Journal of Cell Science eedn nE eo n Ca and redox ER on dependent h RCa ER the ucinb ittn Ca this dictating perform by oxidoreductases function and chaperones ER Various source. Tae(EC)(izt ta. 09.Udrresting Under 2009). al., et Ca (Rizzuto conditions, (SERCA) ATPase el(lnyadBlbn 02 hvtvk n Orrenius, and the Zhivotovsky of 2012; survival Balaban, Ca and ER–mitochondria and 2011), death (Glancy in cell plays metabolism mitochondrial (Ca (TCA) pyruvate acid tricarboxylic enzyme cycle the mitochondrial drives the which (PDH), activate dehydrogenase to needed is matrix h A ne etn odtos rteruhE n Rqaiycnrlcmatet(RC olwn Rsrs.Orrslshave results Our stress. ER Ca following to (ERQC) either compartment to calnexin calnexin control of quality assigns distribution ER that preferential and switch a ER with rough the correlate the identified also or therefore changes conditions, These resting substrates. under known MAM of the control quality and chaperoning towards ucin steitrcino h Rwt iohnra which mitochondria, with ER the of interaction the is functions 00.Hr,teE xhne Ca al., exchanges et Simmen ER ER 2013; the the Simmen, Here, between and a 2010). contacts (Raturi close (MAM), mitochondria makes the that membrane and ER the mitochondria-associated of the subdomain on occurs ml .Lynes M. Ca Emily to ER calnexin or assigns control that quality switch the is Palmitoylation Article Research lw oncin rti odn n rvnsteraggregation binding their This prevents and 2011). folding protein Williams, client down and slows 2005). (Rutkevich al., glycoproteins et (Higo IP3Rs inhibits which ERp44, and Ero1 2009), are Examples 2010). Ca of lipids, the as storage well are as the (ER) proteins reticulum membrane and and endoplasmic secretory the of of production functions main The Introduction Ca of words: regulation Key the from a function Following its mechanism. shifts ER-stress-dependent in which an function palmitoylated, is well-known less Ca palmitoylation its becomes the calnexin performs (SR) quickly affects that and reticulum calnexin show ERp57 signal sarcoendoplasmic stress, Ca also oxidoreductase sorting ER results with ER the (ER)-internal our short-term with determines interacts reticulum Interestingly, interacts interaction calnexin endoplasmic control. calnexin this palmitoylated quality non-palmitoylated this that contrast, that how and In investigated demonstrate 2b crosstalk. have (SERCA) results information we ATPase of Our lack transport finding, a calnexin. Given this (MAM). of membrane of mitochondria-associated functions the significance on calnexin the enrich to on serves calnexin of palmitoylation The Summary 10.1242/jcs.125856 doi: 3893–3903 126, ß Science Cell of Journal 2013 June 10 Accepted ( correspondence for *Author 3 2 1 I3)adtesronolsi eiuu S)Ca (SR) reticulum sarcoendoplasmic the and (IP3R) lkadaJanowicz Aleksandra ead .Lederkremer Z. Gerardo eateto elRsac n muooy ereWs aut fLf cecs e vvUiest,TlAi 97,Israel 69978, Aviv Tel University, Canada Aviv 2G2, Tel T6G Sciences, AB Life Edmonton, of Alberta, Faculty Canada of Wise 2H7, University George T6G Chemistry, Immunology, AB of and Edmonton, Department Research Alberta, Cell of of University Department Biology, Cell of Department 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. anxni nte e Rcaeoeta id onascent to binds that chaperone ER key another is Calnexin ´ dnse l,21) ie h rca oethat role crucial the Given 2010). al., et rdenas nolsi eiuu,Mtcodi,Qaiycnrl Ca control, Quality Mitochondria, reticulum, Endoplasmic 2+ hnln nstl(,,)tihsht receptors (1,4,5)-triphosphate inositol -handling 2+ eieyfo h Rt h mitochondrial the to ER the from delivery 1 rnRaturi Arun , [email protected] 2+ 2+ nitga eurmn o these for requirement integral An . 1 hnesadpmst pno close or open to pumps and channels a ahnMyhill Nathan , 2+ hc ciae PR L tal., et (Li IP3Rs activates which , 2 xhnehst ergltda the at regulated be to has exchange n hmsSimmen Thomas and 2+ 2+ oesai Sme tal., et (Simmen homeostasis 1 aiaShenkman Marina , ihmtcodi through mitochondria with ) 1 ate .Benson D. Matthew , 2+ transport 1, * 2+ 2 aoiaOtzSandoval Ortiz Carolina , 2+ 2+ signaling inln,Ep7 SERCA2b ERp57, signaling, inln rt rti chaperoning. protein to or signaling ointa anxni eta lyrfrterglto fER 2008; of regulation al., the the for et reinforces player further Myhill central a finding 2007; Ca is This calnexin Su, 2009). that and notion al., enriched often (Hayashi et is but MAM Wieckowski ER, rough the the on on found only not is calnexin Williams, and Rutkevich 2007; 2011). al., et (Jessop system secretory this throughthe trafficking on subsequent and specifically folding efficient depend for disulfide partnership (LDLR) receptor low- protein lipoprotein the as density subset such an the certain al., proteins a glycosylated that et heavily shown ERp57, disulfide-bonded of Oliver have of Studies 2010; 1998). with member al., Michalak, et and Zapun partnership 1997; (Coe a family in (PDI) is isomerase 2009). works that known (Lederkremer, calnexin oxidoreductase process (ERAD) along, a degradation proteasome, All the ER-associated by as degradation and the to ER cytosol the are from retrotranslocation enhancing by period followed (EDEM), degradation prolonged protein ER-associated a and for mannosidase I by cycle trimming mannose calnexin extensive to yet subject the have eventually not that in the are Proteins trapped to 2006). they returned Molinari, been if and and re-glucosylated (Ruddock or cycle be apparatus, calnexin can Golgi three they the folded of to correctly Upon exported one they ER. case be which follow in the will correctly, can and in rapidly fold glycoproteins might intermediates they pathways: calnexin, folding from of release retention to owing upiigy u aoaoyadohr aedtrie that determined have others and laboratory our Surprisingly, 2+ 1 oetE Campbell E. Robert , inln,adi locnitn ihtefc htcalnexin that fact the with consistent also is and signaling, 2+ otn n h euaino Rmtcodi Ca ER–mitochondria of regulation the and content 1 ea .Yap C. Megan , 3 u .Berthiaume G. Luc , 1 ihiWu Jiahui , a -mannosidase-like 2+ signaling 1 , 3893 3 , 2+ 2+ Journal of Cell Science odfeetE udmiscreae iha ER-stress- an with correlates Ca ER subdomains in role ER dependent different to al., et (Frenkel proteins folding slowly with 2004). (ERQC) association compartment relocation control its the quality in and ER results the stress towards ER calnexin that of given homeostasis, Ca ER in on calnexin of role the Interestingly, neat ihteSRACa SERCA the with interacts 3894 ta. 08.W a hw ale htteitreec with interference the 2012), al., that et earlier (Lynes shown oxygenase-1 Kang heme this had 2011; relocates We of al., palmitoylation 2008). et consequences al., (Dowal the unknown et but currently are ORAI1, modification and the including proteins, calnexin, oxygenase-1 on ER to several heme rafts addition modify the In to lipid known 2008). with with is al., palmitoylation calnexin and et a (Ferrera 2012) of and membrane al., on plasma interaction et ERp57 depends the (Lakkaraju is partners that translocon mediating