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Journal of Cell Science RESEARCH ARTICLE Ube2g2–gp78-mediated HERP polyubiquitylation is involved in ER stress recovery Long Yan 1 , Weixiao Liu 1 , Huihui Zhang 2 , Chao Liu 1 , Yongliang Shang 1 , Yihong Ye 3 , Xiaodong Zhang 2, * and Wei Li 1, * ABSTRACT A large number of studies have focused on how individual organisms respond to a stress condition, but little attention has been paid to the stress recovery process, such as the endoplasmic reticulum (ER) stress recovery. Homocysteine-induced ER protein (HERP) was originally identified as a chaperone-like protein that is strongly induced upon ER stress. Here we show that, after ER stress induction, HERP is rapidly degraded by Ube2g2–gp78-mediated ubiquitylation and proteasomal degradation. The polyubiquitylation of HERP in vitro depends on a physical interaction between the CUE domain of gp78 and the ubiquitin-like (UBL) domain of HERP, which is essential for HERP degradation in vivo during ER stress recovery. We further show that although HERP promotes cell survival under ER stress, high levels of HERP expression reduce cell viability under oxidative stress conditions, suggesting that HERP plays a dual role in cellular stress adaptation. Together, these results establish the ubiquitin–proteasome-mediated degradation of HERP as a novel mechanism that fine-tunes the stress tolerance capacity of the cell. KEY WORDS: HERP, ER Stress recovery, Ubiquitin–proteasome system, Ube2g2–gp78, Trade-off INTRODUCTION A cell or a multicellular organism often needs to adjust its physiological status to respond to and defend against various stress conditions such as high or low temperature, oxidative and reductive environments. Adaptation to a stress condition can be achieved by enhancement of certain biological traits, and this is often associated with reduction in other traits that are dispensable to survive under this particular stress condition (Bennett and Lenski, 2007). This kind of ‘trade-off’ is often viewed as a cost or constraint associated with adaptation (Bennett and Lenski, 2007; Novak et al., 2006). Thus once stress is attenuated, the cell needs to adjust its status back to the normal physiological condition in order to resume division, growth or prepare to face other challenges (Qian et al., 2006). In general, during stress response, transcription and translation are two major ways to increase cellular stress tolerance capacity (Spriggs et al., 2010), but in the stress recovery stage, post-translational modification and degradation of protein appear to play key roles in eliminating stress-induced proteins (Kamura et al., 2000; Majmundar et al., 2010; Qian et al., 2006; Roobol et al., 2009). There are two major degradation systems in eukaryotic cells: autophagy, which is involved in the degradation of long-lived proteins and organelles, and the ubiquitin–proteasome system, which usually targets short-lived proteins for degradation (Mizushima and Komatsu, 2011). Because stress recovery usually occurs in a short time frame (Kamura et al., 2000; Qian et al., 2006), the ubiquitin– proteasome system is more likely to play essential role in this process. It has been well established that upregulation of some heat shock proteins (HSPs, molecular chaperones) is needed for cells to survive many stress conditions (Monaghan et al., 2009; Qian et al., 2006). But cells seem to maintain some of these chaperones purposely at low levels under physiological conditions to permit constitutive cellular activities to proceed. The degradation of Hsp70 is mediated by carboxy terminus of Hsp70-binding protein (also known as E3 ubiquitin-protein ligase CHIP, and STIP1 homology and U-box containing protein 1; STUB1)-dependent polyubiquitylation during the stress recovery process (Qian et al., 2006). In addition, another short-lived protein, hypoxia-inducible transcription factor HIF1a, is also rapidly ubiquitylated by the ubiquitin ligase VHL (von Hippel-Lindau) and degraded by proteasomes under normoxic conditions (Kamura et al., 2000; Majmundar et al., 2010). However, whether the ubiquitin– proteasome system participates in other stress recovery processes such as ER stress recovery remains unknown. Various stimuli, such as reductive reagents, oxidative reagents and Ca 2+ overload can damage ER functions, leading to the accumulation of misfolded proteins and ER stress (Ellgaard and Helenius., 2001; Rutkowski and Kaufman, 2004; Shen et al., 2004). To cope with ER stress, the cell employs a quality control system named unfolded protein response (UPR) (Kaufman, 1999; Schulze et al., 2005) to attenuate translation and accumulation of misfolded proteins and to increase chaperone expression. HERP (homocysteine-induced ER stress protein) is a chaperone-like protein that is strongly upregulated by ER stress (Kokame et al., 2001; Schro ¨der and Kaufman, 2005) and then rapidly degraded (Hori et al., 2004; Kim et al., 2008; Sai et al., 2003). Unlike other stress-induced cytoplasmic chaperones, HERP is an integral membrane protein with both its N- and C-termini facing the cytoplasm (Nogalska et al., 2006; Sai et al., 2002). The function of HERP is not fully understood, but accumulating evidence suggests that it has an essential role in ER-membrane-associated protein degradation (ERAD), which functions to retrotranslocate ubiquitylated proteins from the ER to proteasomes for degradation (Schulze et al., 2005). Knockdown or knockout of 1 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China. 2 College of Life Sciences, Wuhan University, Hubei 430072, China. 3 Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. *Author for correspondence ([email protected]; [email protected]) Received 16 May 2013; Accepted 8 January 2014 ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 1417–1427 doi:10.1242/jcs.135293 1417

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RESEARCH ARTICLE

Ube2g2–gp78-mediated HERP polyubiquitylation is involved in ERstress recovery

Long Yan1, Weixiao Liu1, Huihui Zhang2, Chao Liu1, Yongliang Shang1, Yihong Ye3, Xiaodong Zhang2,* andWei Li1,*

ABSTRACT

A large number of studies have focused on how individual

organisms respond to a stress condition, but little attention has

been paid to the stress recovery process, such as the endoplasmic

reticulum (ER) stress recovery. Homocysteine-induced ER protein

(HERP) was originally identified as a chaperone-like protein that is

strongly induced upon ER stress. Here we show that, after ER stress

induction, HERP is rapidly degraded by Ube2g2–gp78-mediated

ubiquitylation and proteasomal degradation. The polyubiquitylation of

HERP in vitro depends on a physical interaction between the CUE

domain of gp78 and the ubiquitin-like (UBL) domain of HERP, which

is essential for HERP degradation in vivo during ER stress recovery.

