ORIGINAL PAPER

Ero1α requires oxidizing and normoxic conditions to localizeto the mitochondria-associated membrane (MAM)

Susanna Y. Gilady & Michael Bui & Emily M. Lynes &

Matthew D. Benson & Russell Watts & Jean E. Vance &

Thomas Simmen

Received: 22 December 2009 /Revised: 21 January 2010 /Accepted: 25 January 2010 /Published online: 26 February 2010# Cell Stress Society International 2010

Abstract Protein secretion from the endoplasmic reticu-lum (ER) requires the enzymatic activity of chaperonesand oxidoreductases that fold polypeptides and formdisulfide bonds within newly synthesized proteins. Thebest-characterized ER redox relay depends on thetransfer of oxidizing equivalents from molecular oxygenthrough ER oxidoreductin 1 (Ero1) and protein disulfideisomerase to nascent polypeptides. The formation ofdisulfide bonds is, however, not the sole function of ERoxidoreductases, which are also important regulators ofER calcium homeostasis. Given the role of human Ero1αin the regulation of the calcium release by inositol 1,4,5-trisphosphate receptors during the onset of apoptosis, wehypothesized that Ero1α may have a redox-sensitivelocalization to specific domains of the ER. Our resultsshow that within the ER, Ero1α is almost exclusivelyfound on the mitochondria-associated membrane (MAM).The localization of Ero1α on the MAM is dependent onoxidizing conditions within the ER. Chemical reductionof the ER environment, but not ER stress in generalleads to release of Ero1α from the MAM. In addition,the correct localization of Ero1α to the MAM alsorequires normoxic conditions, but not ongoing oxidativephosphorylation.

Keywords Endoplasmic reticulum (ER) .Mitochondria .

Mitochondria-associated membrane (MAM) .

Oxidative protein folding . Ero1α

Abbreviations2ME 2-mercaptoethanolBiP/GRP78 Immunoglobulin binding protein/glucose-

regulated protein of 78 kDaEro1α ER oxidoreductin 1HEK Human embryonic kidneyMAM Mitochondria-associated membranePACS-2 Phospho-furin acidic cluster sorting

protein 2

Introduction

The endoplasmic reticulum (ER) is the point of origin forprotein secretion. To allow for this crucial function, the ERcontains folding chaperones required for the production ofsecretion-competent, fully folded secretory proteins. Forexample, the hsp70 family member BiP/GRP78 is amongthe first chaperones to interact with hydrophobic surfaces ofnewly synthesized polypeptides. At the expense of ATP,Bip/GRP78 promotes the folding of these hydrophobicamino acids to the interior of the protein structure (Gething1999; Hammond and Helenius 1994; Kim et al. 1992). Thelectin chaperones calreticulin and calnexin also mediate thefolding of newly synthesized proteins by cycling on and offtheir folding substrates, depending on the presence of aglucose linked to the high mannose sugar core (Carameloand Parodi 2008; Michalak et al. 2009). Another importantgroup of ER protein-folding enzymes is oxidoreductasesthat catalyze the formation of disulfide bonds. The best-

S. Y. Gilady :M. Bui : E. M. Lynes :M. D. Benson :T. Simmen (*)Department of Cell Biology, University of Alberta,Edmonton, AB T6G2H7, Canadae-mail: Thomas.Simmen@ualberta.ca

R. Watts : J. E. VanceGroup on the Molecular and Cell Biology of Lipids,Department of Medicine, University of Alberta,Edmonton, AB, Canada T6G 2S2

Cell Stress and Chaperones (2010) 15:619–629DOI 10.1007/s12192-010-0174-1

known member of this group is protein disulfide isomerase(PDI), which recognizes unfolded hydrophobic amino acidson newly synthesized proteins (Freedman et al. 2002;Klappa et al. 1997). Following the docking with itssubstrates, PDI is able to oxidize reduced cysteines orisomerize pre-formed disulfide bonds through transientmixed disulfides with its CXXC active sites and substrates(Horibe et al. 2004; Walker and Gilbert 1995). Aprerequisite for the catalytic activity of PDI is the presenceof proteins of the Ero1 family within the ER (Cabibbo et al.2000; Mezghrani et al. 2001; Sevier and Kaiser 2008).These oxidoreductases, represented by Ero1α and Ero1β inhuman cells, mediate the re-oxidation of PDI after itsenzymatic reaction by transferring oxidative equivalentsusing their CXXCXXC active sites (Benham et al. 2000;Pagani et al. 2000). The oxidative power of Ero1 proteinsoriginate from their flavin adenine dinucleotide (FAD)prosthetic group that transfers electrons from cysteines tomolecular oxygen (Wang et al. 2009). Hypoxia, the absenceof oxygen, impacts on the functioning of Ero1 proteins intwo ways: First, Ero1α, but not Ero1β is a target ofhypoxia-inducible factor (HIF-1α)-mediated transcription(Gess et al. 2003; May et al. 2005). Second, whereas thisinduction may result in the increased production of growthfactors by tumor cells, in vitro experiments suggest thatEro1 proteins cannot promote PDI re-oxidation in theabsence of oxygen (May et al. 2005; Tu and Weissman2002). Another factor that impacts on the catalytic activityof Ero1 proteins is their correct localization to the ER. Bothyeast and rice Ero1 proteins require association with the ERmembrane for their functioning (Onda et al. 2009; Pagani etal. 2001). Human Ero1α requires the interaction with PDIand another oxidoreductase, ERp44, for its retention withinthe ER (Anelli et al. 2003; Otsu et al. 2006).

