Inhibition of protein translocation at the endoplasmic reticulum promotes activation of the unfolded protein response.

Abstract

Selective small-molecule inhibitors represent powerful tools for the dissection of complex biological processes. ES(I) (eeyarestatin I) is a novel modulator of ER (endoplasmic reticulum) function. In the present study, we show that in addition to acutely inhibiting ERAD (ER-associated degradation), ES(I) causes production of mislocalized polypeptides that are ubiquitinated and degraded. Unexpectedly, our results suggest that these non-translocated polypeptides promote activation of the UPR (unfolded protein response), and indeed we can recapitulate UPR activation with an alternative and quite distinct inhibitor of ER translocation. These results suggest that the accumulation of non-translocated proteins in the cytosol may represent a novel mechanism that contributes to UPR activation.

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Biochem. J. (2012) 442, 639–648 (Printed in Great Britain)doi:10.1042/BJ20111220
639
Inhibition of protein translocation at the endoplasmic reticulum promotes
activation of the unfolded protein response
Craig MCKIBBIN*1, Alina MARES*, Michela PIACENTI†, Helen WILLIAMS†, Peristera ROBOTI*, Marjo PUUMALAINEN*,
Anna C. CALLAN*, Karolina LESIAK-MIECZKOWSKA‡, Stig LINDER‡, Hanna HARANT§, Stephen HIGH*, Sabine L. FLITSCH?,
Roger C. WHITEHEAD†2and Eileithyia SWANTON*2
*Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, U.K., †School of Chemistry, University of Manchester, Oxford Road,
Manchester M13 9PL, U.K., ‡Department of Oncology and Pathology, Karolinska Institute, 171 76 Stockholm, Sweden, §Ingenetix GmbH, Simmeringer Hauptstrasse 24, 1110 Vienna,
Austria, and ?School of Chemistry, University of Manchester, Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K.
Selective small-molecule inhibitors represent powerful tools for
the dissection of complex biological processes. ESI(eeyarestatin
I) is a novel modulator of ER (endoplasmic reticulum)
function. In the present study, we show that in addition to
acutelyinhibitingERAD(ER-associateddegradation),ESIcauses
production of mislocalized polypeptides that are ubiquitinated
and degraded. Unexpectedly, our results suggest that these
non-translocated polypeptides promote activation of the UPR
(unfolded protein response), and indeed we can recapitulate
UPR activation with an alternative and quite distinct inhibitor of
ER translocation. These results suggest that the accumulation
of non-translocated proteins in the cytosol may represent a novel
mechanism that contributes to UPR activation.
Key words: eeyarestatin, endoplasmic reticulum, non-translo-
cated protein, Sec61, unfolded protein response.
INTRODUCTION
In eukaryotic cells, the ER (endoplasmic reticulum) is the major
site for the synthesis of membrane and secretory proteins. These
are co-translationally translocated across or integrated into the
membrane through a proteinaceous channel, the Sec61 complex
[1]. Once exposed to the ER lumen, polypeptides are folded,
assembled and may undergo post-translational modification, such
asN-linkedglycosylation,priortobeingtransportedtotheirsiteof
function. However, folding is an intrinsically error-prone process,
and polypeptides may also fail to attain their native state due to
errorsintranslation,geneticmutation,absenceofpartnersubunits
or unfavourable environmental conditions. Such misfolded or
misassembled polypeptides are potentially harmful, and in order
to prevent their deployment within the cell, they are retained in
the ER by a quality control system [2]. To avoid congestion and
potential interference with productive protein folding, misfolded
proteins must be cleared from the ER, and this is achieved
via a process known as ERAD (ER-associated degradation) [3].
ERAD is mediated by the cytosolic proteasome, which means
that proteins selected for ERAD must be retrotranslocated back
across the ER membrane into the cytosol prior to degradation [3].
Ubiquitin ligases at the cytosolic face of the ER membrane ubi-
quitinate the ERAD substrate, directing it to the UPS (ubiquitin–
proteasome system). Ubiquitination also attracts p97, an ATPase
that facilitates extraction of many ERAD substrates from the
ER membrane. The ERAD substrate is then deglycosylated
and deubiquitinated, before entering the catalytic core of the
proteasome where it is broken down into small peptides [3].