MAM 2012) known al., in the et implicated its (Lynes to motif to palmitoylation targeting juxtamembrane calnexin extent Calnexin the of with SERCA2b. correlated binding role a of such whether examined further hrfr,w aeeaie hte h agtn fcalnexin of targeting the whether examined have we Therefore, ora fCl cec 2 (17) 126 Science Cell of Journal 2+ inln oad iohnra We mitochondria. towards signaling 2+ up(oeike l,2000). al., et (Roderick pump 2+ inln ol depend could signaling ult oto ftekonclei usrtsLL and LDLR Results substrates calnexin known the (ASGPR). receptor asialoglycoprotein of control quality ots hte Rsrs nlecstefnto fclei in calnexin of function the Ca influences stress ER ER whether test its To and ER calnexin the of within palmitoylation localization the affects stress ER tt fclei o h eli em fclua Ca cellular of terms in palmitoylation cell the the for functional calnexin of of state consequences the functional investigate the To characterized we calnexin, 2012). of diverse targeting palmitoylation-dependent al., of multiple significance et have (Lakkaraju might effects palmitoylation ER-associated but Rpoen.T oepeieydtrieteoii and origin a used the we stress, determine ER precisely short-term during more calnexin other of such To two destination shown), No of not proteins. 1A). (data h BAP31 (Fig. 1 or ER h PDI after 4 for observed started at was increased that shift and membranes shift treatment light a tunicamycin detected towards we light biochemical calnexin technique, into this of by membranes Using ER membranes. stress separate heavy and ER to decided following We fractionation. calnexin of localization 2+ inln n hprnn,w is xmndthe examined first we chaperoning, and signaling ramn fo 4clsec) cl as 25 bars: Scale each). m cells 14 (from 10 treatment h 4 following endogenous calnexin wild-type, for 0.30 control conditions, under calnexin for endogenous 0.36 wild-type coefficients: mitotracker Manders and (inset). calnexin signals just a of with area right zoomed the on shown are red) blue; mitochondria, PDI, green; (calnexin, merged images PDI The calnexin, (mitotracker). of mitochondria signals and the for analyzed and described as microscopy immunofluorescence for 10 processed with were treated Tunicamycin cells and Control cells stress. HeLa ER on depends mitochondria ( h 4 or 10 h with 1 treatment a following and membranes heavy into light separated were lysates cell HeLa Homogenized stress. ER following LM, respectively) and (HM fractionation membrane internal ER calnexin. the of affects distribution stress ER 1. Fig. nteMMfato fato i)fo three from ** six) experiments. (fraction independent fraction MAM amount the calnexin in the shows as graph well The as calnexin. (MAM), PDI FACL4 for and blotting (pan-ER) western and SDS-PAGE indicated by as analyzed were fractions Membrane treatment h 10 4 with a following Optiprep were by lysates separated cell HeLa Homogenized stress. ER (before control C, ( calnexin. treatment). time and distribution PDI the for shows course graph The 2 2). complex (C. mitochondrial PDI, and for (CNX) blot calnexin western and SDS-PAGE indicated by as analyzed were fractions Membrane C m. h anxnsga rxmlto proximal signal calnexin The ) m uiayi nosxfractions. six into Tunicamycin M B pirpfatoainfollowing fractionation Optiprep ) m uiayi (Tuni). Tunicamycin M ( m A Tunicamycin M P ev n light and Heavy ) 2+ 5 inln and signaling 0.035. m M Journal of Cell Science upeetr aeilFg 1 i.SA.TegnrlER 4 general ligase The acyl-CoA long-chain S2A). MAM-localized Fig. and 1B; S1, PDI (Fig. and marker Fig. medium homogenized gradient material then density supplementary h, stressors, Optiprep 4 ER using al., for different fractionated et two tunicamycin, with (Myhill and treated markers thapsigargin were to ER cells developed other HeLa from had 2008). markers we MAM that separate technique fractionation biochemical le st htpliolto fclei sncsayfor. necessary is calnexin of also palmitoylation depalmitoylation what its to give with could observation as parallel an such clues because in within interactors, key calnexin occurs its of affects that relocation the ER whether test the to aimed we Next, MAM between SERCA2b interactions and of the calnexin affects loss stress ER Short-term a with al., together et depalmitoylation. (Delom 2B; dephosphorylation, (Fig. enrichment. depalmitoylation, stress calnexin calnexin not in stress ER results but to long-term stress ER ER to short-term led ER short-term 2007), contrast both a indeed in that Therefore, observed induce situations We to S2B). or stress tunicamycin Fig. h with material ‘click 4 treated supplementary a cells for of used that DTT we to cells Thus, untreated 2012). signal from palmitoylation calnexin al., the compare its to et approach for chemistry’ responsible (Lynes be in targeting to change that known MAM be palmitoylation, the could its 1999; stress for loses ER calnexin al., explanation short-term during Another et calnexin in of 2000). (Chevet localization al., SERCA2b change et and Roderick ribosomes of a interaction of with the controls reduction which calnexin PACS-2 2A), a controls (Fig. that 584 to which serine and 565, led binding, stress serine stress out that on short-term, phosphorylation ER given not calnexin calnexin, ruled but an of long-term, relocation during only the results behind site is of phosphorylation status Our known the its tested each and we timecourse. ER 2008), for of al., the et within state phosphorylation (Myhill localization PACS-2 phosphorylation its to for the binding important Because is calnexin. calnexin of known to relocation due not ER. is the from that in away function changes calnexin specific structural a of suggests movement MAM this the and however, Csorda time, 2011; us ER same al., between by the et contacts reports (Bravo visible mitochondria increases earlier and first condition given this At surprising, that MAM. others appears the during mitochondria stress at a from away with ER calnexin calnexin consistent of of relocation is the localization result glance, This the mitotracker. in calnexin as against decrease well antibodies as with of PDI, labeled shown). overlap cells and the HeLa signal, in (yellow in not observed box) decrease was inset signals a mitotracker tunicamycin, After (data and with calnexin 1C). the treatment (Fig. of membrane mitochondria h reduced and 4 calnexin plasma suggested between increased similarly to apposition the lead microscopy less not to did Immunofluorescence to diminished calnexin was of as fractions relocation targeting MAM The gradient, not with but 20%. calnexin, FACL4 Optiprep than of after or association However, PDI the the others. of the stressors, and of ER with laboratory with our bottom treatment and by FACL4 shown the previously marker at MAM mitochondria the calnexin of with 40% MAM to the co-fractionates close of conditions, sedimentation control the Under for compartment. control to used were (FACL4) et eamdt dniytemcaimta ol edt this to lead could that mechanism the identify to aimed we Next, ´ ta. 06.At 2006). al., et s eue uigE tes eaclswr nuae o ih10 with h 4 for incubated were cells HeLa (dog 584 stress. 