We further show that although HERP promotes cell survival under

ER stress, high levels of HERP expression reduce cell viability

under oxidative stress conditions, suggesting that HERP plays a

dual role in cellular stress adaptation. Together, these results

establish the ubiquitin–proteasome-mediated degradation of HERP

as a novel mechanism that fine-tunes the stress tolerance capacity of

the cell.

KEY WORDS: HERP, ER Stress recovery, Ubiquitin–proteasome

system, Ube2g2–gp78, Trade-off

INTRODUCTIONA cell or a multicellular organism often needs to adjust its

physiological status to respond to and defend against various

stress conditions such as high or low temperature, oxidative and

reductive environments. Adaptation to a stress condition can be

achieved by enhancement of certain biological traits, and this is

often associated with reduction in other traits that are dispensable

to survive under this particular stress condition (Bennett and

Lenski, 2007). This kind of ‘trade-off’ is often viewed as a cost or

constraint associated with adaptation (Bennett and Lenski, 2007;

Novak et al., 2006). Thus once stress is attenuated, the cell needs

to adjust its status back to the normal physiological condition in

order to resume division, growth or prepare to face other

challenges (Qian et al., 2006). In general, during stress response,

transcription and translation are two major ways to increase

cellular stress tolerance capacity (Spriggs et al., 2010), but in

the stress recovery stage, post-translational modification and

degradation of protein appear to play key roles in eliminating

stress-induced proteins (Kamura et al., 2000; Majmundar et al.,

2010; Qian et al., 2006; Roobol et al., 2009). There are two major

degradation systems in eukaryotic cells: autophagy, which is

involved in the degradation of long-lived proteins and organelles,

and the ubiquitin–proteasome system, which usually targets

short-lived proteins for degradation (Mizushima and Komatsu,

2011). Because stress recovery usually occurs in a short time

frame (Kamura et al., 2000; Qian et al., 2006), the ubiquitin–

proteasome system is more likely to play essential role in this

process.

It has been well established that upregulation of some heat

shock proteins (HSPs, molecular chaperones) is needed for cells

to survive many stress conditions (Monaghan et al., 2009; Qian

et al., 2006). But cells seem to maintain some of these chaperones

purposely at low levels under physiological conditions to permit

constitutive cellular activities to proceed. The degradation of

Hsp70 is mediated by carboxy terminus of Hsp70-binding protein

(also known as E3 ubiquitin-protein ligase CHIP, and STIP1

homology and U-box containing protein 1; STUB1)-dependent

polyubiquitylation during the stress recovery process (Qian et al.,

2006). In addition, another short-lived protein, hypoxia-inducible

transcription factor HIF1a, is also rapidly ubiquitylated by the

ubiquitin ligase VHL (von Hippel-Lindau) and degraded by

proteasomes under normoxic conditions (Kamura et al., 2000;

Majmundar et al., 2010). However, whether the ubiquitin–

proteasome system participates in other stress recovery

processes such as ER stress recovery remains unknown.

Various stimuli, such as reductive reagents, oxidative reagents

and Ca2+ overload can damage ER functions, leading to the

accumulation of misfolded proteins and ER stress (Ellgaard and

Helenius., 2001; Rutkowski and Kaufman, 2004; Shen et al.,

2004). To cope with ER stress, the cell employs a quality control

system named unfolded protein response (UPR) (Kaufman, 1999;

Schulze et al., 2005) to attenuate translation and accumulation of

misfolded proteins and to increase chaperone expression. HERP

(homocysteine-induced ER stress protein) is a chaperone-like

protein that is strongly upregulated by ER stress (Kokame et al.,

2001; Schroder and Kaufman, 2005) and then rapidly degraded

(Hori et al., 2004; Kim et al., 2008; Sai et al., 2003). Unlike other

stress-induced cytoplasmic chaperones, HERP is an integral

membrane protein with both its N- and C-termini facing the

cytoplasm (Nogalska et al., 2006; Sai et al., 2002). The function

of HERP is not fully understood, but accumulating evidence

suggests that it has an essential role in ER-membrane-associated

protein degradation (ERAD), which functions to retrotranslocate

ubiquitylated proteins from the ER to proteasomes for

degradation (Schulze et al., 2005). Knockdown or knockout of

1State Key Laboratory of Reproductive Biology, Institute of Zoology, ChineseAcademy of Sciences, Beijing 100101, China. 2College of Life Sciences, WuhanUniversity, Hubei 430072, China. 3Laboratory of Molecular Biology, NationalInstitute of Diabetes and Digestive and Kidney Diseases, National Institutes ofHealth, Bethesda, MD 20892, USA.

*Author for correspondence ([email protected]; [email protected])

Received 16 May 2013; Accepted 8 January 2014

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 1417–1427 doi:10.1242/jcs.135293

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Herp leads to stabilization of several ERAD substrates (Horiet al., 2004; Okuda-Shimizu and Hendershot, 2007). It has been

reported that upregulation of HERP can protect cells from ER-stress-induced apoptosis, mainly by forming an ERAD complextogether with p97 homohexamer, Derlin1, VIMP and theubiquitin E3 ligase HRD1 (Jarosch et al., 2002; Schroder and

Kaufman, 2005; Schulze et al., 2005; Ye et al., 2001), whichstimulates HRD1-mediated ubiquitylation and degradation ofaberrant ER proteins (Kny et al., 2011).

In contrast to its function in ER stress, the fate of HERP afterER stress is still largely unknown. Here we show that after ERstress, HERP is quickly degraded by the Ube2g2–gp78-mediated

ubiquitin–proteasome system, and the interaction between theubiquitin-like (UBL) domain of HERP and the coupling of theubiquitin conjugation to the ER degradation (CUE) domain of

gp78 is essential for HERP polyubiquitylation and subsequentdegradation. Overexpression of HERP in cells improvedtolerance to ER stress. Surprisingly, HERP overexpressionreduces cell viability under some oxidative stress conditions.

