. Original
magnification 360. e, D3 mouse ES cells were treated with control siRNAs or
IV, green; actin, red; DNA, blue). Original magnification 340.
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deposition during fibrosis or keloid formation36. Given the strong
clustering phenotypes observed in CUL3-depleted mouse ES cells,
inhibition of CUL3–KLHL12 might impair the proliferation of
metastatic cells, which display features of undifferentiated cells37,38.
and function provides an exciting starting point to understand and
therapeutically exploit key events in protein trafficking.
For stem cell culture, mouse D3 ES cells were maintained in GIBCO Dulbecco’s
Modified Eagle ES cell medium containing 15% FBS, 13 sodium pyruvate, 13
inhibitory factor (Millipore), and grown on gelatin-coated culture plates.
Doxycycline-inducible 293T Trex Flag–BTB stable cell lines were made with the
Flp-In T-REx 293 Cell Line system (Invitrogen) and maintained with blasticidin
and hydromycin B.
For screening, two siRNA oligonucleotides were designed against 40 mouse
ubiquitin ligases (Qiagen). siRNA oligonucleotides (10pmol) and Lipofectamine
ES cell colonies was examined by bright-field microscopy 48h after transfection.
To identify CUL3–KLHL12 substrates, doxycycline-inducible 239T cell lines
expressing Flag–KLHL12 or Flag–KLHL9 were induced for 48h. Cleared lysate
was subjectedto anti-FlagM2 affinity gel(Sigma),and precipitationswere eluted
with 33Flag peptide (Sigma). Concentrated eluates were analysed by SDS–
PAGE, and specific bands were identified by mass spectrometry analysis by the
Vincent J. Coates Proteomics/Mass Spectrometry Laboratory.
For in vitro ubiquitylation reactions, CUL3/RBX1 purified from Sf9 cells was
conjugated to NEDD8 using recombinant APPBP1–UBA3, UBC12 (also known
as UBE2M) and NEDD8. KLHL12 purified from Escherichia coli and SEC31A–
SEC13 complexes from Sf9 cells were added together with energy mix, E1,
UBCH5C(alsoknown as UBE2D3) and ubiquitinand incubatedat 30uC for 1h.
For confocal microscopy, cells fixed in paraformaldehyde and permeabilized
with Triton X-100 were incubated with primary antibodies for 2h and Alexa-
labelled secondary antibodies (Invitrogen) for 1h. Pictures were taken on Zeiss
LSM 510 and 710 confocal microscopes and analysed with LSM image browser
and Imaris 3D imaging processing software. Images were processed for contrast
enhancement to remove noise.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 3 August 2011; accepted 3 January 2012.
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We are grateful to J. Schaletzky for critically reading the manuscript and many
discussions. We thank the members of the Rape and Schekman labs for advice and
suggestions, L. Lim for providing Cul3-shRNAs, C. Glazier for contributions on BTB
funded by grants from the Pew Foundation (M.R.), the NIH (NIGMS-RO1, M.R.; NIH
long term post-doctoral fellow.
Author Contributions Experiments were designed by L.J., K.B.P., R.S. and M.R.; L.J.
performed the mouse ES cell screen, identified KLHL12 and SEC31, and analysed the
analysed collagen export in fibroblasts; K.E.W. analysed COPII formation in cells; C.B.
identified inactive KLHL12; A.G. performed electron micrscopy; L.J., K.B.P. and M.R.
prepared the manuscript.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of this article at
www.nature.com/nature. Correspondence and requests for material should be
addressed to M.R. (firstname.lastname@example.org).
5 0 0 | N A T U R E | V O L 4 8 2 | 2 3 F E B R U A R Y 2 0 1 2
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and pcDNA5 vectors for expression in mammalian cells. Cul3, Sec31A and Sec13
were also cloned into pCS2 vector for IVT/T and expression in mammalian cells.
pcDNA4-Cul3N250contains thefirst cullin repeatoftheN-terminal CUL3( amino
as a dominant negative for CUL3/BTB-mediated ubiquitylation. The KLHL12
mutants FG289AA, RL342AA, RGL369AAA, RE416AA, YDG434AAA and
RCY510AAA were made by site-directed mutagenesis.
and UBA1 were purified from Sf9 ES insect cells. UbcH5c and Ubc12 were cloned
into pET and pCS2 vector with a N-terminal 63His tag. The pET-His-ubiquitin
was used for bacterial purification whereas pCS2-His-ubiquitin was expressed in
mammalian cells. Wild-type ubiquitin, APPBP1–UBA3 and NEDD8 were pur-
chased from Boston Biochem.