Situations that perturb the balance between protein synthesis,
folding and degradation pathways (termed proteostasis [4]), can
lead to a build-up of misfolded proteins in the ER, causing
ER stress. Eukaryotic cells possess a signalling network known
as the UPR (unfolded protein response), to detect and manage
such imbalances in ER proteostasis [5]. UPR signalling pathways
function to reduce the rate of protein synthesis and up-regulate
theexpressionofchaperonesandERADfactors,therebyreducing
levelsofmisfoldedproteinsintheERandrestoringhomoeostasis.
If homoeostasis is not re-established, prolonged activation of
the UPR can initiate programmed cell death [5]. Disturbances
in ER proteostasis and UPR signalling are implicated in the
pathogenesis of a diverse range of diseases, including diabetes
mellitus, cancer, neurodegeneration, and bone and joint diseases,
as well as classical ER protein folding diseases such as cystic
fibrosis [6]. As a result, there is considerable interest in the
therapeutic potential of selective small molecules to manipulate
these pathways in order to ameliorate disease [4].
The small molecules ESI(eeyarestatin I) and ESII(eeyarestatin
II) are ERAD inhibitors, first identified in a high-throughput
screen based on stabilization of GFP (green fluorescent protein)-
tagged MHC I heavy chain, which is degraded via an ERAD-
like pathway in cells expressing the viral protein US11 [7]. The
degradation of a conventional ERAD substrate, TCRα (T-cell
receptor α subunit), was also inhibited by ESI[7], and it was
suggested that the compound acts at an early stage in the ERAD
pathway, prior to retrotranslocation. Subsequent work provided
evidence that a later stage in ERAD, namely the p97 ATPase and
an associated deubquitinating activity, may also be inhibited by
ESI[8,9].ESIhasalsobeenshowntoactivatetheUPRandinduce
cell death [10]. Intriguingly, the cytotoxic activity of ESIappears
to be particularly effective against cancer cells, and synergizes
with that of bortezomib (Velcade), a proteasome inhibitor with
proven anticancer properties [10]. Together with accumulating
evidence that UPR activation plays a role in a variety of
Abbreviations used: CHX, cycloheximide; cpd A, translocation inhibitor compound A; DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol;
eIF2α, eukaryotic initiation factor 2α; EndoH, endoglycosidase H; ER, endoplasmic reticulum; EDEM-1, ER degradation-enhancing α-mannosidase-
like 1; ERAD, ER-associated degradation; ES, eeyarestatin; HEK, human embryonic kidney; IP, immunoprecipitation; IRE1, inositol-requiring enzyme 1;
PDI, protein disulfide-isomerase; PERK, PKR (double-stranded-RNA-dependent protein kinase)-like ER kinase; PS2, proteasome inhibitor 2; RT, reverse
transcription; TCRα, T-cell receptor α subunit; UPR, unfolded protein response, UPS; ubiquitin–proteasome system; XBP1, X-box-binding protein 1.
1Present address: Department of Clinical Biochemistry, Royal Surrey County Hospital, Egerton Road, Guildford, GU2 7XX, U.K.
2Correspondence may be addressed to either of these authors (email lisa.swanton@manchester.ac.uk or roger.whitehead@manchester.ac.uk).
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C. McKibbin and others
cancers [11], these observations highlight a potential therapeutic
application for ESI-related compounds. Both activation of the
UPR and perturbation of cellular ubiquitin homoeostasis have
been proposed to cause ESI cytotoxicity [10]. However, the
underlying molecular mechanism(s) through which ESIactivates
the UPR and exerts its effects is not yet clear.
We have previously shown that ESIinhibits Sec61-mediated
translocation of a range of proteins across the ER membrane,
albeit to various degrees depending on the precursor studied
[12]. The consequences of ESI-dependent perturbations of ER
translocation in cells are not known, raising the possibility that
inhibitionoftranslocationcontributestothepotentcellulareffects
of ESI. In the present study we show that ESItreatment results in
the production of mislocalized membrane and secretory proteins,
anddemonstratethataccumulationofthesespeciescorrelateswith
activation of the UPR. Using an alternative inhibitor of transloca-
tion, we provide evidence that accumulation of non-translocated
polypeptides in the cytosol promotes activation of the UPR.