The ER is not a homogeneous organelle, but iscomposed of numerous domains, including the transitionalER that mediates ER export and the rough ER (rER) thathouses the protein translation and import machinery (Voeltzet al. 2002). Besides the rER, the mitochondria-associatedmembrane (MAM) is most highly enriched in ER chaper-ones and oxidoreductases (Hayashi et al. 2009). The MAMis the location where phosphatidylserine biosynthesis andits transfer to mitochondria occur (Shiao et al. 1998; Stoneand Vance 2000), but also contains calcium handlingproteins, such as the inositol 1,4,5-trisphosphate receptor(IP3R) (Csordas et al. 1999; Hajnoczky et al. 2000). On theMAM, ER chaperones and oxidoreductases appear toregulate the ER calcium content. For instance, calnexinreversibly interacts with sarcoplasmic/endoplasmic reticu-lum calcium ATPase (SERCA) 2b to block calcium import(Roderick et al. 2000). Similarly, calreticulin inhibits uptakeof calcium by inhibiting the affinity for calcium of theSERCA2b pump, but also regulates IP3-induced calcium

release (Camacho and Lechleiter 1995; John et al. 1998). Invivo, these functions of calreticulin may very well be morecrucial for survival than its chaperone activity, sincecalreticulin-deficient cells have impaired Ca2+ homoeo-stasis, yet only a modest decrease in protein folding(Michalak et al. 2009; Molinari et al. 2004). Anotherexample of a folding enzyme regulating ER calciumcontent is the oxidoreductase ERp44 that interacts withcysteines of the IP3R type 1, thereby inhibiting calciumtransfer to mitochondria when ER conditions are reducing(Higo et al. 2005). Recent results suggest that Ero1α mightalso perform such a function, since Ero1α interacts with theIP3R and potentiates the release of calcium during ERstress (Li et al. 2009). This function of Ero1α could impactthe interaction of the ER with mitochondria that depends onthe formation of calcium hotspots on the cytoplasmic faceof the ER (Yi et al. 2004) and the induction of apoptosisthat critically depends on ER-mitochondria calcium com-munication (Mendes et al. 2005; Szalai et al. 1999). Toincrease our understanding of the functions of Ero1αduring resting and stressed conditions, we examined itslocalization within the ER by subcellular fractionation andimmunofluorescence microscopy. Our results show thatEro1α is highly enriched on the MAM, consistent with itsrole in the regulation of ER calcium homeostasis. Hypoxialeads to a rapid and eventually complete depletion of Ero1αfrom the MAM. Our findings suggest that hypoxic tumorcells could display high levels of this oxidoreductase ontheir surface or in the space surrounding the tumor.

Materials and methods

Antibodies and reagents

All chemicals were from Sigma (Oakville, ON), exceptfor tunicamycin and thapsigargin (Enzo Life Sciences,Farmingdale, NY). The rabbit anti-calnexin antiserumwas generated using a cytosolic peptide by OpenBiosystems, Huntsville, AL. Antibodies against GAPDHwere provided by Tom Hobman, Edmonton, AB; themonoclonal antiserum against Ero1α was a gift fromRoberto Sitia, Milan, Italy. Antibodies to the humanproteins Ero1α (polyclonal rabbit, Santa Cruz, SantaCruz, CA), PDI, calreticulin, actin (ABR/Pierce, Golden,CO), β-tubulin, BiP/GRP78 (Calbiochem, San Diego,CA), βCOP (Genetex, San Antonio, TX), mitochondrialcomplex 2 (Mitosciences, Eugene, OR), and the myc-tag(Millipore, Billerica, MA) were purchased as indicated.HEK 293 and HeLa cells were from ECACC (PortonDown, UK) and cultured in Dulbecco's modified Eagle'smedium (DMEM, Invitrogen, Carlsbad, CA) containing10% fetal bovine serum (Invitrogen, Carlsbad, CA).