To purify recombinant KLHL12 for ubiquitylation assays, we expressed
pMAL-TEV-KLHL12-his and pMAL-TEV-KLHL12FG289AA-his in BL21(DE3)
cells, purified the proteins on amylose resin, cleaved them by TEV protease,
and re-purified them on Ni-NTA agarose. Wild-type Klhl12 and mutants were
also cloned into pMAL vector and purified as maltose-binding protein (MBP)-
tagged proteins for in-vitro protein binding assays.
All shRNAs were cloned in pSuper-GFP neo vector (from Oligoengine) into
cytochrome b5, was purchased from Clontech.
We raised mouse monoclonal antibodies against human KLHL12 and human
30058 and 30067). We also raised antibodies against SEC13, SEC24C and
SEC24D. Other antibodies used in this study are: CUL3 (Bethyl Laboratories,
catalogue no. A301-109A), SEC31A (BD Biosciences, catalogue no. 612350),
collagen IV (Abcam, catalogue no. ab19808), anti-Flag (Sigma, catalogue nos
F3165, F7425), Ubiquitin (Santa Cruz, catalogue no. sc-8017, P4D1), rhodamine
no. SPA-891), anti LC-3 (Sigma, catalogue no. L-7543), anti-alpha tubulin
(DM1A, Abcam, catalogue no. ab7291), anti-fibronectin (Abcam, ab2413),
anti-GM130 (BD Biosciences, catalogue no. 610822), and anti-EGFR (Ab12,
Neomarkers, MS-400P1). LF-67 (anti-sera for Type I procollagen) was obtained
as a gift from L. Fisher.
in ES cell medium containing 15% FBS, 13 sodium pyruvate, 13 non-essential
amino acids, 1mM b-mercaptoethanol and 1,000Uml21leukaemia inhibitory
factor (Millipore, catalogue no. ESG1107) in GIBCO Dulbecco’s Modified Eagle
FBS was bought from HyClone. The doxycycline-inducible 293T Trex KLHL12-
33Flag stable cell line was made with Flp-In T-REx 293 Cell Line system from
ingly. These cell lines were maintained with 10% TET(2) FBS, blasticidin and
NIA (National Institute on Ageing) Ageing Cell Repository. For generating pro-
collagen stable HT-1080cell lines, we cloned proalpha(1) into a pRMc/CMV-
vector and selected for neomycin resistance39. This vector was provided as a gift
by N. Bulleid. Cells were kept in a 37uC incubator with 5% CO2.
siRNA screen in mouse ES cells. siRNA oligonucleotides against 40 mouse
ubiquitin E3 enzymes were pre-designed by Qiagen and handled as instructed.
screen. 10pmol of siRNA oligonucleotides and 0.25ml of Lipofectamine2000 were
pre-incubated in a 0.1% gelatin-coated 96-well plate in 20ml of OPTIMEM for
15min at room temperature. The D3 mouse ES cells were trypsinized and seeded
were examined using bright-field microscopy at 48h post transfection. Hit valid-
vendors (Qiagen, Dharmacon) and that target different sites of the Cul3 mRNA.
Knockdown efficiency was tested by qRT–PCR and immunoblot.
Rescue of Cul3-siRNA phenotype in mouse ES cells by Matrigel and collagen
IV. D3 mouse ES cells were grown on tissue culture dishes coated with gelatin
(negative control), growth-factor-depleted Matrigel (BD Biosciences, catalogue
no. 356231), or purified collagen IV (BD Biosciences, catalogue no. 354233).
Matrigel and collagen IV were applied at 10mgcm22. CUL3 was depleted 24h
logy was analysed by confocal microscopy against integrin b1, actin and DNA.
Drug treatments of CUL3-depleted cells. To study the synthetic lethal effect of
D3 mouse ES cells with 0, 25, 50 or 100nM of dasatinib for 18h before the
phenotypes were analysed by light microscopy.
phenotype analysis. Alternatively, RHOA was co-depleted using specific siRNAs.
Cell cycle analysis. To assess the division rate of CUL3-depleted mouse ES cells,
we treated cells with control, Cul3-, or Ube2C/Ube2S-siRNA and seeded at
33105cells per well in gelatin-coated six-well plates. The specificity of Ube2S-
and Ube2C-siRNAs was tested before40. The cells were trypsinized at 2, 3 and
4days post transfection and counted by haemocytometer.