EXPERIMENTAL
Materials
TheM2anti-FLAGantibodywasfromSigma–Aldrich.TheP4D1
anti-ubiquitin antibody was from Santa Cruz Biotechnology.
Derlin-1 and PDI (protein disulfide-isomerase) polyclonal rabbit
antisera were made by Eurogentec. Anti-p97 antiserum was from
Professor Bernhard Dobberstein (Center for Molecular Biology,
University of Heidelberg, Heidelberg, Germany). Plasmids
encoding TCRα were from Professor Ron Kopito (Department
of Biology, Stanford University, Stanford, CA, U.S.A.) and
Professor Emmanuel Wiertz (Leiden University, Leiden, The
Netherlands). Bovine opsin cDNA was subcloned from the pZEO
vector into pcDNA5/FRT/TO (Invitrogen). The preprolactin–
mCherry construct was from Professor Viki Allan (Faculty of
Life Sciences, University of Manchester, Manchester, U.K.).
Synthesis of ESI, ESIIand ESR35
Synthesis of the ESs (see the Supplementary methods
section at http://www.BiochemJ.org/bj/442/bj4420639add.htm)
commenced with nitroso-compound 1 (see Supplementary Fig-
ure S1 at http://www.BiochemJ.org/bj/442/bj4420639add.htm),
which was prepared in a straightforward fashion by the reaction
of iso-butene with nitrosyl chloride (generated in situ from iso-
amyl nitrite and HCl) [13]. The reaction of compound 1 with
methyl glycinate proceeded smoothly to give a very good yield of
oximino ester 2 [14]. Exposure of compound 2 to two equivalents
of 4-chlorophenyl isocyanate or 1-naphthyl isocyanate gave the
bis-adducts 3a and 3b [3], which were subsequently converted
into their corresponding acyl hydrazides 4a and 4b by treatment
withanaqueousmethanolicsolutionofhydrazine(acylhydrazide
4a is the compound also referred to as inactive analogue ESR35).
Finally, condensation of hydrazides 4a and 4b with E-3-(5-nitro-
2-furyl)acrylaldehyde in methanol gave the corresponding ESs
(ESIand ESII) in excellent yields. The double-bond geometry of
the final products was indicated as E by the magnitude of the
vicinal coupling constant between the olefinic protons (15.8 Hz
for ESIand 15.4 Hz for ESII).
Cell culture and transfection
HeLacellswereculturedinDMEM(Dulbecco’smodifiedEagle’s
medium) containing 10% (v/v) FBS (fetal bovine serum) and
2 mM L-glutamine.CellsweretransfectedusingLipofectamineTM
2000 (Invitrogen) and used in experiments after 24 h.
Treatment with compounds
Stock solutions (10 mM) of ESR35, ESII, ESI, cpd A (translocation
inhibitor compound A) and PS2 (proteasome inhibitor 2;
benzyloxycarbonyl-Leu-Leu-Phe-aldehyde; Calbiochem) were
made up in DMSO. ESR35, ESIIand ESIwere added to a final
concentration of 8 μM, PS2 and cpd A to 10 μM, and CHX
(cycloheximide)to100 μg/ml,thencellswereincubatedat37◦C.