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Percoll was from GE Healthcare (Piscataway, NJ);Optiprep was from Axis-Shield (Dundee, Scotland).Mitotracker was from Invitrogen (Carlsbad, CA).

Western blotting, immunofluorescence microscopy

Western blotting was performed according to standardprotocols using goat-anti mouse/rabbit secondary antibod-ies conjugated to Alexafluor 680/750 (Invitrogen, Carlsbad,CA) on an Odyssey infrared imaging system (LICOR,Lincoln, NE). Processing of samples for immunofluores-cence microscopy was performed as follows: HEK 293cells were grown on coverslips for 24 h and preloaded withMitotracker for 30 min according to the manufacturer'sinstructions. Cells were washed with phosphate-bufferedsaline (PBS) containing 1 mM CaCl2 and 0.5 mM MgCl2(PBS++) and fixed with 4% paraformaldehyde for 20 min.After washing with PBS++, cells were permeabilized for1 min with 0.1% Triton X-100, 0.2% bovine serum albuminin PBS++. Immunofluorescence preparations with the anti-βCOP antibody involved fixation by incubation in metha-nol for 2 min at −20°C. Cells were then incubated withprimary antibodies (1:100 dilution for PDI monoclonal andEro1α polyclonal) and secondary antibodies in PBS++,0.2% bovine serum albumin (BSA) for 1 h each, interruptedwith three washes using PBS++. Secondary antibodies wereAlexaFluor-conjugated 350 and 488 (Invitrogen, Carlsbad,CA) and used at a dilution of 1:2,000. Samples weremounted in Prolong AntiFade (Invitrogen, Carlsbad, CA).Images were obtained with an Axiocam on an Axioobservermicroscope (Carl Zeiss, Jena, Germany) using a 100× plan-Apochromat lens. All images were iteratively deconvolvedusing the Axiovision 4 software. Deconvolved images wereenhanced with Photoshop (Adobe, San Jose, CA) using thelevels functions only, until reaching saturation in the mostintense areas of the image.

Membrane fractionation

Mitochondria and light membranes were separated asfollows. HEK 293 cells were homogenized with 15passages through a ball-bearing homogenizer (Isobiotec,Heidelberg, Germany, ball clearance 18 μM) in homogeni-zation buffer (0.25 M sucrose, 10 mM HEPES–NaOH, pH7.4, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid(EGTA), containing Complete protease inhibitor, Roche,Mississauga, ON). The resulting homogenates were centri-fuged for 10 min at 800×g to remove unbroken cells andnuclei. Post-nuclear supernatants were centrifuged for10 min at 10,000×g to yield heavy membrane fractions(HM). The supernatants were then centrifuged for 60 min at100,000×g to separate the cytosolic fraction and lightmembrane fraction.

Membranes of the ER (MAM and rER) werefractionated on a continuous OPTIPREP gradient using25%, 20%, 15%, 10%, and 5% OPTIPREP. HEK 293and HeLa cells, treated as indicated, were homogenizedas indicated above. For non-reducing conditions,10 mM N-ethylmaleimide was added. Cell debris andnuclei were pelleted by centrifugation at 1,000×g for10 min. The post-nuclear supernatant was overlayed ontothe continuous gradient and centrifuged at 32,700 rpm for3 h at 4°C in a SW55Ti rotor (Beckman Coulter,Mississauga, ON). Six equal fractions were collected fromthe top of the gradient and precipitated with acetone.Fractions were probed on a Western blot for the followingmarkers of ER domains according to Myhill et al. 2008:calnexin (mostly MAM), acyl-CoA/cholesterol acyltrans-ferase (ACAT1, MAM), PDI, calreticulin, BiP/GRP78(pan-ER), ERp57, eIF2α (rER), and mitochondrial com-plex 2 (mitochondria). Markers of the Golgi and theplasma membrane have also been described previously(Myhill et al. 2008).

Mitochondria were separated from the MAM asfollows: HEK 293 cells were grown to confluency on15 20-cm dishes and homogenized using a ball-bearinghomogenizer as above in 4 ml isolation buffer (250 mMmannitol, 5 mM HEPES, pH 7.4, 0.5 mM EGTA, 0.1%BSA). Debris and nuclei were removed by 5 mincentrifugation at 600×g in 15 ml Corex tubes in a JA 20rotor. The supernatant was centrifuged at 8,500 rpm in aJA-12 rotor for 10 min to pellet crude mitochondria.Subsequently, microsomes were pelleted at 100,000×g for1 h in a TLA120.2 rotor. The previously isolatedmitochondria were resuspended in 1 ml isolation mediumand layered on top of 8.5 ml Percoll isolation medium(225 mM D-mannitol, 25 mM HEPES, pH 7.4, 1 mMEGTA, and 20% Percoll (v/v)) in a 10-ml ultraclearpolycarbonate Beckman tube. The tube was centrifugedfor 30 min at 30,500 rpm (95,000×g) in a Ti-70 rotor withslow acceleration and deceleration, after which purifiedmitochondria (3/4 down the tube) and MAM wereremoved from the Percoll gradient (located above themitochondria). Percoll was removed from the mitochon-dria fraction and the MAM fraction was diluted 5-foldwith fresh isolation medium and re-centrifuged at60,000 rpm in a TLA120.2 rotor for 1 h. Equalproportional amounts were loaded for all fractions.