ES cell differentiation analysis. To differentiate mouse ES cells into embryoid
bodies (EB), we trypsinized undifferentiated D3 mouse ES cells, washed once with
leukaemia inhibitory factor-free ES cell media, and seeded the cells at 23106cells
and the cells were re-seeded onto 10-cm Corning Ultra-Low-Attachment Dishes
changed every other day for a total of 6 or 9days. Total RNA of ES cells and EB
samples was extracted using TRIzol (Invitrogen, catalogue no. 15596-026) and
chloroform. The expressionofpluripotent markersand BTB genes at various time
points during differentiation was analysed using quantitative real-time PCR.
As a complementary experiment, D3 mouse ES cells were treated with control
or Oct4 siRNA. 48h after transfection, cells were collected and total RNA was
extracted using TRIzol as above. The expression of pluripotent markers, tissue
specific genes and BTB genes in control and OCT4-depleted cells were analysed
Quantitative real-time PCR analysis. WeusedTRIzol(Invitrogen,catalogue no.
were synthesized by using RevertAid first strand cDNA synthesis kit (Fermentas,
SYBR Green/Rox qPCR system (Fermentas, catalogue no. K0221).
Identification of CUL3–KLHL12 substrates. To identify CUL3–KLHL12 sub-
strates, we generated a doxycycline-inducible human KLHL12–33Flag stable cell
line using the Flp-In T-REx 293 Cell Line system (Invitrogen). As controls, we
generated stable cell lines expressing other BTB proteins including KLHL9.
KLHL12–33Flag and KLHL9–33Flag expression was induced in 30315cm
plates by 1mgml21of doxycycline for 48h, and cells were collected by centrifu-
gation and lysed by douncing 40 times in PBS10.1%NP40. The cell lysate was
cleared by centrifugation and then subjected to anti-Flag M2 affinity gel (Sigma,
in PBS.The elution was repeated three times for1h at room temperature. Eluates
were pooled, concentrated to 100ml using Amicon Ultra-0.5, Ultracel-10
Membrane (Millipore, catalogue no. UFC501008) and run on a SDS–PAGE gel.
The gel was stained by SimplyBlue SafeStain (Invitrogen, catalogue no. LC6060),
andspecificgelbandswere cutoutandsentformass spectrometry analysisbythe
Vincent J. Coates Proteomics/Mass Spectrometry Laboratory at UC Berkeley.
action of endogenous proteins, we lysed HeLa cells or D3 mouse ES cells by
freeze-thaw twice in 20mM HEPES buffer pH7.5, 5mM KCl, 1.5mM MgCl2,
13 protease inhibitor cocktail (Roche). Specific antibodies against CUL3, SEC13
or SEC31 conjugated to protein G agarose beads were added to the cleared cell
lysate and incubated at 4uC for 4h. Protein complexes were eluted with gel-
loading buffer at 95uC. Endogenous proteins in complexes were detected by
immunoblot using specific antibodies against CUL3, SEC13, SEC31 or KLHL12.
To detect ubiquitylation of endogenous COPII components, we incubated
HeLa cell extract with pre-immune serum or antibody against SEC13 conjugated
gel-loading buffer at 95uC. Ubiquitylated proteins in the complex were detected
by immunoblot against ubiquitin.
In vitro protein interaction assays. To dissect the KLHL12 and SEC31A inter-
action, we coupled 20mg recombinant MBP–KLHL12, various mutants or MBP
as a control to 15ml amylose resin by incubating at 4uC for 1h. CUL3, SEC31A
and mutants were expressed from pCS2 and labelled with [35S]-Met using TnT
Sp6 Quick Coupled Trsnc/trans Syst (Promega, catalogue no. L2080). The
labelled CUL3 or SEC31A were incubated with MBP-KLHL12 or mutants at
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4uC for 3h. Beads were washed four times with TBST and twice with TBS, and Download full-text
incubated in SDS loading buffer at 95uC. Samples were run on SDS–PAGE and
results were visualized by autoradiography.
In vitro ubiquitylation assays with CUL3–KLHL12. CUL3/RBX1 was conju-
gated to NEDD8at 30uC for 1h with the following conditions: 2.5mM Tris/HCl
pH7.5,5mMNaCl,1mM MgCl2, 1mMDTT,13energymix40, 1mM APPBP1–
UBA3, 1.2mM UBC12, 4mM CUL3/RBX1, and 60mM NEDD8. For in vitro
ubiquitylation of SEC31A, we set up a 10ml reaction as follows: 2.5mM Tris/
HCl pH7.5, 5mM NaCl, 1mM MgCl2, 1mM DTT, 13 energy mix, 100nM
UBA1, 1mM UBCH5C, 1mM CUL3,NEDD8/RBX1, 1mM KLHL12, 150mM
ubiquitin, 0.05mg SEC13/31A. The reaction was carried out at 30uC for 1h
and stopped by adding SDS gel loading buffer.