Metabolic labelling and IP (immunoprecipitation)
Cells were incubated in DMEM lacking methionine and cysteine
(Invitrogen) for 20 min, and were labelled with 22 μCi/ml
EasyTag [35S]Met/Cys (PerkinElmer) for 40 min at 37◦C. Cells
were washed twice in PBS and either harvested immediately
or chased in DMEM supplemented with 2.5 mM unlabelled
methionine/cysteine plus the indicated compounds. Cells were
harvested in 50 μl of ice-cold IP buffer (140 mM NaCl, 10 mM
Tris/HCl, pH 7.5, 1 mM EDTA and 1% Triton X-100) plus
proteaseinhibitorcocktail(Sigma).ForIP,lysatesweredenatured
in 1% SDS, then 5 vol. of IP buffer containing 10 mM non-
radioactive methionine/cysteine, 1 mM PMSF and 4% pansorbin
(Calbiochem) was added. Samples were rotated for 1 h at 37◦C,
centrifuged at 15000 g for 10 min, and the supernatant was
incubated overnight at 4◦C with an anti-FLAG antibody. Immune
complexeswerecollectedonProteinA–Sepharosebeads,washed
with IP buffer, eluted in reducing SDS/PAGE sample buffer and
resolved by SDS/PAGE. Radioactive gels were analysed by FLA-
3000 phosphorimaging (Fuji) and quantified using AIDA v3.52
software (Raytest Isotopenmessgerate).
Western blotting
To detect polyubiquitinated material, cells cultured in 12-well
dishes were lysed in 50 μl of IP buffer and then denatured
in SDS/PAGE sample buffer at 70◦C for 10 min. Half of the
sample was run on SDS/10% PAGE gels, analysed by blotting
with an anti-ubiquitin antibody and then visualized by enhanced
chemiluminescence.
XBP1 (X-box-binding protein 1) splicing
TotalRNAwasextractedusingTRIzol®reagent(Invitrogen),and
first-strand cDNA was synthesized with an oligo-p(dT)15primer.
cDNA was used as a template for PCR using primers flanking
the XBP-1 intron (FWD, 5?-ACAGCGCTTGGGGATGGATG-3?;
REV, 5?-TGACTGGGTCCAAGTTGTCC-3?), and PCR products
were analysed on 2% agarose gels.
Fluorescence microscopy
HeLa cells incubated with 8 μM ESI in the medium for 0–
8 h were fixed in methanol for 4 min at −20◦C, then probed
with primary antibodies against PDI and fluorophore-conjugated
secondary antibodies (Molecular Probes). For visualization
of preprolactin–mCherry, HeLa cells transfected using JetPEI
reagent (Peqlab), were incubated with 100 μg/ml CHX for 3 h
at 37◦C. Cells were washed three times for 1 min with 1 ml
of PBS, then treated with 8 μM ESI, 10 μM PS2 or 8 μM
ESI and 10 μM PS2 for 6 h. Following treatment, cells were
washed in PBS and fixed in 3% paraformaldehyde in PBS for
25 min at room temperature (21–23◦C). Images were obtained
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Inhibition of translocation promotes the UPR
641
using an Olympus BX60 upright microscope with a MicroMax
cooled charge-coupled device camera (Roper Scientific) driven
by Metamorph software (Universal Imaging Corporation).
Subcellular fractionation and EndoH (endoglycosidase H) treatment
Cells in 10 cm dishes were rinsed twice in PBS, harvested by
scraping and resuspended in 100 μl of hypotonic buffer (20 mM
Hepes, pH 7.6, 5 mM KCl, 2.5 mM EDTA and 1 mM PMSF) on
ice. Cells were sonicated for three 10 s pulses in a sonicating
waterbath. Lysates were centrifuged at 50000 g for 30 min at
4◦C.Themembranepelletwasresuspendedin100 μlof100 mM
Na2CO3, pH 11.5, incubated on ice for 20 min, then centrifuged
at100000 gfor1 hat4◦C.Thesupernatantwasremoved,andthe
pellet was solubilized in 100 μl of IP buffer for 10 min at 4◦C,
followed by a final centrifugation step at 100000 g for 1 h at 4◦C.
The supernatant was removed and the Triton X-100-insoluble
pellet was resuspended in IP buffer containing 0.1% SDS. Opsin
was immunoprecipitated from the carbonate supernatant, Triton
X-100 supernatant and Triton X-100-insoluble fractions, and was
resolved by SDS/PAGE. Where indicated, immunoprecipitated
material was incubated with 500 units of EndoH (New England
Biolabs) for 2 h at 37◦C.