We provided further evidence to show that this mechanism isevolutionarily conserved from yeast to mammalian cells. Ourresults suggest that the ubiquitin–proteasome system plays an

important role in the ER stress recovery process, which isessential for adjusting the cellular physiological status to survivemultiple challenges in changing environments.

RESULTSHERP is quickly degraded by the ubiquitin–proteasomesystem after ER stressIt was reported that HERP is strongly upregulated both at themRNA and protein level by homocysteine and other ER stressinducers (Hori et al., 2004; Kokame et al., 2000; Rubel et al.,

2013). We firstly confirmed that ER stress inducers such ashomocysteine, b-mercaptoethanol, tunicamycin, thapsigargin anddithiothreitol (DTT) but not oxidative ER stress inducers such as

H2O2 and Paraquat caused the accumulation of HERP protein inHEK293T, HeLa and HCT116 cells by immunoblotting(Fig. 1A,B; supplementary material Fig. S1A). HERP was rapidly

induced and the protein level peaked at 4 hours after DTTtreatment in both HEK293T and HeLa cells, but subsequently, theprotein level reduced quickly (Fig. 1C; supplementary materialFig. S1B). Presumably this was due to the inactivation of DTT by

oxidation, because the HERP level did not decrease at all duringthe experiment when the cells were induced by relatively stablereductive inducers such as thapsigargin and homocysteine

(compare supplementary material Fig. S1B–D,F). Thus, thisstage could represent the ER stress recovery phase. To test thispossibility, we first treated HEK293T cells with different

reductive ER stress inducers for 4 hours, then transferred cellsto fresh medium without any reductive ER stress inducers andtested the HERP protein level (Fig. 1D; supplementary material

Fig. S1E,G). We found a similar result in all of the tested celllines: HERP was quickly degraded and returned to the normallevel in 8 hours after ER stress (Fig. 1D; supplementary materialFig. S1C), suggesting that after 4 hours incubation, cells

consumed DTT and started the recovery process. Because ERstress can be easily induced by DTT without changing themedium, it was selected for most of the following experiments.

To test whether autophagy or the ubiquitin–proteasomepathway mediates the HERP turnover during ER stressrecovery, we treated HEK293T cells with inhibitors that

specifically block these two pathways. Only the proteasome

inhibitor MG132 was found to strongly inhibit HERP degradationunder normal condition as well as during ER stress recovery

(Fig. 1E,F). By contrast, none of the four autophagy or lysosomeinhibitors such as ammonium chloride (NH4Cl), 3-methyladenine, bafilomycin A1 or chloroquine affected HERPdegradation. These results suggest that HERP is degraded through

the ubiquitin–proteasome system rather than by autophagy.

E2–E3 (Ube2g2–gp78)-complex-mediated HERPubiquitylationBecause HERP is an ER-membrane-associated protein, the E2and E3 enzymes that mediate HERP polyubiquitylation should

also be ER-membrane-associated proteins. It has been reportedthat HRD1 and gp78 are two ERAD-related E3 ubiquitinligases that are associated with the ER membrane (Chen et al.,

2006; Kostova et al., 2007). Hrd1 forms a complex with SEL1Land mainly mediates the degradation of soluble, ER-luminalsubstrates and integral membrane proteins (Rubenstein et al.,2012; Shmueli et al., 2009). gp78, which was the first

described and is the best documented human ERAD E3, is amulti-spanning membrane protein with its catalytic domainfacing the cytosol (Chen et al., 2006; Fang et al., 2001; Li

et al., 2009). Both proteins were shown to interact with HERP,but only gp78 could ubiquitylate HERP efficiently in vitro

(Kny et al., 2011; Li et al., 2007; Schulze et al., 2005), raising

the possibility that gp78 mediates HERP ubiquitylation anddegradation. To test this idea, we created a HCT116 gp78knockout (KO) cell line by homologous recombination and

subsequent Cre–loxP-based selection marker excision (Zhanget al., 2011). The lack of gp78 expression in these cells wasverified by immunoblotting (supplementary material Fig. S2)and HERP degradation during ER stress recovery were

examined by immunoblotting. gp78 knockout significantlyreduced HERP turnover compared with that in the wild-typecell line, regardless of whether or not DTT was present

(Fig. 2A,B). Thus, we conclude that gp78 is an E3 required forHERP turnover during ER stress recovery.

To identify the cognate E2 of gp78 for HERP degradation

during ER stress recovery, we tested a collection of mammalianE2s using an in vitro ubiquitylation assay (Jin et al., 2007), andfound that at least six of them were able to cooperate with gp78 inpolyubiquitylation (supplementary material Fig. S3). Because

only E2G1, E2G2, E2J1 and E2J2 are ER-membrane-associatedE2s (Ye and Rape, 2009), we focused on these four E2s. Inagreement with previous results (Cao et al., 2007; Li et al., 2007),

only Ube2g2 could function with gp78 but not HRD1 toefficiently assemble either free polyubiquitin chains, or onHERP (Fig. 2C,D and supplementary material Fig. S4). These

results suggest that the gp78–Ube2g2 pair are involved in HERPpolyubiquitylation during the ER stress recovery process.