In vivo ubiquitylation assays with CUL3–KLHL12. 293T cells grown in 10-cm
Klhl12-FLAG, pcDNA4-Cul3-FLAG, or pcDNA4-Cul3N250-FLAG, as indicated,
using calcium phosphate. 24h later, 1mM MG132 was added and cells were incu-
buffer A (6M guanidine chloride, 0.1M Na2HPO4/NaH2PO4and 10mM imida-
zole, pH8.0). Cells were lysed by sonication for 10s and incubated with 25ml Ni-
A, twice with buffer A/TI (1 volume buffer A and 3 volumes buffer TI), once with
buffer TI (25mM Tris-Cl, 20mM imidazole, pH6.8), and incubated in 60ml SDS
95uC. Samples were separated by SDS–PAGE and ubiquitylated SEC31A was
detected by immunoblot using antibody against SEC31A.
To detect SEC31A ubiquitylation upon CUL3/KLHL12 depletion, we co-
transfected 100nM siRNAs against CUL3 or KLHL12 with pCS2-HA-Sec13/
with 0.5% Triton X-100 in 13 TBS, 2% BSA. Cells were incubated with primary
antibodies against SEC31A, SEC13, SEC24C, ERGIC53, CD63, BiP (also known as
goat anti-rabbit IgG (H1L); Alexa Fluor 488 goat anti-mouse IgG (H1L);
HOECHST 33342,) for 1h at room temperature followed by extensive washing.
Pictures were taken on Zeiss LSM 510 and 710 Confocal Microscope systems and
analysed with LSM image browser and Imaris 3D imaging processing software.
Transmission electron microscopy. Mock- and KLHL12-transfected HeLa cells
were grown to 70% confluence as a monolayer on an Aclar sheet (Electron
Microscopy Sciences). The cells were fixed for 30min in 0.1M cacodylate buffer,
post-fixation with 1% osmium tetroxide on ice. This was followed by staining
with 1% aqueous uranyl acetate for 30min at room temperature. For dehydration
exposure to 35% ethanol at 4uC, to 50% ethanol and 70% ethanol at 220uC, and
95%, and 100% ethanol at 235uC. Cells were restored to room temperature in
100% ethanol before flat embedding in an Epon resin. Thin (70–100nm) sections
aqueous uranyl acetate and 2% tannic acid. The sections were imaged at 120kV
using a Tecnai 12 Transmission Electron Microscope (FEI).
For the purpose of immunolabelling, HeLa cells expressing Flag–KLHL12 or
doxycycline-inducible 293T Trex Flag–KLHL12 stable cell lines were fixed in 2%
paraformaldehyde and 0.5% glutaraldehyde and embedded in LR white resin.
Ted Pella). 70-nm thick sections were picked on 100-mesh nickel grids coated
with Formvar film and carbon, incubated in blocking buffer (5% BSA, 0.1% fish
Flag antibody at a dilution of 1:40 for 1h. Goat anti-mouse IgG conjugated with
10-nm gold (BD Biosciences) was used as the secondary antibody at a dilution of
1:40 for 1h. Sections were post stained in 2% uranyl acetate for 5min.
further purified using RNeasy Mini Kit (Qiagen, catalogue no. 74104).
Microarray analysis was performed by the Functional Genomics Laboratory
(UC Berkeley) using Affymetrix Mouse 430A 2.0 chip.
Analysis of collagen export from cells. IMR-90 human lung fibroblasts grown
on 100-mm dishes in DMEM/10% FBS were transfected with Flag–KLHL12,
Flag–KLHL12(FG289AA), Flag–KEAP1 and pcDNA5-flag using nucleofection
on six-well plate with 25-mm coverslips. When indicated, co-transfections with
2mg each of Flag–KLHL12 and dominant-negative CUL3 were performed.
Dialysed 10% FBS media was used for ascorbate free transfections. Brefeldin A
(Sigma) was used at a concentration of 2.5mgml21and cells were incubated for
30min. MG132 was used at 20mM for 2h, chloroquine was used at 200mM for
1h. Media was collected the next day and cells on coverslips were fixed with 3%
lysates. Cells on coverslips were permeabilized with 0.1% Triton for 15min at
Fluor 488 goat anti-rabbit IgG (diluted 1:200). After staining cells with appropriate
primary and secondary antibodies, we fixed coverslips on slides using mounting
reagent containing DAPI. Images were analysed with a Zeiss LSM710 confocal
microscope and captured with Zen10 software. Merges of images were performed
with ImageJ and LSM image Browser. Media collected from six-well plates was
normalized with respect to lysate protein concentration estimated using BCA
method. Media and lysates of each reaction were checked by immunoblot analysis.