RESULTS
ESIinduces accumulation of polyubiquitinated proteins and
induces ER stress
ESI, ESIIand a related compound ESR35, designed to provide a
negative control treatment (cf. [9]), were synthesized according
totheschemeoutlinedinSupplementaryFigureS1.Theinhibitory
effect of ESI on the ERAD pathway was confirmed in two
different human cell lines (see Supplementary Figure S2 at
http://www.BiochemJ.org/bj/442/bj4420639add.htm).
we found that acute (< 1 h) treatment of cells with ESIinhibited
degradation of the model substrate TCRα (Supplementary Figure
S2A), showing that the action of ESI on ERAD is rapid
and does not require prolonged treatment. In contrast, ESII
treatment did not reproducibly delay TCRα degradation, and
ESR35had no detectable inhibitory effect (Supplementary Figure
S2). ESItreatment also resulted in the appearance of substantial
amounts of higher-molecular-mass forms of the model ERAD
substrate (Supplementary Figure S2B). Less pronounced high-
molecular-mass species were also visible in cells treated with
a proteasome inhibitor (PS2) and at the start of the chase in
untreated cells (Supplementary Figure S2B), suggesting that
they might represent polyubiquitinated forms of TCRα en
route to degradation. Consistent with this interpretation, the
total levels of polyubiquitinated proteins detected in cells were
also markedly increased upon ESI treatment (Figure 1A, lane
4, and Supplementary Figure S2C). A similar, although less
pronounced, effect was observed following treatment with ESII
and PS2 (Figure 1A, lanes 3 and 5), whereas ESR35 had no
discernableeffect(Figure1A,lane2).Thesehigh-molecular-mass
polyubiquitin conjugates accumulated with time, and became
apparent after 1 h of exposure to ESI (Figure 1B). Increased
levels of polyubiquitinated material following ESItreatment have
been observed previously and have been proposed to reflect an
inhibition of deubiquitination [8].
Consistent with previous work [10], we also found that ESI
induced ER stress in cultured cells. Splicing of Xbp1 mRNA,
an early event in the IRE1 (inositol-requiring enzyme 1) branch
of the UPR can be measured using RT (reverse transcription)–
PCR. A single PCR product, representing the unspliced
Notably,
mRNA, was observed in DMSO-treated cells (Figure 1C,
lane 2). A faster-migrating product, representing spliced Xbp1
mRNA, was apparent in cells treated with the reducing
agent DTT (dithothreitol) to perturb oxidative protein folding
(Figure 1C, lane 1). ESIalso induced Xbp1 mRNA splicing (Fig-
ure 1C, lane 5), showing that treatment with this compound
resulted in activation of IRE1. In contrast, no spliced Xbp1 was
detected in cells treated with ESII or ESR35 (Figure 1C, lanes
3 and 4), consistent with the minor effect of these compounds
on ERAD and polyubiquitination (Supplementary Figure S2 and
Figure 1A). The response to ESI did not require prolonged
treatment with ESI, and spliced Xbp1 was evident after 4 h
(Figure 1D). Activation of the PERK [PKR (double-stranded-
RNA-dependent protein kinase)-like ER kinase] branch of the
UPR results in phosphorylation of eIF2α (eukaryotic initiation
factor 2α), resulting in a decrease in the rate of protein synthesis
under conditions of ER stress, as observed following treatment
with the reducing agent DTT to perturb oxidative protein folding
(Figure 1E, cf. lanes 1 and 2). Phosphorylation of eIF2α occurred
rapidly upon ESItreatment (Figure 1E, bottom two panels), and
this effect correlated with a decrease in total protein synthesis
(Figure 1E, top panel, lanes 3–8), showing that the PERK
branch of the UPR was also activated. Similar effects of ESI
were also observed in HEK (human embryonic kidney)-293
cells (see Supplementary Figure S3 at http://www.BiochemJ.
org/bj/442/bj4420639add.htm).
resulted in a more pronounced phosphorylation of eIF2α and
greater translational attenuation in HEK cells than in HeLa cells
(Supplementary Figure S3), suggesting that sensitivity to ESIcan
vary between cell types (cf. [10]).