Secretion assays

For secretion assays, HEK 293 cells were grown toconfluency in 6-well dishes (900,000 cells/well) for 24 hin DMEM/10%FBS. Subsequently, the medium was ex-changed to OPTIMEM for the indicated times and themedium was precipitated using acetone.

Oxygen and Ero1α localization 621

Results

For most experiments, we used HEK 293 cells, whichexpress high levels of Ero1α. Since over-expression of ERproteins can lead to their mis-localization within the

expansive ER network (Registre et al. 2004), we decidedto exclusively analyze the distribution of endogenousEro1α. To test whether Ero1α localizes to a specific sub-domain of the ER, we first fractionated cellular membranesinto heavy and light membranes (Fig. 1a). This fraction-

Fig. 1 Ero1α co-fractionates with the MAM in HEK 293 and HeLacells. a Ero1α fractionates into heavy membranes. Membranes fromHEK 293 cells were fractionated into low (HM) and high speed pellets(LM), which were analyzed by Western blotting for complex 2(mitochondria), ACAT1 (MAM), calnexin (rER/MAM), PDI (all ER),and Ero1α. b Ero1α distribution between mitochondria and theMAM. HEK 293 homogenates were fractionated into cytosol (Cyt.),microsomes (Micro), crude mitochondria (MC), purified mitochondria(MP), and MAM according to Materials and methods. Marker proteinsindicate mitochondria (complex 2), MAM (ACAT1), and ER mem-branes (PDI and calnexin). c Ero1α distribution within ER domains.HeLa and HEK 293 cell homogenates were fractionated on adiscontinuous 10–30% Optiprep gradient. Marker proteins indicate

mitochondria (complex 2), MAM (ACAT1, calnexin), pan-ER (PDI),and Ero1α. Lane numbers refer to the Optiprep fractions. Thelocalization of Ero1α from three independent experiments is quanti-fied in Fig. 3a. d Presence of Ero1α oxidative forms on domains ofthe secretory pathway. HEK293 homogenates were fractionated andanalyzed under non-reducing conditions. Ero1α was detected byWestern blot. e Analysis of the ER domain distribution of selectedcomponents of the ER protein folding machinery and the Golgicomplex. HEK 293 cell homogenates were fractionated on a discontin-uous 10–30% Optiprep gradient. eIF2α indicates the position of the rER(fractions 3 and 4), β-COP indicates the cis-Golgi (fraction 2), ACAT1indicates the MAM (fractions 5 and 6). Calreticulin (CRT), BiP/GRP78(BiP), ERp44, and ERp57 were analyzed for their intra-ER localization

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ation protocol showed that the majority of the Ero1α signalwas found on heavy membranes that comprise mitochon-dria, the rER and the MAM (Myhill et al. 2008). We nextanalyzed the distribution of Ero1α on a Percoll gradient,which can distinguish between MAM and mitochondrialocalization in liver tissue (Stone and Vance 2000) and celllines (Wieckowski et al. 2009). On the Percoll gradient,membranes isolated as crude mitochondria are resolved intopure mitochondria that are largely devoid of ER markersand a MAM fraction that is enriched in calnexin and acyl-CoA/cholesterol acyltransferase 1 (ACAT1, Fig. 1b,(Myhill et al. 2008; Rusinol et al. 1994). Conversely,mitochondrial complex 2 is found in the crude and puremitochondria fraction, but not the MAM fraction (Fig. 1b).This fractionation showed that Ero1α was present virtuallyexclusively on the MAM in HEK 293 cells (Fig. 1b). Wecomplemented this analysis with our MAM isolationprotocol based on Optiprep that separates the MAM fromother domains of the ER, such as the rER and the latesecretory pathway, but does not distinguish between theMAM and mitochondria (Myhill et al. 2008; Roth et al.2010). On the Optiprep gradient, markers of the MAM peakat the bottom in fraction 6, whereas ER proteins that are notenriched on the MAM, such as PDI are found in allfractions, including fractions containing the rER markersERp57 and eIF2α (Fig. 1c, e). With this protocol, weconfirmed that in HeLa and HEK 293 cells, the bulk of theEro1α signal co-fractionated with markers of the MAM,but not the rER or the late secretory pathway (Fig. 1c).When analyzing our fractions for the presence of thecharacteristic oxidative states of Ero1α, we found that fullyoxidized OX1 and OX2 Ero1α was found at the bottom ofthe gradient, whereas the late secretory pathway contained amixture between oxidized and reduced Ero1α (Fig. 1d). Weconfirmed that the localization of Ero1α on our biochem-ical fractionations was unique by comparing its distributionin HeLa and HEK 293 cells to the chaperones andoxidoreductases calreticulin, ERp57, ERp44, PDI, andBiP/GRP78. Of these chaperones, BiP/GRP78 was theonly one which showed a significant enrichment onfractions of the MAM. Conversely, PDI and, in particular,ERp57 showed high amounts on fractions of the rER(Fig. 1c, e). The distribution of ERp44 was unique, butentirely consistent with the functions described for thisoxidoreductase: it is predominantly found in fractions co-migrating with the ERGIC and cis-Golgi marker βCOP,where it functions as a scavenger of proteins with exposedthiols (Anelli et al. 2003), but also on the MAM, where itregulates the activity of the IP3R (Higo et al. 2005).Therefore, our three subcellular fractionation protocolsshowed that Ero1α is highly enriched on the MAM, incontrast to numerous other ER oxidoreductases andchaperones that are present in higher amounts on the rER.