Tubulin was used as loading control for lysates. Ascorbate chase experiments were
done by adding ascorbate (0.25mM ascorbic acid and 1mM asc-2-phosphate) to
KLHL12-transfected cells, followed by incubation for 5, 10, 30 and 60min.
A human fibrosarcoma cell line (HT1080) stably transfected with proalpha1(1)
was used for CUL3 knockdowns. Cul3- and Klh12-shRNAs targeting two different
regions in both genes were cloned into pSuperGFP and transfected using
Lipofectamine 2000. pSuper GFP was used as negative control. Cells were grown
on 25-mm coverslips in six-well plates and fixed 2days post transfection. Collagen
anti-PDI (1:1,000) antibody. Fibronectin and EGFR were stained in parallel experi-
ments. Fibronectin expression was induced in HT1080 using 1mM dexamethasone
in cells expressing GFP shRNAs. Cells without GFP shRNAs and transfected with
pSUPER GFP were quantified as well. Images were taken on a Zeiss LSM 710
from remaining cells on six-well plates and checked for knockdown efficiency.
siRNA oligonucleotides used in this study. RNA interference oligonucleotides:
mCul3 #1, GAAGGAATGTTTAGGGATA; mCul3 #2, GGAAGAAGATGCAG
CACAA; mCul3 #3, GGTGATGATTAGAGACATA; mCul3 #4, CAACTTTCT
AAGCAGAGAGAAA; mKlhl12, CCTTGAGAGTGGAGCAGAA; hKlhl12,
CCAAAGACATAATGACAAA; mKBTBD8, GAACATGAGCAGAGTGAAA;
mOct4, AGGCAAGGGAGGTAGACAA; hSec31, CCTGAAGTATTCTGAT
AAA; mSec13 (pool of 4 oligonucleotides), CCATGTGTTTAGTAATTTA,
GGCAATATGTGGTCACCTA, GCTGAAAGTATTCATGTAA and GGAAC
AAATGACTATTATT; mCdc42 (pool of 4 oligonucleotides), GATCTAATT
TGAAATATTA, GGATTGAGTTCCTAATTAA, AGAGGATTATGACAGAC
GACTAATAGTCTACATTTA, GGAGGTGTCTCGTCCAATA, CTATGACA
cleotides), CCCTTGTGTCCATATTTAA, CCACGAGGGTTGCCATCAA, CA
GACTTGTTGTACATATT and GCAACAAGAGCAAGCCCAA; mRhoG (pool
of 4 oligonucleotides), GGTTTACCTAAGAGGCCAA, GCTGTGCCTTAAG
GACTAA, GCACAATGCAGAGCATCAA and GGCGCACCGTGAACCTA
AA; mRhoA (pool of 4 oligonucleotides), GGATTTCCTAATACTGATA,
GAAAGTGTATTTGGAAATA, AGCCCTATATATCATTCTA, CGTCTGCCA
TGATTGGTTA; mRac1 (pool of 4 oligonucleotides), GGTTAATTTCTGTCA
AACA, GCGTTGAGTCCATATTTAA, GCTTGATCTTAGGGATGAT and
GCTGTATTCTA and GGGACAATGTGTATTACTA; mIqgap1 (pool of 4
oligonucleotides), ACATGATGATGATAAACAA, GGTTGATTTCACAGAAGAA,
GTATAAATTTATTTCTTAA and GGTGGATCAGATTCAAGAA; mCul1
(pool of 2 oligonucleotides), GCATGATCTCCAAGTTAAA and CGTGTAATC
TGCTATGAAA; mCul2 (pool of 2 oligonucleotides), GCGCTGATTTGAAC
GTGTGATTACCATAATAAA and CCAGGAAGCTGGTCATCAA; mCul5
TATAATGAA; mCul7 (pool of 2 oligonucleotides), GCATCAAGTCCGTTAA
TAA and GGATGTGATTGATATTGAA.
39. Geddis, A. E. & Prockop, D. J. Expression of human COL1A1 gene in stably
transfected HT1080 cells: the production of a thermostable homotrimer of type I
collagen in a recombinant system. Matrix 13, 399–405 (1993).
40. Williamson, A. et al. Identification of a physiological E2 module for the human
anaphase-promoting complex. Proc. Natl Acad. Sci. USA 106, 18213–18218
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