Our recent work suggests that ESIdelays certain intracellular
trafficking pathways [13], and we therefore examined the effect
of the ES compounds on cell morphology. Interestingly, large
vacuole-like structures developed within 4–8 h of treatment
with ESI (Figure 1F, arrows), and these were also apparent
by immunofluorescence microscopy using an antibody specific
for ER-localized proteins (Figure 1G), suggesting that these
structures were derived from the ER. ESR35had no obvious effect
on cell morphology (Figure 1F), and although no effect of ESII
was visible within 8 h (Figure 1F), prolonged treatment (12–
16 h) did cause the appearance of such vacuoles (results not
shown). A similar swelling of the ER occurs during paraptosis
[14], a non-apoptotic form of cell death, and has also been
observed under conditions of chronic ER stress [15,16]. Hence
these changes in ER structure may reflect ESI-induced ER stress
and/or commitment to cell death.
Interestingly,ESI
treatment
Inhibition of translocation promotes ER stress
The mechanisms through which ESI induces ER stress are
not known, although inhibition of ERAD and/or perturbation
of ubiquitin homoeostasis could potentially play a role [10].
Indeed, inhibition of the proteasome is able to induce
activation of the UPR in a variety of cell types (e.g. [17]).
Interestingly, however, treatment of HeLa cells with PS2
for 8 h failed to induce Xbp1 mRNA splicing (Figure 2A,
lane 3), despite causing a clear build-up of polyubiquitinated
species and profound inhibition of ERAD (Figure 1A and
Supplementary Figure S2). Similarly, Xbp1 splicing was not
observed in cells treated with an alternative proteasome inhibitor
MG132 (Figure 2A, lane 4), or in response to a small
molecule that inhibits protein deubiquitination (NSC687852)
(Figure 2A, lane 5), and causes a pronounced accumulation of
polyubiquitin adducts [18,19] (see Supplementary Figure S4A
at http://www.BiochemJ.org/bj/442/bj4420639add.htm). Thus
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C. McKibbin and others
Figure 1Cellular effects of ES compounds
(A) HEK cells were treated for 8 h with DMSO, 8 μM ESR35,ESIIor ESI, or 10 μM PS2, and lysates were analysed by blotting with an anti-ubiquitin (Ub) antibody. (B) HeLa cells were treated with
DMSO for 4 h or 8μM ESIfor the time indicated, and lysates were analysed by blotting with an anti-ubiquitin antibody. (C) HeLa cells were treated with 2 mM DTT for 2h, DMSO, or 8 μM ESR35,
ESIIor ESIfor 8h. Xbp1 mRNA splicing was determined by RT–PCR. PCR products corresponding to unspliced (u) and spliced (s) Xbp1 mRNA are indicated. (D) HeLa cells were treated with 8μM
ESIfor the time indicated and Xbp1 splicing was determined as above. (E) HeLa cells treated with 10mM DTT for 30min or 8μM ESIfor the time indicated were labelled with [35S]Met/Cys for
40min. Lysates were analysed by phosphorimaging (top panel), or blotting with anti-peIF2α (phospho-eIF2α) or anti-eIF2α antibodies (bottom two panels). In (B and E) the molecular mass in kDa
is indicated on the left-hand side. (F) HeLa cells were treated with DMSO or 8 μM ESR35, ESIIor ESIfor 8h then visualized by phase-contrast microscopy. Arrows indicate vacuolar structures. (G)
HeLa cells were treated with 8 μM ESIfor the time indicated, fixed, stained with anti-PDI and fluorescently labelled secondary antibodies, and examined by fluorescence microscopy.