565, ER 555, ( during serine treatment). reduced – (before phosphorylation control of C, sites against nomenclature). known antibodies three phospho-specific the using of blotting each western by analyzed and anxndpliolto usqetyrslsi reduced in dephosphorylation. results its SERCA2b. with not subsequently calnexin of but association depalmitoylation calnexin, Calnexin short- of that our depalmitoylation together, demonstrate Taken S2C). Fig. results material had (Lynes (supplementary 2012) calnexin we al., non-palmitoylatable et where in alanine resulting mutant with (CCAA), residues a residues cysteine SERCA2b–calnexin membrane-proximal in two the calnexin the replaced of of reduction phosphorylation to reduced thes led with that Consistent within interaction. residue we frame this However, on time phosphorylation 2000). the of al., reduction detect et not (Roderick this could of SERCA2b regulation the with in implicated interaction been previously had 584 serine phosphorylation the on of Calnexin reduction 3C). similar (Fig. a in interaction resulted SERCA2b–calnexin 503 CCAA) calnexin, (cysteine calnexin dog residues in of alanine 504 with residues and palmitoylation cysteine to or two subject the are DTT of that replacement S either The with 3B). that with found (Fig. calnexin we treated of conditions, stress interaction cells ER these the Under HeLa h. 4 and for tunicamycin cells HeLa control stress. these could, ER under during SERCA2b we and fates SERCA2b ER, calnexin different that of undergo the suggested This movement of 3A). any (Fig. membranes conditions detect the heavy not and as examined however, well MAM ER away as the moved we with calnexin gradient, Whereas from membranes. Optiprep Thus, treatment light our versus heavy whether on SERCA2b. on SERCA2b test al., of affects first et distribution also to (Roderick SERCA2b decided stressors with we interacts 2000), calnexin Because niiulyatrddrn nE testm ore eaclstetdfrthe for treated cells 10 HeLa with course. times time indicated stress ER an during during altered course. individually palmitoylation time and stress phosphorylation ER calnexin an of Analysis 2. Fig. uiayi n hnpoesdfrcikceitya ecie previously described 2012). as chemistry al., click et for (Lynes processed then and Tunicamycin ots hshptei,w muorcpttdclei from calnexin immunoprecipitated we hypothesis, this test To h ucino anxnpliolto 3895 palmitoylation calnexin of function The m uiayi eesa-ye ihsml buffer sample with snap-lysed were Tunicamycin M ( A anxnpopoyaina t he ie is sites three its at phosphorylation Calnexin ) idns ecudas o detect not also could we findings, e emE tesrslsi the in results stress ER term RAbdcesdsignificantly decreased ERCA2b B anxnpliolto is palmitoylation Calnexin ) m M Journal of Cell Science 00.Tu,w efre netniesre fCa of series extensive an performed al., et we (Roderick SERCA Thus, on role 2000). its through mitochondria and ER rnt(i.4) ocag nteacmlto fcytosolic of accumulation the in change No 4A). Ca (Fig. not present was or calnexin non-palmitoylated or palmitoylated whether i.SA.Teerslswr ossetwt ale reports Ca earlier ER measure with to decided consistent we However, were 2002). al., results et (Zuppini These S3A). Fig. Rsoe,orfnig ugse htmtcodilCa mitochondrial that suggested findings our stores, ER el i o hwatrdacmlto fctslcCa with cytosolic of not these accumulation Because altered but 4B). show (Fig. calnexin, not palmitoylated did wild-type be cells cannot with that transfected calnexin MEFs Montero, in and showed measurements (Alvarez our aequorin Surprisingly, ER-targeted 2002). using source the at the in differences any detect Ca to cytosolic unable of were accumulation We Fluo8. with regulate might Ca calnexin palmitoylated ER that suggested results Our ER– Ca controlled mitochondria for allows palmitoylation Calnexin ihtasgri n esrdteices fctslcCa cytosolic of increase the measured and thapsigargin with Kase l,21)ta etasetdwt idtp n non- and Ca wild-type SERCA with inhibited transfected we First, we calnexin. that (MEFs) palmitoylatable 2010) fibroblasts al., et embryonic (Kraus mouse calnexin-knockout using 3896 ol lob ifrn nclsepesn anxncompared calnexin expressing cells in different be also could amtyae anxnt euaetefu fCa of flux the regulate to calnexin palmitoylated 2+ a bevduigFR- ihr(upeetr material (supplementary either FURA-2 using observed was 2+ ora fCl cec 2 (17) 126 Science Cell of Journal inln.I hswr orc ewudexpect would we correct were this If signaling. 2+ transfer 2+ ihti sa,rgrls of regardless assay, this with , 0 oeCa more 80% 2+ ewe the between 2+ 2+ 2+ 2+ uptake pumps assays from 2+ 2+ 2+ htclei-ncotMF eeual obotteipr of import the boost Ca to IP3R-released unable were MEFs calnexin-knockout that el omlyices iohnra Ca mitochondrial when stress, increase ER of condition normally the under cells true also was this whether rt n crao 08.Teeoe etse whether tested we Ca Therefore, in 2008). differences show Scorrano, de mitochondria 2001; and al., et (Arnaudeau Brito MEFs calnexin-knockout in that with ore Baoe l,21;Csorda 2011; al., et (Bravo sources rsneo hpiagnso httepeec fwild-type Ca of mitochondrial presence the dampened that show calnexin thapsigargin of presence niiino RCa ER of inhibition ae oehr u eut ugs htclei utbe the must thus calnexin and SERCA that of Ca activity suggest of the rate results control to our again. palmitoylatable stress-responsive together, MEFs 4D), Taken calnexin-knockout (Fig. making deficiency this thus of rescued not but calnexin calnexin, wild-type non-palmitoylatable of transfection the However, stress. eut ihtemtcodilCa mitochondrial the with our results Moreover, S3B). Fig. material (supplementary MEFs knockout nwl-yeMF aeu infcnl esCa less significantly up take MEFs wild-type in niaeta h rsneo amtyae anxnrdcdthe reduced Ca calnexin import to palmitoylated results mitochondria in of of our ability presence Because that the calnexin. that was non-palmitoylated indicate with effect using this seen Importantly, compared not 4C). (Fig. when MEFs calnexin-knockout administration, thapsigargin 2+ motit mitochondria. into import 2+ 2+ *** experiments. independent three from results shows graph The SERCA2b. co- immunoprecipitating and calnexin FLAG-tagged for were analyzed FLAG) (IP: immunoprecipitates FLAG and inputs) (5% lysates DSP-crosslinked calnexin. or CCAA wild-type FLAG-tagged with transfected were cells HeLa SERCA2b. with immunoprecipitation co- CCAA) (CNX mutant palmitoylation ( Tunicamycin). for rp hw eut rmtreindependent three from results shows graph The SERCA2b. co-immunoprecipitating and for calnexin analyzed were CNX) (IP: calnexin immunoprecipitates and inputs) (5% lysates 10 eitherDSP-crosslinked or with (DTT) h Dithiothreitol 4 mM for 2 treated were cells HeLa stress. ER following co-immunoprecipitation SERCA2b xeiet (*** experiments P ** treatment). control (before C, experiments. independent fraction three membrane from heavy the in and amounts calnexin SERCA2b the shows and graph (CNX) The calnexin SERCA2b. for blotting western SDS-PAGE and by and indicated as analyzed were fractions 10 with treatment h a following 4 respectively) LM, light and and (HM heavy membranes into separated were HeLa lysates homogenized cell panel: left Lower panel). SERCA2b (upper for blotting western by and indicated SDS-PAGE as analyzed were fractions Membrane eaae sn pirp olwn treatment h 10 4 with a following Optiprep, were using lysates separated cell HeLa Homogenized stress. ER ( SERCA2b. the and affect calnexin palmitoylation between and interaction stress ER 3. Fig. nomtcodi fe tunicamycin h 4 a after mitochondria into A up.Ide,w on htmitochondria that found we Indeed, pumps. 