Interaction between gp78 CUE and the HERP UBL domain isessential for HERP polyubiquitylationSubstrate specificity is usually determined by E3 (Pickart, 2001;Rubel et al., 2013). We therefore tested whether there was a

physical interaction between gp78 and HERP. GST–gp78c (thecytosolic domain of gp78) purified from Escherichia coli was usedto pull down His-tagged recombinant HERPc, the cytosolic domain

of HERP. After Coomassie staining, we found HERPc wasefficiently co-precipitated, indicating a direct interaction betweenthese proteins (Fig. 3B, lane 3). The cytosolic segment of gp78

contains four domains: RING, CUE, G2BR and VIM (Chen et al.,

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2006; Donaldson et al., 2003; Shih et al., 2003). To identifywhich domain was responsible for its interaction with HERP, we

generated gp78 mutants as indicated in Fig. 3A. We purified theseproteins as GST-tagged proteins from E. coli. GST pull-downexperiments showed that when the CUE domain was deleted gp78ccould not bind HERPc. By contrast, deletion of either G2BR or

VIM did not affect gp78 binding to HERPc (Fig. 3B). These resultssuggested that the CUE domain in gp78 is required for theinteraction between gp78 and HERP. Importantly, when the CUE

domain was deleted, gp78c could not efficiently assemblepolyubiquitin chains on HERPc (Fig. 3C,D), suggesting thisinteraction is functionally important. Furthermore, gp78 appeared

to assemble lysine-48-linked ubiquitin chains on HERPc becauseubiquitylation with a K48R ubiquitin mutant only supportedmonoubiquitylation of HERPc (Fig. 3E). It has been reported that

Lys48-linked ubiquitin chains target proteins for degradation bythe 26S proteasome (Chau et al., 1989; Ye and Rape, 2009), which

agrees with our in vivo studies showing that HERP is an unstableprotein degraded by the proteasome.

To identify the domain in HERP that is responsible forinteraction with gp78, we generated several HERPc mutants(Fig. 3F) and purified them from E. coli. The binding to gp78cwas tested by a GST pull-down experiment. We found that

deletion of either the UBL domain or a small C-terminal fragmentabolished the binding between HERPc and gp78c (Fig. 3G). Thisis consistent with the established ubiquitin-binding function of

the CUE domain (Chen et al., 2006; Donaldson et al., 2003; Shihet al., 2003). An in vitro ubiquitylation experiment showed thatthese mutants could not be ubiquitylated by gp78c (Fig. 3H). This

result, together with our previous observations that lysine 61 inthe UBL domain is the ubiquitylation site for gp78-mediatedpolyubiquitylation (Li et al., 2007), indicates that the UBL

domain of HERP is one of the key sites to interact with gp78,further resulting in the ubiquitylation of HERP.

Fig. 1. HERP was eliminated through the ubiquitin–proteasome system after ER stress. (A) HERP was strongly induced by reductive stress inducers butnot oxidative stress inducers. HEK293T cells were treated with 10 mM homocysteine, 10 mM b-mercaptoethanol, 12 mM tunicamycin, 1 mM thapsigargin,500 mM DTT, 200 mM H2O2 and 500 mM Paraquat for 4 hours. Cell extracts were subjected to western blotting with the indicated antibodies. Con, untreated.(B)HERP was induced by DTT in various cell lines. Different cell lines were treated with or without 500 mM DTT for 4 hours, then the cell extracts were subjectedto western blotting with the indicated antibodies. (C) After 4 hours induction with DTT, HERP was quickly degraded in HEK293T cells. HEK293T cells weretreated with 500 mM DTT for different times, cell extracts from different time points were subjected to western blotting with the indicated antibodies. (D) HERPwas degraded in the ER stress recovery process. HEK293T cells were treated with 500 mM DTT, then transferred to fresh medium without DTT and sampleswere taken at different time points. Con, untreated. (E,F) HERP was degraded through the ubiquitin–proteasome system. (E) HEK293T cells were treatedwith 25 mM NH4Cl, 10 mM 3-methyladenine (3-MA), 10 mM MG132, 100 nM bafilomycin A1 (BAFA1) or 25 mM chloroquine (CQ) for 24 hours. Cell extractswere subjected to western blotting with the indicated antibodies. Con, untreated. (F) HEK293T cells were treated with 500 mM DTT for 4 hours, then transferredto new medium containing different inhibitors, which were the same with those described in E, and cultured for another 8 hours. Con, HEK293T cells wereharvested directly after 4 hours induction with 500 mM DTT and not treated with any of the inhibitors.

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The gp78 CUE domain and the HERP UBL domain areessential for HERP degradation in vivo

To test whether the interaction between the CUE and UBLdomains is physiologically important for HERP turnover in cells,we reintroduced wild-type gp78 or the gp78 DCUE mutanttogether with a control E3 HRD1 into gp78-deficient HCT116

cells and tested whether they could rescue the HERP degradationdefect (Fig. 4A). We treated these transfected cells with DTT for4 hours, then transferred them to fresh medium without DTT. The

turnover of HERP was monitored by immunoblotting. Wild-typegp78 transfection successfully rescued the HERP degradationdefect, whereas the gp78 DCUE mutant and the control E3 HRD1

failed to do so (Fig. 4B). These results indicate that the CUEdomain is essential for gp78 function in the degradation of HERP

during ER stress recovery.To test the function of HERP UBL domain on its degradation,

we overexpressed wild-type HERP, HERP DUBL or HERP K61Rmutants in HEK293T cells (supplementary material Fig. S5).

Cycloheximide-chase experiment showed that wild-type HERPwas rapidly degraded in HEK293T cells, whereas the HERP DUBLand HERP K61R mutants were significantly stabilized (Fig. 4C).

These results suggest that the UBL domain regulates HERPstability in cells and that gp78-dependent polyubiquitylation onlysine 61 of HERP mediates its proteasome degradation.

Fig. 2. Ube2g2- and gp78-mediated HERP ubiquitylation. (A) HERP degradation was disrupted by gp78 knockout in the HCT116 cell line. HCT116 wild-type(WT) and HCT116 gp78 KO cells were treated with 500 mM DTT for different times. Cell extracts were subjected to western blotting with the indicated antibodies.(B) HERP degradation was disrupted by gp78 knockout during the ER stress recovery process. After treatment with 500 mM DTT for 4 hours, HCT116 WTand HCT116 gp78 KO cells were then transferred to fresh medium and cultured for different times. Con, untreated. (C) Ube2g2–gp78 assembled polyubiquitinchains efficiently in the in vitro ubiquitylation assay. Reactions were conducted with UbE1, ER-related E2s, gp78 and FLAG–Ub. The formation of ubiquitinchains was analyzed by immunoblotting with anti-FLAG antibody. Con, reaction without E2. (D) HERPc was ubiquitylated by Ube2g2–gp78. Ubiquitylationreactions were conducted with UbE1, ER-related E2s, gp78c, RGS-His HERPc and FLAG–Ub. The reactions were analyzed by immunoblotting withanti-RGS–His antibody. Con, reaction without E2. *Nonspecific band.