To further examine the localization of Ero1α, weanalyzed the distribution of this oxidoreductase in HEK293 and HeLa cells by immunofluorescence microscopy.Ero1α and PDI showed distinct distributions within HEK293 (Fig. 2a) and HeLa cells (Fig. 2b). Whereas PDIlocalized to a dispersed reticular staining pattern thatconcentrated on the nuclear envelope in both cell lines(Fig. 2a, b), the staining pattern of Ero1α was patchier andtended to show more apposition with mitochondria (com-pare green arrowheads for PDI/Ero1α/mitochondria overlapand red arrowheads for Ero1α/mitochondria overlap inFig. 2a, b). As typical for proteins of the MAM, this closeapposition did not translate into full overlap between Ero1αand mitochondria (Myhill et al. 2008; Rizzuto et al. 1998).Together, these results are consistent with the fractionationdata that showed that Ero1α and PDI have a distinctintracellular localization.

The localization of ER proteins to the MAM is aregulatable mechanism, as shown in the case of calnexinthat can relocate from the MAM to the rER underconditions of extended ER stress (N. Myhill and T.Simmen unpublished observations). Another example isthe cytosolic connector protein PACS-2 that temporarilyshifts from light membranes to membranes of the MAMand mitochondria during ER stress (Simmen et al. 2005).We hypothesized that Ero1α, being an ER luminal protein,would exhibit intra-ER motility. To test this idea, we firsttreated HEK 293 cells with reducing agents known to leadto secretion of over-expressed, fully reduced Ero1α via adisruption of the Ero1α ERp44 retention mechanism(Anelli et al. 2002, 2003). As expected, treatment of cellswith 1 mM 2-mercaptoethanol (2ME) and 1 mM dithio-threitol (DTT) for 2 h led to a shift of the Ero1α signal onthe Optiprep gradient that monitors the localization ofEro1α among the MAM, the rER, the Golgi, and theplasma membrane. Under these conditions, the amount ofEro1α on the MAM decreased from roughly 75% toapproximately 25% of the total signal, whereas its amountson fractions of the late secretory pathway increasedreciprocally (Fig. 3a). We verified whether the propertiesof secretory pathway membranes remained the same in thisexperiment by confirming the sustained presence of PDI,ACAT1, and βCOP in their respective fractions (Fig. 3b).Since we could not detect significant relocation for calnexinor calreticulin either, our results mean that during our timeframe, only Ero1α shifted away from the MAM (Fig. 3b).Moreover, Ero1α did not relocate from the mere exposureto ER stress, as indicated by the gradients of homogenatesfrom HEK 293 cells exposed to tunicamycin, thapsigargin,or glucose deprivation (Fig. 3c). Nevertheless, this incuba-tion with reducing agents led to the induction of XBP-1 atthe protein level within our 2-h time frame (Fig. 3d).Therefore, the complete reduction of the ER environment,

Oxygen and Ero1α localization 623

but not the exposure of cells to ER stress led to therelocation of Ero1α from the MAM to cellular fractions ofthe late secretory pathway.