neither blockade of ERAD nor perturbation of global ubiquitin
homoeostasis (using a variety of different inhibitors) activated
the UPR in HeLa cells over this time course. In contrast,
parallel treatment with ESIinduced a robust ER stress response
(Figure 2A, lane 2). These results are significant as they suggest
that ESIdoes not induce ER stress solely by blocking degradation
of ERAD substrates or disrupting the UPS [10]. We therefore
considered the alternative possibility that the ability of ESI
to activate UPR was related to its ability to perturb protein
translocation [12]. In order to test this hypothesis, we used a
second small-molecule inhibitor of co-translational translocation
(referred to in the present study as cpd A), derived from
a naturally occurring cyclic depsipeptide [20]. Like ESI, the
cyclodepsipeptide inhibitors cause a wide-ranging inhibition
of translocation when used at low micromolar concentrations
[21,22], but are structurally distinct and target a different stage
of Sec61-mediatedprotein translocation acrossthe ERmembrane
[20,23].Strikingly,treatmentofHeLacellswiththistranslocation
inhibitor also induced Xbp1 mRNA splicing on a timescale
comparable with ESI treatment (cf. Figures 1C and 2B). In
addition,cpdAinducedphosphorylationofeIF2αandreducedthe
rate of translation (Figures 2C and 2D), demonstrating that both
the IRE1 and PERK branches of the UPR were activated. This
effect of the cyclodepsipeptide inhibitors has not previously been
reported and supports the view that inhibiting Sec61-mediated
translocation promotes ER stress. This effect could potentially
be caused by the reduced biogenesis of specific ER factors such
as chaperones, some of which are known to be relatively short-
lived [24,25]. However, the global inhibition of protein synthesis
using CHX did not induce Xbp1 splicing (Figure 2E, lane 5),
suggestingthatalackofERcomponentsresultingfromadefectin
co-translational translocation is unlikely to underlie the ability of
ESIand cpd A to activate the UPR. These observations raised the
possibility that the induction of ER stress by these translocation
inhibitors might instead be related to the production of non-
translocated forms of membrane and secretory proteins. In order
to address this issue further, we examined the fate of various
ER-targeted proteins in the presence of ESI.
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Inhibition of translocation promotes the UPR
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Figure 2Inhibition of translocation promotes ER stress
(A) HeLa cells were incubated with DMSO, 8 μM ESI, 10μM PS2, 10μM MG132 or 0.4μM of the deubiquitinase inhibitor NSC687852 (NSC) for 8 h. Xbp1 mRNA splicing was determined by
RT–PCR. Products corresponding to unspliced (u) and spliced (s) Xbp1 mRNA are indicated. (B) HeLa cells were incubated with DMSO or the indicated concentration of the translocation inhibitor
cpdAfor8 h,andXbp1splicingwasdeterminedasabove.(C)HeLacellswereincubatedwithDMSOor10μMcpdAfor8h,andlysateswereanalysedbyblottingwithanti-peIF2α (phospho-eIF2α)
or anti-eIF2α antibodies. (D) HeLa cells were incubated with DMSO or 10μM cpd A for 8h, pulse-labelled with [35S]Met/Cys for 40min, and lysates were analysed by phosphorimaging(top panel)
or blotting with an anti-tubulin antibody (bottom panel). Molecular mass in kDa is indicated on the left-hand side. (E) HeLa cells were incubated with 2mM DTT for 2h, DMSO for 8 h, 8μM ESIfor
4 or 8 h, or 100μg/ml CHX for 8 h. Xbp1 splicing was determined as above.
ESIinduces mislocalization and degradation of membrane and
secretory proteins in cultured cells
Addition of N-linked glycans to polypeptides occurs within the
ER lumen and was thus used to provide a readout of TCRα
insertion into the ER (cf. [12,22]). In DMSO- and ESR35-treated
cells, FLAG–TCRα was efficiently N-glycosylated and migrated
as a species of approximately 48 kDa (Figure 3A, lanes 1 and 2).
As observed previously [12], levels of the N-glycosylated form
of TCRα were reduced in ESI-treated cells (Figure 3A, lane 4).
In addition, a more rapidly migrating species of the size expected
for the unglycosylated protein was found to accumulate after 8 h
of treatment with ESI(Figure 3A, lane 4). A similar, although less
pronounced, effect was seen upon ESIItreatment (Figure 3A, lane
3). These observations indicate that ESItreatment results in the
production of a form of TCRα lacking N-glycosylation.
We next examined the effect of ESI on the subcellular
localizationofa chimaeric
of fluorescent mCherry fused to the ER-targeting sequence of
preprolactin (ppl–mCherry). In untreated cells, ppl–mCherry
was predominantly localized to the secretory pathway, as
demonstrated by the reticular and ribbon-like pattern of
fluorescence, typical of the ER and Golgi apparatus respectively
(Figure3B,panelA).CellswerethentreatedwithCHXtoprevent
further protein synthesis and allow existing protein to exit the ER.