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Herp plays a key role in the trade-off between reductive andoxidative stress adaptationWe next wished to study why HERP is rapidly eliminated duringER stress recovery. In the natural condition, only when a cell canperform multiple tasks, can it survive numerous stresses (Shoval

et al., 2012). In general, adaptation to a specific stressenvironment is often accompanied by increased sensitivity toanother stressor (Ackerman and Gems, 2012; Bennett and Lenski,

2007; Casanueva et al., 2012; Shoval et al., 2012). We

hypothesized that ER stress survival is accompanied bydeterioration in other traits that render cells sensitive to adifferent stress inducer. To test this idea, we ectopicallyexpressed HERP and an irrelevant control membrane protein,

gp78, in which the trans-membrane domain was fused with GFP(gp78-TM–GFP) in HEK293T cell. The overexpression level ofHERP is roughly equal to that induced by ER-stress inducers

Fig. 3. Interaction between theCUE domain of gp78 and the UBLdomain of HERP was essential forHERPc ubiquitylation.(A) Schematic representation thegp78 variants tested in B. (B) TheCUE domain of gp78 controlled theinteraction with HERPc. Theindicated GST fusion proteins wereimmobilized on glutathione beadsand incubated with purified HERPc(lanes 2–7). Bound proteins wereeluted and analyzed by SDS-PAGEand Coomassie Blue staining. Lane 1shows HERPc input (20%).(C) Deletion of the Cue domain ofgp78 affected polyubiquitin chainassembly on the active site ofUbe2g2. Polyubiquitylation reactionsconducted with E1, Ube2g2, FLAG–Ub, ATP, gp78 WT or gp78 DCUEwere analyzed under non-reducingcondition by immunoblotting.(D) gp78 DCUE is defective inHERPc polyubiquitylation.Polyubiquitylation reactions wereconducted as in C but with HERPc.*A nonspecific band. (E) Ube2g2–gp78 assembled Lys48-linkedpolyubiquitin chain on HERPc.Polyubiquitylation reactions wereconducted as in D, but with K48RFLAG–Ub. *A nonspecific band.(F) Schematic representation ofHERP variants used in G and H.(G) The UBL domain of HERPparticipates in the interaction withgp78c. GST or GST–gp78c proteinswere immobilized on glutathionebeads and incubated with purifiedHERPc variants (lanes 5–12). Boundproteins were eluted and analyzed bySDS-PAGE and Coomassie Bluestaining. Lanes 1–4 show HERPmutant input (20%). *The pulldownprotein. (H) Ubiquitylation of HERPvariants in vitro. Polyubiquitylationreactions were conducted asdescribed in D, but using HERPvariants. *Nonspecific band.

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(supplementary material Fig. S6A), and the transfection of gp78-TM–GFP did not affect HERP degradation during the ER stress

recovery process (compare supplementary material Fig. S6B withC). We then treated the cells with DTT at various concentrationsand found that the viability of HERP-overexpressing cells wassignificantly higher than the gp78-TM–GFP-overexpressing cells,

consistent with the notion that HERP overexpression improvesadaptation to ER stress (Fig. 5A). Interestingly, when HERP-expressing cells were treated with hydrogen peroxide (H2O2) to

induce oxidative stress, they were more sensitive to H2O2-induced cell death compared with the gp78-TM–GFP-over-expressing cells (Fig. 5B). We then tested some other stress

conditions such as reductive stress (homocysteine treatment),oxidative stress (Paraquat treatment) and DNA damage (UVirradiation). Consistent with our model, the ability to grow underreductive stress condition was significantly higher in HERP-

overexpressing cells than in gp78-TM–GFP-overexpressing cells(Fig. 5C and supplementary material Fig. S6D), but the oxidativestress tolerance ability of HERP-overexpressing cells was

impaired (Fig. 5C and supplementary material Fig. S6E).However, no difference was found in viability between HERP-

expressing and the gp78-TM–GFP-overexpressing cells after UV

irradiation (Fig. 5C). Furthermore, we expressed HERP DUBL inHEK293T cells and treated them with H2O2. We found thatHERP DUBL-overexpressing cells were even more sensitive to

oxidative stress than those overexpressing full-length HERP

(Fig. 5D). This result suggests that the proteotoxicity of HERP

under oxidative stress condition is independent of the UBLdomain. Because the UBL domain promotes gp78-mediateddegradation of HERP, the increased cell death of HERP DUBL-expressing cells under oxidative stress conditions is probably due

to higher expression of this mutant. Together, our results suggestthe existence of a trade-off during cellular adaptation to reductivestress and oxidative stress: the induction of HERP by reductive

stress improves cell viability under this particular stresscondition, but compromises the ability of the cell to deal withoxidative stress. This model provides a plausible explanation for

why HERP needs to be rapidly returned to its basal level after ERstress.

Evolutionarily conserved ER stress recovery mechanism inyeastWe next tested whether the aforementioned mechanismis evolutionarily conserved (Fig. 6A,B). In yeast, a UBL-

containing protein named Usa1p was proposed to be the HERPhomolog (Carvalho et al., 2010). To test whether Usa1p andHERP are really functional homologs, we created a USA1 (yeast

HERP homolog)-deleted strain. As expected, the USA1 knockoutstrain was sensitive to ER stress compared with an isogenic wild-type strain (supplementary material Fig. S7A). We then expressed

Fig. 4. The CUE domain of gp78 and the UBL domain of HERP were essential for HERP degradation in vivo. (A) HCT116 gp78 KO cells weretransiently transfected with WT gp78 or gp78 CUE mutant together with a control E3 HRD1 and the levels of gp78 were compared with those in the HCT116 WTcells, by immunoblotting. (B) The CUE domain of gp78 was essential for HERP degradation in vivo. The gp78 knockout HCT116 cell line was transfectedwith WT or CUE-domain deleted gp78 together with HRD1, then treated with 500 mM DTT for 4 hours, The cells were then transferred to fresh medium withoutDTT and cultured for the indicated time, after which the level of HERP was determined. The control cells were transfected with the indicated plasmids butnot treated with DTT. (C) Lysine 61 of the UBL domain in HERP was essential to its degradation in vivo. HEK293Tcells transfected with pRK-HERP, pRK-HERPDUBL or pRK-HERP K61R were treated with 10 mg/ml cycloheximide for different times, and their degradation were analyzed by immunoblotting with theindicated antibodies. GFP was used as a transfection control. Con, without transfection.