5 pirpadmmrn rcinto following fractionation membrane and Optiprep ) .9 N)frSRAb ( SERCA2b. for (NS) 0.395 P 5 0.006. m uiayi Tn) nosxfractions. six into (Tuni), Tunicamycin M ´ ta. 06.Orrslsshow results Our 2006). al., et s 2+ 2+ P 5 niao y hd nthe in Rhod2 dye indicator C rmteE,w ettested next we ER, the from .03frDT *** DTT, for 0.0073 anxnadtecalnexin the and Calnexin ) P 2+ m 5 uiayi.Membrane Tunicamycin. M 2+ .1 o calnexin; for 0.013 paefloigthe following uptake 2+ paefollowing uptake B motfo ER from import 2+ Calnexin– ) m Tunicamycin. M hncalnexin- than P , 0.001 Journal of Cell Science EsadK el rnfce ihFA-agdwl-yeo CAclei eelsdadpoesdfrwsenbotn sn h LGantibody. FLAG the using blotting western for processed and lysed were calnexin CCAA or wild-type FLAG-tagged with transfected cells KO and MEFs * wild-type, calnexin P ncot(O EsadK el rnfce ihFA-agdwl-yeo CAclei eelae ihRo2 el eete rae ih50 with treated then ( were cytometry Cells flow Rhod2. by with treatment loaded histamine were after calnexin and CCAA before or recorded wild-type was FLAG-tagged fluorescence with probe transfected and cells histamine KO and MEFs (KO) knockout hpiagnmdae RCa ER thapsigargin-mediated anxn(N)koku K)MF n Oclstasetdwt LGtge idtp W)o CAclei eelae ihFu8 el eethen were Cells Fluo8. with loaded were calnexin CCAA or (WT) wild-type FLAG-tagged with transfected cells 1.5 KO with and treated MEFs (KO) knockout (CNX) Calnexin rnfce ihFA-agdwl-yeo CAclei eec-rnfce ihapamdecdn Rtree eurn uiecnewsplott * Methods. was Luminescence and aequorin. Materials ER-targeted the encoding in plasmid outlined a protocol with the co-transfected following were experiments calnexin independent CCAA three or wild-type FLAG-tagged with transfected rdetars h iohnra ebae wn oterhigh their Ca to owing constitutive membranes proton mitochondrial high the abnormally across an gradient exhibit could cells calnexin-knockout wild-type, calnexin for hd.Clswr hntetdwt 1.5 with treated then were Cells Rhod2. sapeeust o h iohnra ebaepotential membrane mitochondrial the for prerequisite (Ca a is i.4 amtyae anxnrgltsE Ca ER regulates calnexin Palmitoylated 4. Fig. htol idtp anxntaseto eutdi a in resulted transfection calnexin demonstrating by wild-type hypothesis this only counterparts confirm to we that non-palmitoylatable able their (TMRM), ester indeed and methyl were and calnexin tetramethylrhodamine with wild-type calnexin cells with transfected calnexin-knockout labeling 5 ie htefcetCa efficient that Given ´ .49frclei idtp (NS), wild-type calnexin for 0.8419 dnse l,21) enx etdtehptei that hypothesis the tested next we 2010), al., et rdenas m hpiagnadpoefursec a eoddbfr n fe hpiagntetetb lwctmty ,cnrl(eoetreatment). (before control C, cytometry. flow by treatment thapsigargin after and before recorded was fluorescence probe and thapsigargin M 2+ P 5 rnfrfo h Rt iohnra By mitochondria. to ER the from transfer P .8 o anxnCA) ( CCAA). calnexin for 0.088 5 .53frclei CA(S.( (NS). CCAA calnexin for 0.8583 2+ 2+ ees.Clei ncot(O EsadK el rnfce ihFA-agdwl-yeo CAclei eelae with loaded were calnexin CCAA or wild-type FLAG-tagged with transfected cells KO and MEFs (KO) knockout Calnexin release. rnfrfo h Rt mitochondria to ER the from transfer m P hpiagnadpoefursec a eoddbfr n fe hpiagntetetb lwctmty ** cytometry. flow by treatment thapsigargin after and before recorded was fluorescence probe and thapsigargin M 5 .86frclei CA(S.( (NS). CCAA calnexin for 0.8816 2+ E signaling. anxnepeso eeso ersnaieclsfo lwctmtyeprmns anxnkoku (KO) knockout Calnexin experiments. cytometry flow from cells representative of levels expression Calnexin ) D esrmn fmtcodilCa mitochondrial of Measurement ) ( A esrmn fctslcCa cytosolic of Measurement ) B esrmn fE Ca ER of Measurement ) fiin nteasneo anxn u h ose Ca boosted the but calnexin, of absence the in efficient n nrae h RCa ER the calnexin increases palmitoylated requires and stress ER under observed Ca transfer Fig. ER–mitochondria material (supplementary Therefore, response induction the protein in S3D). result unfolded we not even did the was experiments, transfections of our constructs that our calnexin and two 4E) Throughout the (Fig. of shown). expression that not verified of uptake (data the membrane potential in plasma case TMRM equilibrates membrane the which also KCl, was of mitochondrial This presence S3C). the Fig. material of (supplementary reduction specific h ucino anxnpliolto 3897 palmitoylation calnexin of function The P 5 2+ .8( 0.08 2+ olwn hpiagn(g-eitdE Ca ER (Tg)-mediated thapsigargin following olwn itmn-eitdE Ca ER histamine-mediated following C P 2+ esrmn fmtcodilCa mitochondrial of Measurement ) 5 otn.Clei ncot(O EsadK cells KO and MEFs (KO) knockout Calnexin content. .51frclei ncot(S,*** (NS), knockout calnexin for 0.2581 2+ content. 2+ 2+ rnfri more is transfer ees.Calnexin release. 2+ 2+ following release. P , .0 for 0.001 dfrom ed P m 5 M 0.02 2+ Journal of Cell Science fclswspoesdfrsraeboiyaina ecie nteMtrasadMtosadpoe o itnltdsraeLL.Tebrgahsho graph bar 25 The bars: LDLR. Scale surface images. biotinylated lower for the probed in and shown Methods as and th calnexin Materials parallel, In FLAG-tagged the antibody. * transfected in anti-LDLR experiments. intracellular described the independent using as blotting three biotinylation western of surface for means for processed and processed lysed was were cells calnexin of CCAA or wild-type FLAG-tagged with transfected CAclei.DPcosikdlsts(%ipt)adFA muorcpttswr nlzdfrFA-agdclei n co-immunoprecipitati and calnexin FLAG-tagged for *** analyzed experiments. were independent three immunoprecipitates from ERp57. FLAG results co-immunoprecipitating and shows and graph inputs) The calnexin (5% ERp57. for lysates analyzed DSP-crosslinked were calnexin. CNX) CCAA (IP: immunoprecipitates calnexin and ( inputs) (5% lysates DSP-crosslinked ( niae ySSPG n etr ltigfrEp7 D n anxn(N) h rp hw h R5,PIadclei mut nteheavy the in amounts calnexin and PDI ERp57, the shows * graph treatment). The (without (CNX). control calnexin C, and experiments. 