Since Ero1α requires oxygen for its enzymatic reaction(Tu and Weissman 2002), we examined whether or not thedeprivation of atmospheric oxygen during hypoxia influ-enced its localization. Thus, we incubated HEK 293 cells ina hypoxic chamber containing 1% oxygen. Since reducingagents such as 2ME or DTT cause a shift of the oxidationstate of Ero1α from the fully oxidized OX1 and OX2 forms

to the reduced form, we first aimed to determine whetherhypoxia could have a similar effect (Anelli et al. 2002).Indeed, the incubation in hypoxic atmosphere temporarilytransformed a portion of Ero1α from the fully oxidizedOX2 to the fully reduced form after 30 min of hypoxia(Fig. 4a). Moreover, starting as soon as 15 min after theexposure to the hypoxic environment, Ero1α shifted fromthe MAM to membranes of the late secretory pathway(Fig. 4b). This translocation appeared complete after about2 h of incubation in hypoxia, when HIF1α protein

Fig. 2 Intracellular localization of Ero1α to the vicinity of mitochon-dria. a HEK 293 cells were grown on coverslips for 24 h andprocessed for immunofluorescence microscopy. Ero1α was detectedwith a rabbit polyclonal antibody, PDI with a mouse monoclonalantiserum and mitochondria were visualized with Mitotracker.Immunofluorescence images were acquired and deconvolved. Inserts

show a magnified area, indicated by white frames on the biggerpictures. The red arrowheads point out Ero1α/mitochondria overlap,whereas the green arrowheads point out triple PDI/Ero1α/mitochondriaoverlap. Scale bar=25 μm. A representative image is shown. b HeLacells were processed and imaged as in (a)

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expression started to increase (Fig. 4f). Under hypoxicconditions, the amount of Ero1α on the MAM decreasedfrom about 75% of total to about 20%. Since we detectedEro1α in fractions 1 and 2 of our Optiprep gradient, weexamined whether this translated into an overlap withβCOP. Indeed, our immunofluorescence experimentsshowed accumulations of paranuclear staining for Ero1αat 16 h hypoxia, which however only partially overlappedwith the crescent-shaped βCOP pattern (Fig. 4c). Undercontrol conditions, we could not detect accumulations(Fig. 2 and data not shown). Interestingly, we neitherdetected relocation of other ER protein folding chaperonesincluding PDI, calnexin, and BiP/GRP78 nor of the MAMmarker ACAT1 and the cis-Golgi marker βCOP during theincubation under hypoxic conditions (Fig. 4d). The Ero1αrelocation that occurred under hypoxia was not dependenton the correct functioning of mitochondria, since neither theelectron transport chain inhibitors rotenone and antimycin,nor the ATP synthase inhibitor oligomycin when added for2 h led to a shift of Ero1α from the MAM to membranes ofthe late secretory pathway (Fig. 4e). Our results therefore

show that oxygen supply is required to maintain the normalredox state of Ero1α and its retention on the MAM.

Our fractionation and immunofluorescence experimentsdid not allow us to conclusively determine the destinationto where Ero1α migrates under conditions of disrupted ERredox or low oxygen. Therefore, we tested the hypothesisthat Ero1α undergoes secretion by probing the supernatantsof DTT- and 2ME-treated HEK 293 cells for Ero1α. Asdemonstrated earlier for over-expressed Ero1α, the incuba-tion of cells with the reducing agent 2ME, and even moreremarkably with DTT, led to the secretion of Ero1α startingat 10 min of incubation (Fig. 5a). Consistent with a slightincrease of the calreticulin signal in late secretory pathwaymembranes (Fig. 3b), we also detected some secretion ofcalreticulin under these conditions (Fig. 5a). These resultssuggested that hypoxia leads to secretion of endogenousEro1α. We tested this possibility, but only detectedsecretion of endogenous Ero1α, but not calreticulin, afterlong periods of hypoxia, i.e., after 16 h (Fig. 5c) despite theinduction of HIF1α at the protein level starting at 2 h ofhypoxia (Fig. 4f). Together, our results show that Ero1α,

Fig. 3 Ero1α MAM retention isredox-sensitive. a Optiprepfractionation of HEK 293 con-trol cells and cells treated for 2 hwith 1 mM 2ME and 1 mMDTT. Three independent experi-ments were probed for Ero1αand quantified by LICOR. Theindividual amounts of Ero1α perfraction are plotted in the middlegraph (error bars omitted forclarity, but shown on the rightpanel). Right panel: the amountof Ero1α on the MAM wasdetermined as the sum of signalfound within fractions 5 and 6(n=3). b Optiprep fractionationof HEK 293 cells as in a.Western blots were probed forcalnexin (CNX) and calreticulin(CRT). c Optiprep fractionationof HEK 293 cells treated withthe ER stressors tunicamycin(Tuni., 10 μM), thapsigargin(Thaps., 1 μM), and in mediumdepleted of glucose, but supple-mented with galactose (-Gluc.,see Materials and methods). dProtein levels of the ER stressassociated transcription factorXBP-1 after 2 h treatment withreducing agents. Loading equal-ized using α-tubulin as aloading control