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either USA1 or HERP in the USA1-deficient strain under thecontrol of USA1 promoter. The expression of USA1 rescued thereductive stress sensitivity of Dusa1 cells back to the wild-type

level, whereas HERP could only partially rescue the ER stresssensitivity (supplementary material Fig. S7B), suggesting thatUsa1p is a functional ortholog of HERP.

Next we tested whether Usa1p also plays an important role inthe trade-off during cellular adaptation to reductive and oxidativestresses. We then overexpressed USA1 under the controlof a strong TDH3 promoter in the wild-type strain. The

overexpression of USA1 slightly retarded the growth of yeastcells (supplementary material Fig. S7C), suggesting there is someproteotoxicity of Usa1p. We then treated these cells with either

DTT or H2O2, and consistent with the function of HERP inmammalian cells, USA1 overexpression also helped yeast cellsresist the reductive stress, but impaired their oxidative stress

tolerance ability (Fig. 6C,D). These results suggest that the roleof Usa1p and HERP in the trade-off during cellular adaptation toreductive and oxidative stresses is evolutionarily conserved.

Because the UBL domain is conserved in all the homologs ofHERP, it raises the possibility that Usa1p may also be degraded bythe ubiquitin–proteasome system during the ER stress recovery

process. To test this idea, we added a GFP tag to endogenousUsa1p at the C-terminus. After treating with an ER stress inducersuch as DTT, Usa1p was induced and reached the peak at around

4 hours and then degraded rapidly, which was the same as thebehavior of HERP in mammalian cells (Fig. 6E). The degradationof Usa1p could also be significantly inhibited by MG132 (Fig. 6F).

Thus, we conclude that HERP degradation during the ER stressrecovery process is also evolutionarily conserved.

DISCUSSIONIn the present study, we investigated the fate of HERP after ERstress. We found that HERP was quickly eliminated by anubiquitin–proteasome-dependent pathway during the ER stress

recovery process. It has been reported that HERP can bepolyubiquitylated by an E3 ubiquitin ligase POSH, but theubiquitin chains formed by POSH were lysine-63-linked and they

seem to regulate HERP localization in response to ER stress(Tuvia et al., 2007). Our previous work showed that gp78 canubiquitylate HERP in vitro (Li et al., 2007; Li et al., 2009), but

whether it can catalyze HERP turnover during ER stress recoveryis unclear. Here we show that gp78 is a major E3 that mediateslysine-48-linked ubiquitylation of HERP and its degradation in

Fig. 5. HERP plays a key role in the trade-off between reductive and oxidative stress adaptation. (A) HERP overexpression increased HEK293T cellviability in the reductive stress condition. HEK293T cells transfected with the control, gp78-TM–GFP or HERP were treated with DTT for 24 hours and theviability of the cells was monitored by MTT assay. (B) HEK293T cells were sensitive to oxidative stress when HERP was overexpressed. HEK293T cellstransfected with gp78-TM–GFP or HERP were treated with H2O2 for 24 hours, and the viability of the cells was tested as in A. (C) HERP overexpressionincreased cells ability to adapt to reductive stress but impaired their tolerance to oxidative stress, but did not change their sensitivity to UV irradiation. Cellviability was tested as in A. HEK293Tcells transfected with gp78-TM–GFP, but not treated by these reagents were used as the control. (D) UBL domain of HERPattenuated the deterioration of HERP by promoting its degradation. HERP DUBL-overexpressing HEK293T cells were even more sensitive to oxidative stressthan cells overexpressing full-length HERP. Values shown are means (6 s.e.m.) of six experiments. *P,0.05, **P,0.01.

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cells. gp78-mediated HERP turnover represents another means ofcontrolling the HERP protein level in addition to transcriptional

upregulation by ER stress inducers. Unlike other molecularchaperones such as Hsp70, which could be directly ubiquitylatedby an associated E3 ligase CHIP after substrate is depleted (Qian

et al., 2006), HERP associates with the HRD1–SEL1L complex,stimulating HRD1-mediated ubiquitylation and degradation ofaberrant ER proteins (Kny et al., 2011), but itself is ubiquitylated

by another E3, gp78. Thus, when compared with the CHIPsystem, the mechanism that regulates HERP expression andactivity involves another layer of complexity.

Previously, it was reported that the yeast homolog of HERP,Usa1p, is involved in eliminating un-partnered Hrd1p by mediatingits self-ubiquitylation and ultimate degradation (Carroll andHampton, 2010). gp78-mediated HERP polyubiquitylation and

subsequential degradation is conceptually similar to the abovementioned ERAD components controlling the degradation ofeach other. The self-degradation of Hrd1p requires the UBL

domain of Usa1p in yeast, but at present we do not know whetherthe degradation of excess HRD1 depends on HERP or not inmammalian cells. However, the ubiquitylation of HERP also

depends on a physical interaction between its UBL domain and theCUE domain of gp78. The primary function of the CUE domain isto bind ubiquitin and preferentially polyubiquitin chains (Chen

et al., 2006; Donaldson et al., 2003; Shih et al., 2003). Thus, it ispossible that during ER stress HERP assists HRD1 in mediating

ubiquitylation of aberrant ER proteins. gp78 may cooperate withHRD1 in ubiquitin chain assembly. After substrates are eliminated,the UBL domain in HERP then works as a ubiquitin moiety and is

recognized by the CUE domain of gp78. As a result, HERP isubiquitylated and degraded, thus initiating the ER stress recoveryprocess.