After washing to remove CHX, cells were incubated with ESI,
PS2 or a combination of ESIand PS2 for a further 6 h, prior to
imaging. This treatment regime (outlined in Figure 3B) allowed
us to determine the localization of ppl–mCherry synthesized in
the presence of ESI. ppl–mCherry fluorescence was considerably
lower after 3 h of CHX treatment, confirming that much of the
protein had been secreted from the cells (Figure 3B, panel B).
In cells treated with PS2 alone following CHX treatment, the
ppl–mCherry distribution was similar to that in untreated cells
(Figure3B,cf.panelsAandC),suggestingthatthemajorityofthe
proteinsynthesizedunderconditionsofproteasomeinhibitionwas
correctly targeted to the ER. A different pattern of fluorescence
was seen in cells treated with ESI(Figure 3B, panel D). Under
these conditions, the reticular distribution of ppl–mCherry was
replaced by more diffuse fluorescence distributed throughout the
cell,suggestingthataproportionofthenewlysynthesizedprotein
had not entered the ER, but was instead located in the cytosol. A
secretory proteincomposed
variableamountofmCherryfluorescentproteinwasalsoobserved
in punctate structures adjacent to the nucleus (Figure 3B, panels
D and E). Co-treatment with PS2 resulted in a much clearer
accumulation of fluorescent protein in the cytosol (Figure 3B,
panel E), suggesting that ppl–mCherry which failed to enter the
ER in the presence of ESIwas degraded via the proteasome.
To validate these data, we took a complementary biochemical
approach to explore the fate of a polytopic membrane protein
synthesized in the presence of ESI. Membranes isolated from
HeLa cells transiently expressing opsin were extracted with
sodium carbonate, pH>11, to remove peripherally associated
proteins [26]. Membrane-integrated proteins are resistant to such
treatment,andwerere-isolatedbycentrifugation,thensolubilized
in Triton X-100. This method allowed separation of the ER lu-
menal chaperone PDI (Figure 3C, lane 3), from the integral ER
membrane protein derlin-1, which remained in the membrane-
associated pellet after carbonate extraction (Figure 3C, lanes
4 and 5). p97, which is peripherally associated with the ER
membrane,wasremovedinthecarbonatesupernatant(Figure3C,
lane 3), confirming that this treatment effectively strips non-
integral proteins from the ER membrane. Next, cells treated with
DMSO, ESI, ESII or ESR35 for 1 h were pulse-labelled in the
continuedpresenceofthecompoundsplusPS2,priortocarbonate
extractionandopsinIP.Inuntreatedcells,thelargemajorityofthe
radiolabelled opsin was present in the carbonate-resistant pellet
(Figure 3D, lanes 2 and 3, DMSO), demonstrating that it was
stably integrated into the ER membrane. A smaller proportion
of the radiolabelled non-glycosylated opsin was extracted in
the carbonate supernatant (Figure 3D, lane 1, DMSO), probably
reflecting protein that failed to be correctly integrated into the ER
membrane. A similar distribution of opsin was seen in ESII- and
ESR35-treated cells (Figure 3D, ESIIand ESR35). In the presence of
ESI,however,radiolabelledopsinwasalmostexclusivelyfoundin
the carbonate supernatant and was non-glycosylated (Figure 3D,
lane 1, ESI). Virtually no radiolabelled opsin could be detected in
the sodium carbonate-extracted pellet and there was no indication
of any glycosylated protein chains (Figure 3D, lanes 2 and 3,
ESI), suggesting that ESIinhibited the integration of opsin into
the ER membrane, leading to production of non-glycosylated
protein. Notably, the population of non-glycosylated opsin was
onlyreadilyapparentinHeLacellswhenPS2wasincludedduring
the pulse-labelling period (Figure 3E, cf. lanes 5 and 9), showing
c ?The Authors Journal compilation c ?2012 Biochemical Society
© 2012 The Author(s)
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