Oxygen and Ero1α localization 625

Fig. 4 Ero1α MAM retention is oxygen-sensitive. a Analysis of theEro1α oxidation state when incubated under hypoxic conditions (1%oxygen) for the indicated times. Lines point to fully reduced Ero1α (red)and the two oxidized forms OX1 and OX2. b Optiprep fractionation ofHEK 293 control cells and cells incubated for the indicated times (inminutes and hours, see numbers on the left) in hypoxic environment (1%oxygen). Three independent experiments were probed for Ero1α andquantified by LICOR. The individual amounts of Ero1α per fraction atthe 1 and 16 h time points are plotted in the middle graph (error barsomitted for clarity, but shown on the right panel). Right panel: theamount of Ero1α on the MAM was determined as the sum of signalfound within fractions 5 and 6 (n=3). c Immunofluorescence co-localization of Ero1α and βCOP at 16 h of hypoxia. HEK 293 cells were

grown on coverslips for 24 h, followed by incubation in hypoxia for 16 hand processed for immunofluorescence microscopy. Ero1α was detectedwith a rabbit polyclonal antibody and βCOP with a mouse monoclonalantiserum. Immunofluorescence images were acquired and deconvolved.Inserts show a magnified area, indicated by white frames on the biggerpictures. Scale bar=25 μm. A representative image is shown. Partialoverlap is seen in the magnified area, but also on numerous spotselsewhere. d Optiprep fractionation of HEK 293 cells as in (b). Westernblots were probed for calnexin (CNX) and BiP/GRP78 (BiP). e Optiprepfractionation of HEK 293 cells treated for 2 h with the mitochondrialstressors rotenone (1 μM), oligomycin (5 μM), and antimycin (100 μM).f HIF1α protein levels after 2 h in hypoxic environment (Hyp, 1%oxygen). Loading equalized using actin as a loading control

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which is normally confined to the MAM, leaves thatdomain of the ER and enters the secretory pathway,resulting in a partial co-localization with βCOP, butultimately leading to its secretion under conditions ofdisrupted cellular redox and prolonged hypoxia.

Discussion

Our results show for the first time the specific enrichmentof an ER oxidoreductase to the MAM. The enrichment ofEro1α on the MAM appears to be specific for this ERoxidative folding enzyme, since PDI, ERp57 and thechaperones BiP/GRP78 and calreticulin do not show such

an enrichment. Nevertheless, all of these proteins are foundto some extent on the MAM (Fig. 1), thus still allowingthem to perform calcium-regulatory roles on this domain ofthe ER. In contrast to calnexin, another MAM-enriched ERfolding enzyme, the localization of Ero1α to this signalinghub of the ER is highly susceptible to the redox state of theER and to oxygen supply, since incubation in reducingmilieu and under hypoxic conditions lead to the rapiddepletion of Ero1α from the MAM (Figs. 3 and 4).Subsequently, this oxidoreductase appears in the growthmedium of cultured cells, in a much more pronounced waythan other ER chaperones such as calreticulin (Fig. 5a, c).Interestingly, the relocation of Ero1α happens typicallyafter 1–2 h of treatment, much earlier than the induction oftranscriptional responses to reducing milieu and hypoxia(Fig. 5b, d). A determining factor for the Ero1α shiftappears to be the Ero1α oxidation state. Characteristically,Ero1α appears in two oxidized states, OX1 and OX2,which are sensitive to incubation with DTT (Anelli et al.2002). We detected a partial, temporary shift of the Ero1αoxidation state to the reduced form after 30 min of hypoxia(Fig. 4a), correlating with the mixed ratio of oxidized andreduced Ero1α in fractions of the late secretory pathway(Fig. 1d) and indicating that, as for incubation with DTT,hypoxia leads to a disruption of the thiol-mediated retentionmechanism of Ero1α (Anelli et al. 2003).

The localization of Ero1α to the MAM is surprising fortwo reasons. First, Ero1α is expected to play an importantrole in the re-oxidation of the thioredoxin-related oxidore-ductase PDI, which is classically thought to occur on therER (Fassio and Sitia 2002; Sevier and Kaiser 2008). Thealmost exclusive presence of Ero1α on the MAM suggeststhat Ero1α-mediated PDI re-oxidation occurs on the MAM,rather than on the rER. Such a scenario would require thetransport of PDI/peptide or at least PDI/Ero1α complexesfrom the rER to the MAM, so far unobserved. However, theshuttling of PDI/Ero1α complexes into the vicinity ofmitochondria would result in the higher availability ofmitochondrial ATP and FAD, both of which are required forefficient oxidative protein folding (Papp et al. 2005).Alternatively, PDI could be re-oxidized by the other Ero1family member, Ero1β, which is present in non-detectablelevels in HEK 293 and HeLa cells (Dias-Gunasekara et al.2005, 2006). Since PDI is found abundantly in all domainsof the ER, such a mechanism definitely remains apossibility. Clearly, the formation of PDI/Ero1α complexesis not a determinant of PDI localization, since none of theconditions described in this paper led to a significantrelocalization of PDI. Instead, PDI, like calreticulin andBiP, could predominantly depend on the Lys-Asp-Glu-Leu(KDEL)-receptor for its ER retention. Notably, Ero1α lacksthe KDEL sequence at its C-terminus. ERp57 and ERp44show yet other types localizations of intra-ER localization