In sharp contrast to the many studies about how cells respondto various stresses, the processes of stress recovery are still farfrom well characterized. CHIP-mediated Hsp70 degradation is

one of the best investigated. CHIP is involved in proteasome-dependent clearance of unfolded cytosolic proteins during stressresponses. At the same time, it is also required for recovery fromthe stress response by promoting Hsp70 ubiquitylation and

subsequent degradation (Qian et al., 2006). Another analogousexample can be found in hypoxia. After stress, the SUMOylatedhypoxia-inducible factor 1a (HIF1a) binds to a ubiquitin ligase,

von Hippel-Lindau (VHL) protein, leading to its rapidubiquitylation and degradation by the proteasome (Kamuraet al., 2000). Recently, it was reported that in response to DNA

damage, cells inhibit protein synthesis by activating theeukaryotic elongation factor 2 kinase (eEF2K), whereas duringthe recovery process, eEF2K is ubiquitylated by the SCF (Skp,

Fig. 6. Evolutionarily conserved role of HERP and USA1 in the trade-off during adaptation to reductive and oxidative stress conditions.(A) Phylogenetic tree of HERP homolog proteins from Saccharomyces cerevisiae, Drosophila melanogaster, Danio rerio, Mus musculus and Homo sapiens.(B) Sequence alignment showing the UBL domain of HERP homolog proteins as in A. (C) USA1 overexpression increased the viability of yeast cells whenexposed to reductive stress. USA1 was overexpressed under the control of a strong TDH3 promoter in the wild-type strain and grown on SC platescontaining 2.5 mM DTT. (D) USA1 overexpression impaired the oxidative stress tolerance of yeast. USA1 was overexpressed under the control of a strongTDH3 promoter in the wild-type strain and grown on SC plates containing 1.5 mM H2O2. (E) Usa1p was strongly induced by DTT in BY4742. Yeast cellswere cultured to the exponential phase and then induced with 500 mM DTT for the times indicated. Cell extracts were subjected to western blotting with theindicated antibodies. (F) MG132 blocked Usa1p degradation in BY4742. Yeast cells were cultured to the exponential phase, then treated with 500 mM DTTand50 mM MG132 for the times indicated. The Usa1p level was monitored as in E.

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Cullin, F-box-containing) bTrCP (b-transducin repeat-containingprotein) E3 ubiquitin ligase and then degraded by the proteasome,

thus allowing the restoration of peptide chain elongation(Meloche and Roux, 2012). In this study, we found that duringthe ER stress recovery process, HERP was quickly degradedthrough a Ube2g2–gp78-dependent ubiquitin–proteasome system,

and the yeast homolog of HERP, Usa1p, was similarly eliminatedby the ubiquitin–proteasome system. Based on these results, weproposed that, ubiquitin–proteasome-mediated degradation of

certain stress response molecules may generally occur duringstress recovery processes, which may involve different E2 and/orE3 enzymes under different stress conditions.

The assumption of cost associated with gain or trade-offsbetween traits has been postulated for centuries as either aphilosophical or a biological premise (Bennett and Lenski, 2007),

but the evidence in support of this notion is still lacking becausemost comparative studies on different taxa are essentiallycorrelational without functional validation. Recently, somebacteria such as E. coli were used to prove the trade-off

concept by imposing selection on one trait and then measuringcorrelated changes in other traits such as adaption to either low orhigh temperature (Bennett and Lenski, 2007; Ibarra et al., 2002;

Novak et al., 2006). In the last year, it was reported that atrade-off occurs in C. elegans between stress resistance andreproduction/growth fitness (Ackerman and Gems, 2012;

Casanueva et al., 2012). However, most of these studies arestill far from determining the molecular mechanism underlyingthese trade-offs. In the current study, we not only show a clear

trade-off between reductive and oxidative stress tolerance fromyeast to mammalian cells, but also demonstrate HERP and itshomologs play a key role in this trade-off. In addition, we foundthat the Ube2g2–gp78-mediated ubiquitin–proteasome system

regulates this trade-off by promoting HERP polyubiquitylationand subsequent degradation, thus providing an elegant molecularmechanism that modulates two stress adaptation programs.

MATERIALS AND METHODSAntibodies and proteinsAnti-FLAG, anti-Myc and anti-b-actin antibodies were purchased from

Abmart, and anti-RGS-His antibody was purchased from Qiagen.

Ube2g2, gp78, HERP and GFP polyclonal antibodies were generated in

rabbits using the corresponding recombinant proteins as antigen. FLAG–

ubiquitin and GST–E1 were purchased from Boston Biochem.

PlasmidspET28-Ube2g2, pQE9-HERPc constructs have been described previously

(Li et al., 2007). All the other 24 mammalian E2s were constructed by

amplifying the ORFs from mouse cDNA and cloning into the pET28a

vector. The gp78 deletion mutants and HERPc mutants were generated by

site-directed mutagenesis from pGEX-gp78 or pQE9-HERPc vectors. pRK-

HERP and pRK-HERP DUBL were constructed by cloning the coding

sequence into the SalI and NotI sites of the pRK vector. pRS425-USA1 and

-HERP were constructed by cloning the coding sequence of USA1 or Herp

+ 850 bp 59 and 600 bp 39 of USA1 into the XhoI and SacI sites of the

pRS425 vector. pRS315-TDH3 USA1 and pRS315-TDH3 HERP were

constructed by cloning the TDH3 promoter and USA1 or HERP coding

sequence, digestion (XhoI–EcoRI for TDH3, EcoRI–SacI for USA1 and

HERP) and ligating into the pRS315 vector.

Recombinant protein purificationPurification of Ube2g2 and gp78c has been described previously

(Ye et al., 2003). Purified E2 and E3 variants were further fractionated

by size exclusion chromatography on Superdex200 and Superrose 6

columns, respectively in 50 mM Tris-HCl (pH 8.0), 150 mM potassium

chloride, 5% glycerol and 2 mM magnesium chloride. RGS-His-tagged

HERPc mutants were purified under native conditions using Ni-NTA

beads.