10 PDI lysate with independent cell treatment ERp57, three HeLa h for homogenized from 4 panel: blotting fraction a left western following membrane Lower respectively) panel). and LM, (upper SDS-PAGE calnexin and by and (HM PDI indicated membranes ERp57, light for and blotting heavy western into and separated SDS-PAGE by indicated as analyzed were ooeie eacl yae eesprtduigOtpe,floiga4htetetwt 10 with treatment ERp57. h and 4 calnexin a following between Optiprep, interaction using the separated were affect lysates palmitoylation cell and HeLa Homogenized stress ER 5. Fig. light and distribution the heavy the 3A), examined into (Fig. and SERCA2b we membranes to gradient contrast Optiprep First, cellular In by membranes. is stress. ERp57 of ERp57 and ER fractionation and calnexin of calnexin by distribution between affected interaction also similarly to the decided protein slow- we whether Thus, mediate for 2004). test important al., to particularly et ER (Frenkel is substrates calnexin the folding of in interacts role calnexin This ERp57 folding. SERCA2b, oxidoreductase with the interaction with its to addition In its ERp57 regulates with calnexin interaction of state palmitoylation The 3898 C B anxnEp7c-muorcptto olwn Rsrs.HL el eetetdfr4hwt ihr2m ihohetl(T)o 10 or (DTT) Dithiothreitol mM 2 either with h 4 for treated were cells HeLa stress. ER following co-immunoprecipitation Calnexin–ERp57 ) anxnadteclei amtyainmtn CA)Ep7c-muorcptto.HL el eetasetdwt LGtge idtp ( wild-type FLAG-tagged with transfected were cells HeLa co-immunoprecipitation. (CCAA)–ERp57 mutant palmitoylation calnexin the and Calnexin ) ora fCl cec 2 (17) 126 Science Cell of Journal P 5 .7 el eeas rcse o DRsraebnig olwdb emaiiainaddtcinof detection and permeabilization by followed binding, surface LDLR for processed also were Cells 0.07. P 5 .0.( 0.003. anxnCA uatta antb amtyae indeed palmitoylated be cannot the 5C, that Fig. mutant in shown CCAA As this palmitoylated. calnexin test mutant be the To cannot and that chaperones. calnexin calnexin other FLAG-tagged expressed with we could association which possibility, itself, its calnexin trigger of misfolding then ER the share 5B). to of lead (Fig. amount could increased the might stress ERp57 h, cells and 4 calnexin when for proteins tunicamycin between that or interaction observed DTT two either we stress with Indeed, treated these ER are stress. an ER that following under functions membranes suggesting light to 5A), out to (Fig. turned heavy both and from calnexin of move that matched closely ERp57 of D DRsraeboiyain anxnkoku K)MF n Ocells KO and MEFs (KO) knockout Calnexin biotinylation. surface LDLR ) P 5 .5 o R5,* ERp57, for 0.056 m ( A m. m pirpadmmrn rcinto olwn Rstress. 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Likewise, Fig. an the material well. in (supplementary 2-bromopalmitate, doubled as approximately of was twice ERp57 presence to almost calnexin of ERp57 binding with interacted ooei F HaRP,acrigt h Manders dissociation with where ERQC, the calnexin the to non-palmitoylated extends which to of to substrates, demonstrate ERAD results interaction linked our according preferential together, the H2a Taken (H2a–RFP), 6C–F). with (Fig. coefficient RFP colocalization the and to monomeric relocation pronounced ERQC a more for a showed juxtanuclear showed inhibition CCAA proteins proteasomal calnexin short-term h, both 3 upon cells, pattern, ER untreated their disperse in Kamhi- wild-type of Whereas with 2004; compared mutant we calnexin. non-palmitoylatable al., Thus, inhibition the 2007). et of al., localization et (Frenkel the upon Kondratyev 2001; stress al., accumulate ER et Nesher under H2a or ERAD degradation and like calnexin where ERQC, substrates pericentriolar the to targeting shifts of degraded. species trimming quickly the is glycosylated species underglycosylated of the lower whereas fully because N-glycans, its migration on the residues faster mannose a 2004), to al., progressively previously shown et had we (Frenkel of As (one unoccupied). species sites the underglycosylated glycosylation pulse molecules, the an precursor the to H2a For corresponding for time. one seen lower of be periods can bands extended two for calnexin samples, H2a with H2a, wild-type complex substrate in a remained ERAD and Whereas the reassociated calnexin transfected from CCAA non-palmitoylatable time 6B). we with (Fig. efficiently when dissociated from calnexin of pattern H2a transfection upon wild-type different of with compared H2a, dissociation calnexin a nonpalmitoylatable total that followed in showed calnexin increase ERAD. analysis for slight 6A). protein this (Fig. Pulse-chase a of targeting H2a decreased caused of with because probably also CNX interacted wild-type CCAA CCAA than Calnexin Calnexin robustly 1997). al., more et (Kamhi-Nesher much Shenkman ER 2001; the al., in retained et completely normally is the we H2a,which or, substrate, (ASGPR) ERAD receptor ER asialoglycoprotein an Thus, of with precursor the uncleaved calnexin (ERAD). from of interaction exit the degradation analyzed await with ER-associated that interacts proteins calnexin alternatively, of of form domains this unfolded that indicate could calnexin ER in palmitoylated partially stringent least at calnexin. more the wild-type than indeed retention that control suggesting is quality 5D), (Fig. calnexin expressed non-palmitoylated was surface decreased calnexin cell but the CCAA expressed, on was when LDLR unaltered calnexin of being wild-type amount when lysates the increased that cell found in Despite we LDLR 5D), mutant. the (Fig. CCAA of calnexin level transfected LDLR expression the we the or that whether calnexin MEFs calnexin-knockout wild-type determine palmitoylated the with used is To we calnexin not, 2007). when or calnexin differently al., on proceeds depend et maturation by and folding (Jessop first ERp57 protein with we undergo folding disulfides to purpose, known mixed is this forming which the LDLR, For on the dependent chose pathway. particularly is folding that calnexin–ERp57 substrate folding a identify fiin nrclua eeto fLL ynon-palmitoylated by LDLR of retention intracellular Efficient interaction the in change the that suggested results Our enx etdwehrpliolto nlecscalnexin influences palmitoylation whether tested next We rti AS2(yile l,20) amtyainmight palmitoylation 2008), al., et sorting palmitoylation cytosolic (Myhill the in PACS-2 by state mediated changes phosphorylation protein relocations calnexin the intra-ER in the in to whereas results Changes Moreover, compared way. 2). when (Fig. dynamic stress less to exposure ER a longer significantly which to in require another phosphorylation phosphorylation, and calnexin BAP31, calnexin ribosome, unlike chaperone, the reassigns ER, with ER that interaction the calnexin signal within determines quick restrict tasks a a specific identify can is therefore observations palmitoylation as Our palmitoylation that signaling. results palmitoylation stress propose how ER we Our of and time mechanism key role, 7). first either (Fig. to the calnexin control protein for in role quality its illustrate fulfill and to ERp57 with folding interact to tends calnexin euaeteclei oaiainb loig(rnt its Ca not) of are which (or sites ERp57, allowing and to SERCA2b by effectors localized localization its with calnexin interaction the regulate motn o rti odn,cnitn ihispredominant its with consistent folding, protein the for facilitates of important expression palmitoylation