Fig. 5 Disruption of cellular redox and oxygen supply leads to Ero1αsecretion. a Ero1α content of cellular growth medium (supernatants)treated for the indicated times with the indicated reducing agents(1 mM 2ME, 1 mM DTT). Ero1α secretion, detected by the sharpincrease in the growth medium (supernatants), is observed after15 min. b Ero1α content of cellular growth medium (supernatants)incubated for the indicated times in hypoxic atmosphere. Ero1αsecretion is observed after 16 h. For both a and b, calreticulin (CRT)serves as a secretion control

Oxygen and Ero1α localization 627

patterns, suggesting the presence of additional intra-ERlocalization mechanisms. Consistent with their role in theregulation of ER calcium homeostasis, both oxidoreduc-tases show, nevertheless, portions of their respective signalson the MAM.

Second, hypoxia leads to the induction of expression ofEro1α (Gess et al. 2003; May et al. 2005), which,according to our results, is subsequently secreted. Onepossible explanation for these observations is that hypoxiccells need to compensate for constant loss of Ero1α duringhypoxia. Alternatively, Ero1α may perform other functionsoutside the cell during conditions of prolonged hypoxia.

With our results, the classes of identified proteins on theMAM increases by another type, the oxidoreductases. Infact, Ero1α emerges quite surprisingly as one of the best-known MAM markers to date. Supporting our data, Ero1αhas also been implicated in the regulation of ER-mitochondria calcium communication by promoting IP3R-mediated calcium efflux during ER stress (Li et al. 2009).Since the IP3Rs are enriched themselves enriched on theMAM (Hajnoczky et al. 2000), our results raise thepossibility that the majority of human Ero1α, at least inthe tumor-derived HEK 293 and HeLa cells, might regulateIP3R-mediated calcium signaling on the MAM. In thiscase, Ero1α would not be the first ER oxidative proteinchaperone involved in the regulation of ER calciumhomeostasis. These observations have a precedent in thefunctions of calreticulin (Michalak et al. 2009; Molinari etal. 2004), but the presence of a calcium binding domainwithin Ero1α itself also supports such a possibility. Furtherresearch is required to address these possibilities and also toclarify the functional relationship between the two humanEro1 protein family members.

Despite Ero1α being an ER luminal protein, the targetingof Ero1α to the MAM is quite stringent (>75%), comparedwith the chaperone calnexin, whose targeting mechanism tothe MAMwe have previously analyzed (about 2/3 of calnexinlocalizes to the MAM under resting conditions, Myhill et al.2008). Similar to calnexin, the relatively low, but detectableamounts of Ero1α on the rER might be sufficient to performits enzymatic role in ER oxidative protein folding. Furtherelucidation of the Ero1α targeting mechanism to the MAMwill provide more insight into this question. What are thepossible mechanisms by which Ero1α is targeted to theMAM? In the case of calnexin, interaction with the cytosolicconnector protein PACS-2 contributes to its localization tothe MAM (Myhill et al. 2008). Such a mechanism cannotaccount for the MAM-localization of Ero1α directly, sinceEro1α is not in contact with the cytosolic face of the ER.Besides the known interaction of Ero1α with PDI andERp44, interaction with an as-yet unknown receptor maymediate retention of Ero1α on the MAM. Another possibil-ity could be a tight association of Ero1α with the MAM

membrane, which has been shown to exhibit specific lipidcomposition (Sano et al. 2009). Similar to the characteriza-tion of the rice Ero1α ER targeting mechanism (Onda et al.2009), we expect a detailed analysis of the Ero1α peptidesequence to yield information concerning a novel targetingmechanism for ER oxidoreductases and chaperones to theMAM. We further expect such a targeting mechanism tohave important implications on cell survival and proteinsecretion.

Acknowledgements Work in the Simmen laboratory was supportedby Alberta Health Services (Bridge Grant #24170) and by the NCIC/CCSRI (Grant #17291) EML and MDB are supported by ACRIstudentships. T. S. is supported by the Alberta Heritage Foundation forMedical Research (200500396). The authors thank Richard Rachu-binski and Roberto Sitia for comments, support, and reagents.

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