Establishment of the gp78 knockout HCT116 cell lineSomatic cell gene targeting was conducted as described previously

(Zhang et al., 2011). Briefly, the targeting AAVs were packaged in

HEK293T cells by transfecting equal amounts of gp78 KO targeting

vector (supplementary material Fig. S2), pHelper and pRC plasmids

(1 mg each). After 72 hours, the transfected cells were scraped from the

plates and suspended in sterile phosphate-buffered saline. The suspension

was then centrifuged at 500 g, and the pellet was frozen and thawed three

times. Finally, the lysate was centrifuged to remove cell debris and the

supernatant containing rAAV was divided into several aliquots and

frozen at 280 C. HCT116 cells were infected with the gp78-targeting

viruses and selected with neomycin for 2 weeks. The Geneticin-resistant

clones were then screened for homologous recombination by genomic

PCR with primers derived from the neomycin resistance gene (59-

GTTGTGCCCAGTCATAGCCG-39) and the upstream region of the left

homologous arm (59-GGGCCGTATAAGGAATTTGC-39). Positive

clones were confirmed by genomic PCR, with primers derived from

the neomycin resistance gene (59-TCTGGATTCATCGACTGTGG-39)

and the downstream region of the right homologous arm (59-AAC-

ACCTAACTTCGGCATGG-39). Correctly targeted clones were infected

with adenoviruses expressing Cre recombinase to delete the selectable

drug marker. To select clones with successful deletion of the selectable

drug marker, genomic PCR was employed to amplify an approximate

250 bp fragment in which the loxP site was inserted, using specific

primers (59-CATGATGCCACATTCACTGC-39 and 59-GCCCAGTTT-

TACCTGTGTAGGA-39).

The heterozygous KO clones were infected with the same targeting

virus to target the second allele, and the neomycin resistance gene was

excised as described earlier. Final confirmation in the generation of the

KO cell lines was performed using western blotting.

Inhibition assayTo identify intracellular protein degradation systems, cells were treated

with 25 mM NH4Cl, 10 mM 3-methyladenine, 10 mM MG132, 100 nM

bafilomycin A1 or 25 mM chloroquine for 8 hours or 24 hours to inhibit

autophagy or ubiquitin–proteasome pathways. Then the HERP level was

measured by western blotting (Mizushima et al., 2010).

Chase assayIn other cases, HEK293T cells transfected with HERP, HERP DUBL or

HERP K61R were treated with cycloheximide (10 mg/ml) for the

indicated times to inhibit de novo protein synthesis, and then

degradation of the HERP mutants was examined by western blotting.

In vitro ubiquitylation assayUbiquitylation experiment was performed as described previously

(Ye et al., 2003). Briefly, E1 (60 nM), Ube2g2 (200 nM) and gp78c

(300 nM) were incubated with FLAG-tagged ubiquitin (10 mM) at 37 C

in buffer containing 25 mM Tris-HCl (pH 7.4), 2 mM magnesium

ATP and 0.1 mM DTT. Samples taken at different time points were

quenched with Laemmli buffer in the absence of reducing reagent. For

reducing condition, samples were treated with 100 mM DTT before SDS-

PAGE. Ub chains were detected by immunoblotting with anti-FLAG

antibody. Ubiquitylation of HERPc was conducted using the conditions

described above with the addition of HERPc (500 nM). Ubiquitylated

HERPc was detected by immunoblotting with anti-RGS-His antibody

(Qiagen).

Cell culture and ER stress conditionsHEK293T and HeLa cells were maintained in DMEM containing 10%

FBS. HCT116 WT and HCT116 gp78 KO cells were cultured in

McCoy’s 5A medium containing 10% FBS. ER stress was induced by

treating the cells with 10 mM homocysteine, 10 mM b-mercaptoethanol,

12 mM tunicamycin, 1 mM thapsigargin or 500 mM dithiothreitol for

4 hours.

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MTT assayCell viability and cell death under reductive stress or oxidative stress

conditions were measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,

5-diphenyltetrazoliumbromide (MTT) assay. HEK293T cells were

placed into 96-well plates, and transfected with pRK-HERP, pRK-

HERP DUBL or pRK vector. Different concentrations of reductive or

oxidative stress inducers were added for 24 hours, then MTT was added

and the mixture incubated for 2 hours. DMSO was then added to dissolve

the formazan. After 10 minutes the OD was measured at 490 nm.

Western blottingCells were lysed in RIPA buffer containing 50 mM Tris pH 7.5, 150 mM

NaCl, 0.1% SDS, 1% NP40, 2 mM EDTA, 0.5% sodium deoxycholate

and protease inhibitors (Roche). Samples were then subjected to western

blotting with anti-HERP antibody, anti-b-actin antibody, anti-FLAG

antibody or anti-RGS-His antibody.

Yeast sensitivity assaysYeast strains were grown to high density (A600.1.2). Beginning with an

A600 of 0.126, 10-fold serial dilutions were made and spotted onto the

appropriate selected SC plates followed by incubation at 30 C for 2 days.

AcknowledgementsWe thank Chunsheng Han and Qing Li for critical reading of the manuscript.

Competing interestsThe authors declare no competing interests.

Author contributionsL.Y. planned and performed most of the experiments and helped to write themanuscript. W.X.L. performed and analyzed some biochemical experiments. H.Z.established the gp78KO cell line. C.L. and Y.S. performed some biochemicalexperiments. Y.Y. analyzed some of the data. X.Z. and W.L. supervised theproject, designed the experiments and wrote the article.

FundingThis work was supported by the National Natural Science Foundation of China[grant number 30970603 to W. L.]; the Knowledge Innovation Program [grantnumber KSCX2-YW-N-071 to W. L.]; the Intramural Research Program of theNational Institute of Diabetes and Digestive and Kidney Diseases (to Y.Y.); andthe One Hundred Talents Program of the Chinese Academy of Sciences.Deposited in PMC for release after 12 months.

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.135293/-/DC1

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RESEARCH ARTICLE Journal of Cell Science (2014) 127, 1417–1427 doi:10.1242/jcs.135293

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