intermediates. folding upon that from calnexin LDLR of suggest surface dissociation surface calnexin, the of on amount CCAA LDLR with decreased together mature findings, the of These S4B). amount Fig. material the (supplementary calnexin reduces with with ERp57 and association of the H2a binding increases S4A), the Fig. 2BP in material with increase (supplementary palmitoylation 2-fold of a inhibition to more an the leads such that of become stress with observed effects Consistent we the would stressor. idea, mitigate ER ER calnexin hence introduced would scenario, additionally and any of this chaperone a (Baldwin In as induction apoptosis efficient 2012). of al., progression the the et and attenuates factors (2BP) transcription 2-bromopalmitate which could of results function these treatment, anti-stress the Interestingly, the to ER, 2013). explain proteins rough al., help misfolded the et or to (Leitman unfolded close ERQC of or targeting at the control including quality and folding protein h neato ihSRAbadarl nteregulation the in role We a stress. and ER to SERCA2b Ca calnexin during with of assigns interaction palmitoylation palmitoylation the reversible calnexin that of demonstrate of role description mechanistic a the uncover herein presented results The of ability Discussion the require would palmitoylation. undergo glycoprotein to calnexin misfolded the from eedn yl ihnteE n htlsbc n ot from forth and back shuttles and new Ca ER Our regulating the palmitoylation- triggered. within a is undergoes cycle itself ERAD dependent calnexin or that indicate calnexin reached now with is data of substrates folding folding action of proper interaction until enzymatic cyclic a to the and leads I II with glucosidases and together glucosyltransferase (Lederkremer, UDPGlc:glycoprotein property, substrates This calnexin that monoglucosylated 2009). given view, exclusively of point binds substrate’s a the 2011; or from explained al., MAM et the (Bravo at contacts proteins stress, Csorda ER–mitochondria ER ER a during ER of of other since follow tightening enrichment each unique, to an to relatively closer suggesting probably move appears mitochondria is and that calnexin pattern targeting Interestingly, respectively. codnl,plioltdclei per ob less be to appears calnexin palmitoylated Accordingly, ofr h neato fclei ihissbtae a been has substrates its with calnexin of interaction the far, So ´ 2+ h ucino anxnpliolto 3899 palmitoylation calnexin of function The ta. 2006). al., et s rnfrt iohnra hra non-palmitoylated whereas mitochondria, to transfer 2+ inln to ls oteMMadmediating and MAM the to close or at signaling 2+ inln n rti folding, protein and signaling Journal of Cell Science ornfce nNH33cls t1dyatrtaseto el eeicbtdfr3hi h bec CE rpeec f25 of were presence or (E,F) (C,E) CCAA absence CNX the in or h (C,D) 3 WT for inde CNX incubated three were FLAG-tagged of cells and transfection average (H2a–RFP) after CCAA gels, day RFP CNX the 1 monomeric and At of to cells. WT quantifications 3T3 linked CNX phosphorimager NIH H2a with in from coimmunoprecipitated for cotransfected pulse, H2a encoding ones fas the of Plasmids lower a to percentage inhibition. the to the relative proteasomal molecules, progressively shows chase precursor shifts graph after H2a species The anti-H2a), (mean for N-glycans. glycosylated (IP experiments seen its fully amounts on be The total residues can unoccupied). to immunoprecip mannose bands sites All normalized of two glycosylation (Cont.). trimming sample samples, the the control pulse of a of the in (one because anti-FLAG For species wi 15 of phosphorimaging. re-immunoprecipitation underglycosylated instead from by and immunoprecipitated used to was was elution followed correspond H2a antibody by SDS-PAGE lysed, Control followed panels). were 12% antibody (left cells by anti-FLAG Methods the separated with and periods, immunoprecipitated Materials chase the the were in or remainders described chase) the as h and (0 panels) pulse (right the lysates After cell MG-132. inhibitor proteasome the of presence 9 el eetasetda nA t2dy ottaseto h el eeplelbldfr2 i ih[ with min 20 for pulse-labeled were cells the post-transfection days 2 At A. in as transfected were cells 293 n nuae ihmueat-LGadC2cnuae otat-os g.Tesmlswr nlzdi nLMcnoa irsoe Representativ microscope. confocal LSM an in analyzed were 10 samples bar: The Scale IgG. shown. goat-anti-mouse are Cy2-conjugated slices and optical anti-FLAG confocal mouse with incubated and tt fsrs omtcodi,poal oalvaethe alleviate al., to et (Bravo probably ER the mitochondria, within a proteins to signal unfolded can of stress ER accumulation the of that calnexin state palmitoylated of presence the eesbepliolto r eurdt lo h Rt properly to Ca ER its via the and allow signal expression to calnexin required are example, palmitoylation specific reversible our our (Simmen In metabolism folding 2010). 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Fig. 3900 ora fCl cec 2 (17) 126 Science Cell of Journal 2+ 6 oad iohnra ti paetyoigto owing apparently is It mitochondria. towards ....( s.e.m.). C – F nrae RDsbtaeclclzto ttejxaula RC ihCXCA scmae ihCXW upon WT CNX with compared as CCAA CNX with ERQC, juxtanuclear the at colocalization substrate ERAD Increased ) m .Mnescefcet:07 o N idtp,08 o N CA(rm2 cells). 21 (from CCAA CNX for 0.88 wild-type, CNX for 0.77 coefficients: Manders m. csa nihbtro EC2 in SERCA2b of Ca ER inhibitor of measurement an calnexin. calnexin that palmitoylated as reports of acts previous contradict absence results mitochondria our the Surprisingly, mitochondria high in abnormally for potential the crucial membrane by also suggested is as function metabolism, this However, 2011). anxnCA uat(i.4) oeta xlnto for explanation potential non-palmitoylated A the 4B). 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(Axis Billerica, Optiprep Millipore, except (EMD ON) lactacystin (Oakville, and Sigma MA) from Norton, were chemicals All reagents and Antibodies Methods and Materials characteristics new palmitoylation with avenue protein opportunities. therapeutic and ER new a with unknown provides and now interference (Wang yet applications 2012), clinical be various with Kaufman, in interference response of to stress efficacy ER MAM demonstrated the has the the Given between ER. it shuttling the and calnexin shown), mediate our that not influence thioesterases not APT did response (data 2011) the al., stress because observations et (Rusch ER However, B are the palmostatin 2009). inhibitor family Kanaani, of and thioesterase regulators (Baekkeskov important the also of predict results probably enzymes our 2012), depalmitoylation al., et that (Lakkaraju DHHC6 as identified colleagues calnexin- and Foskett confirm mitochondria (Ca by on of findings demonstrated regulation elegantly the our findings as in Rather, plays metabolism, ER earlier 2008). the role al., with paramount the et the (Coe consistent in cells is differences knockout Fig. material This (supplementary detect on calnexin dependent S3D). of to are absence that or response able presence protein the unfolded been the of not induction however, have, Ca calnexin cytosolic of of influence measurement of the the of properties via determination special SERCA2b the in on in lie or could oocytes discrepancy this Rdrc ta. 00,rte hndrc esrmnso ER of measurements direct study than earlier rather the 2000), Ca in al., used et as (Roderick overexpression, calnexin following [ hlot uknhmhr,U) rmxcl aeigmx([ mix labeling cell (Little Healthcare Promix GE from UK). obtained Buckinghamshire, were standards Chalfont, protein methylated labeled 35 ]y) ( S]Cys), lhuhteezm htplioltsclei a been has calnexin palmitoylates that enzyme the Although ept hsrte akddfeec nE Ca ER in difference marked rather this Despite ´ 2+ dnse l,2010). al., et rdenas rasy fE–iohnraCa ER–mitochondria of assays or . 00C/ml a rmPriEmrLf n Analytical and Life PerkinElmer from was Ci/mmol) 1000 2+ cross-talk. 2+ otn,we content, 35 2+ ]e plus S]Met Xenopus waves 14 C]- Boiin ipts A,ga niFC4(ba,Cmrde K,mouse UK), Cambridge, (Abcam, anti-FACL4 goat CA), Milpitas, (Biovision, g nioycnuae oC2wsfo ako as(etGoe PA). Life Grove, (West Labs from anti-mouse Jackson Goat from 1996). were was al., Cy2 et carboxyterminal (Tolchinsky to antibodies anti-H2a studies conjugated earlier antibody polyclonal in fluorescent IgG used rabbit as The was secondary antibody CA). anti- anti-rabbit (P534, (Carlsbad, Goat us Sigma. Technologies from of and was behalf agarose or to on conjugated respectively) mouse MA) IgG human, anti-mouse (Marlboro, Goat P583 Biochemicals dog). dog, P544 Century (P563 21st Abcam by anti-phospho- The from generated 2012). (Mitosciences, were al., et antibodies anti-complex-2 (Lynes calnexin antibody mouse anti-calnexin rabbit IL), OR), Eugene, Rockford, (Thermo-Pierce, anti-PDI eaclstasetdfr4 ihFA–anxnwr rae with treated were FLAG–calnexin with h 48 for (10 transfected tunicamycin cells chemistry click HeLa by palmitoylation of Detection Ca with PBS cold with twice washed proteins surface were of Cells pulldown and labeling Biotin 3T3 NIH 37 at and grown (FCS) were cells serum All calf serum. fetal calf born 10% new plus 10% plus DMEM DMEM (HEK) in in kidney cells grown embryonic were human and cells (MEFs) 293 fibroblasts embryonic mouse HeLa, transfections and culture Cell efre 44 fe transfection. after h 24–48 performed ape eete nuae t4 biotinylated The at above. incubated as then prepared were were condition samples experimental each from cells h auatrrsisrcin.Tasettaseto fHK23clswas cells 293 Ca HEK the of to transfection NIH according to Transient according of Bio) performed instructions. 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Laemmli in in resuspended prepared PBS, MO) with once Louis, washed St were (Sigma, beads streptavidin–agarose ih2m BA 0m CuSO mM 50 TBTA, mM 2 with blotting. western and similar SDS-PAGE a by in analyzed denatured then were cells were non-biotinylated samples the All of manner. lysates The minutes. 5 for opeepoes niios(oh,Bsl wteln)adsrpdinto with scraped centrifuging buffer and by mRIPA obtained in Switzerland) 800 and at were lysed Basel, PBS++, lysates supernatants then the in were Post-nuclear (Roche, glycine cells tubes. mM inhibitors The microcentrifuge 50 PBS. protease with cold minutes Complete 5 with for twice quenched rinsed then was reaction namshr f5 CO 5% of atmosphere an LGatbd Rcln) h cik ecinwste are u nthe on 37 at out minutes carried 30 anti- then for the them was incubating using reaction by immunoprecipitated ‘click’ proteins was labeled The EDTA- calnexin immunoprecipitated (Rockland). inhibitor, and protease antibody QC) CPI FLAG Laval, (containing Roche, buffer SDS-RIPA free, 0.1% in harvested m lyy-amtt rpliaecnuae oBAfr3h el were Cells h. 3 for BSA to conjugated palmitate or alkynyl-palmitate M h ucino anxnpliolto 3901 palmitoylation calnexin of function The m g )o T 2m)fr4h et el eelbldwith labeled were cells Next, h. 4 for mM) (2 DTT or M) o iue t4 at minutes 5 for oisrl nCa in role its back to calnexin ERQC. chaperoning the redirects on Palmitoylation substrates ERAD with interact control. can quality calnexin and non-palmitoylated folding Additionally, protein the mediate on to ERp57 rER Non- with translocon. interacts the calnexin with palmitoylated (rER) ER rough the i.7 oe o h anxnsutigbetween shuttling calnexin the Ca for Model 7. Fig. anxnitrcswt EC nteMMto MAM Ca the ER on regulate SERCA with interacts calnexin 2 2+ eaclswr rninl rnfce with transfected transiently were cells HeLa . inln n ult control. quality and signaling 2+ ˚ vrih narce ih25 with rocker a on overnight C 4 hsht ehd h xeiet were experiments The method. phosphate 0m CPad2m Biotin-azide. mM 2 and TCEP mM 50 , ˚ .I aall yae fnon-biotinylated of lysates parallel, In C. 2+ 2+ inln n,t esretn,on extent, lesser a to and, signaling signaling. 2+ n Mg and ˚ 2+ .Tebiotinylation The C. PS+ n then and (PBS++) Palmitoylated m f40% of l ˚ under C ˚ ˚ C C Journal of Cell Science spbihd(lae n otr,20) olwn h elto fE Ca ER of expressing depletion the constructs Following (and 2002). Montero, transfection plasmid following and h (Alvarez this 24 measurements published with for as processed and transfected indicated) as were calnexin Cells Spain). ih05m GAad1 10 and with reconstituted EGTA was aequorin mM 0.5 with Ca Ca ER mitochondria and ER Plasmid-based 1.5 with treated were indicated as response transfected protein unfolded cells the assay Calnexin-knockout to PCR transcriptase Reverse with labeled were dishes 100-mm in monolayers cell [ (90%) Subconfluent labeling Metabolic then were cells NH The room mM proteins. 10 at in interacting minutes incubated crosslink 30 and to more for Scientific, twice PBS++ (Thermo incubated washed in proprionate) and (succinimidyl IL) PBS++ Dithiobis Rockford, mM with 2 twice with temperature washed were Cells experiments Co-immunoprecipitation (10 tunicamycin with treated were cells HeLa microscopy Immunofluorescence HRP- with membranes via both ECL. specificity probing proteins the using by ensuring Neutravidin into detected to was conjugated contributes incorporated Palmitoylation and signal. acids bonds the amide fatty of via not removes but bonds treatment thioester alkali This KOH. 3902 uliwr eltdi irfg o 0mntsa 4 at and minutes debris 30 and for ice, on microfuge min a 30 for in cholate cells, pelleted sodium 293 2% were HEK in nuclei by from performed analyzed was immunoprecipitation and washed lysis For buffer then cell Laemmli blotting. were in beads western resuspended The and and h. SDS-PAGE buffer 1 further CHAPS a in for times incubated three lysates the and added then imuigatasre-Efo oa Vnovr C.Qatfcto was Quantification (Japan). BC). phosphorimager (Vancouver, 5100 MS FLA Kodak Biomax Fujifilm from a to and in transcreen-LE exposed HBS performed a were in and using deoxycholate by 2,5-diphenyloxazole film sodium analyzed 20% were 0.5% diluted gels using X-100, then SDS-PAGE fluorography Triton antibody. SDS, anti-H2a 1% 1% with were of reimmunoprecipitated in lysates volumes cell boiled cholate, 10 antibody, sodium with 2% anti-FLAG containing cells 7.5, labeling, with pH metabolic immunoprecipitated buffer after HBS Briefly, in 2004). lysed al., were et (Frenkel previously described olwn iioi emaiiainwsasydo ua luminometer Ca Lumat mitochondrial a For on Germany). assayed Wildbad, was Bad permeabilization (Berthold, digitonin following 5 MNC,1m DA otiigCmlt rtaeihbtr (Roche, 4 the at inhibitors centrifuging 7.4, in minutes by protease pH time 5 obtained Tris Complete for were final mM containing lysates supernatants 10 a EDTA) Post-nuclear CHAPS, washed mM Switzerland). (1% 1 Basel, then buffer NaCl, lysis were CHAPS mM cells 150 in The harvested and reaction. PBS++ crosslinking the quench niae nioisfr1ha 4 at h 1 for antibodies indicated sdsrbdpeiul Aeo ta. 08.Clclzto nlss(Manders) software. Imaris analysis and Colocalization Germany) ImageJ 2008). Jena, The using al., Zeiss, performed et Carl was X-100. (Avezov 510; a previously on (LSM Triton described performed (Avezov microscope was as previously confocal microscopy 1% Confocal scanning described 2001). laser as al., with Zeiss were et Kamhi-Nesher cells permeabilization 2008; FLAG-tagged 3T3 al., Intracellular et NIH after BSA. with 1% anti- employed detected of and procedures dilution FBS was 1:100 10% a indicated of DMEM, calnexin al., binding in et the by (Gilady antibody detected using previously was LDLR binding described performed surface protocol LDLR was the 2010). to microscopy according antibodies, immunofluorescence primary and h, 4 h el o tro eprtr ntedr,clswr efsdwt medium with perfused were cells dark, the in Ca temperature mM room 1 at containing h 1 for cells the (5 Xbp-1 TRIzol for using primers isolated was with RNA amplified Total and controls. CA) as Carlsbad, left Technologies, or h (Life 16 for thapsigargin loecnemaueet eeiiilzdatretbiheto baseline, a of ON). establishment Hill, and after Richmond microscope (Olympus, initialized FV1000 software were Fluoview 3.1 measurements Olympus FV10-ASW Fluorescence a the whose using on R-GECO1, quantified mitochondrial measured encoding knockout was plasmid and a wild-type fluorescence Calnexin with 2011). transfected al., were et MEFs (Zhao used was R-GECO1 targeted olwdb h diino 1 of addition the by followed olwdb ahsadeuinb oln ihsml ufrcontaining buffer sample with boiling by elution and mercaptoethanol. washes overnight, by anti-mouse-IgG–agarose goat followed and anti-FLAG with immunoprecipitated CCTTGTGGTTGAGAACCAGG-3 n AD,rsetvl (5 respectively GAPDH, and ACACATTGGGGGTAGGAACA-3 n %arlmd es respectively. gels, acrylamide 1% and 35 ]y.C-muorcptto ihat-anxnatbd a efre as performed was antibody anti-calnexin with Co-immunoprecipitation S]Cys. 2+ a esrdwt HVrE gf rmJve lae,Valladolid, Alvarez, Javier from (gift pHSVerAEQ with measured was ora fCl cec 2 (17) 126 Science Cell of Journal 2+ uiecnefo hscniinadttlluminescence total and condition this from Luminescence . ˚ t800 at C m m ,-itr-uy-ezhdounn (Sigma), 2,5-di-tert-butyl-benzohydroquinone M thapsigargin. 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Fig. UPR material the chemistry’; supplementary performed and light in ‘click used S3A and the plasmid R-GECO1 with Fig. heavy the assisted material performed M.C.Y. supplementary C.O.S. S5A; in 6; fractionations Fig. membrane in experiments control O n nuae nDE Lf ehoois albd A o 0minutes Louis, 30 St for (Sigma, CA) Carlsbad, TMRM Technologies, nM (Life 20 37 DMEM at or in incubated CA) and Carlsbad, MO) Technologies, (Life 1 Rhod2 either with loaded were Cells cytometry Flow 100 resulting (with The medium enzymes. restriction overnight. same DH10B the transformed into transformed with were products digested ligation previously vector for gene the pTorPE R-GECO1, mitochondria-targeted in expressing R-GECO1 plasmid a construct plasmid To R-GECO1 mitochondria-targeted of Construction ...i irI aaaRsac hi nBioanalytical in Chair Research 25370); Canada and II by 24136 Tier supported a (studentships Health was is Foundation Innovates E.M.L. R.E.C. Alberta Cancer Institute 200500396); the Alberta Research [grant and (scholarship Foundation Society 2010-700306; Cancer Solutions Cancer number Alberta Canadian the [grant the by 25018]; supported number is work This Funding performed the performed A.R. design, manuscript; the the of of experimental design writing the the Ca in as the well participated as and figures analysis, in data and experiments participated E.M.L. contributions from Author gift generous a Spain. was The Valladolid, material. Alvarez, aequorin especially supplementary Javier ER-targeted We the with encoding discussions. help plasmid helpful for for Ballanyi Klaus Michalak thank Marek thank We Acknowledgements for centrifuged subsequently were cells was 800 The suspension at cell times. minutes The 10 EGTA). 15 18 HEPES in mM Germany) 1 mM twice an EDTA, (10 Heidelberg, washed mM through buffer then 1 sucrose, homogenization and passed mM stressors mitochondria 250 indicated 7.4, in pH the collected with and treated PBS++ were cells HeLa fractionations gradient Optiprep rmtetpo h rdetadaaye ySSPG n etr blotting. western and SDS-PAGE by taken rotor analyzed were Ti and Fractions gradient rpm. SW55 gradient the 32,700 an density at of in h top Optiprep 3 centrifuged the of for from was ON) gradient and Mississauga, Coulter, continuous MA), (Beckman Norton, 10–30% (Axis-Shield, a medium over layered was rmr:‘CM_WBm1mt’(5 CATCACGTC-3 ‘GCaMP_FW_BamH1_mito’ primers: S)adsbetdt lwctmtyuigaFC cnctmtr(BD cytometer Scan FACS (50 10 a and using treatment cytometry before flow ON) to Mississauga, subjected Biosciences, and BSA) lsiswr eiidb euniga h nvriyo let Molecular Alberta of University the DNA at R-GECO1 Kit sequencing Unit. mitochondria-targeting Miniprep by Services Plasmid Biology purified verified GeneJET the were the plasmids and using Scientific), performed (Thermo was purification plasmid 0scns(1.5 seconds 20 CMV-mito-GEM-GECO1 the the into ligated and above, described as repurified GCTGTCATCATTTGTAC-3 prpit e lc sn h eee N uiiainKt(hroScientific). the with (Thermo digested from Kit then Purification extracted was DNA was product GeneJet PCR DNA the The electrophoresis. using Co.) slice gel Gene appropriate Nippon S, (Agarose gel 2+ ˚ .Clswr hnhretdi EE-ufrdsln 01 lcs,0.1% glucose, (0.1% saline HEPES-buffered in harvested then were Cells C. esrmnsi i.4–;MS efre h quality the performed M.S. 4A–D; Fig. in measurements .coli E. .coli E. m 9 n GaPR_idI’(5 ‘GCaMP_RV_HindIII’ and ) hpiagn fe rgadministration. drug after thapsigargin) M ooiso grpae eete ikdu n utrdi LB in cultured and up picked then were plates agar on colonies g m t4 at /lapcli,20rmsaig o 2t 6h DNA h. 16 to 12 for shaking) rpm 250 ampicillin, g/ml eegono grpae wt 400 (with plates agar on grown were 1 a sda epae C a are u yusing by out carried was PCR template. a as used was m ˚ ,t eltnce n nrkncls h supernatant The cells. unbroken and nuclei pellet to C, laac albaighmgnzr(Isobiotech, homogenizer ball-bearing clearance M 9 .PRpout eeprfe sn %agarose 1% using purified were products PCR ). m lo ATBous,Snyae A,1 CA), Sunnyvale, Bioquest, (AAT Fluo8 M Bam 9 -GAGGATCCAACCATGGTCGACT- 1and H1 .coli E. 9 Hin -CGCAAGCTTCTACTTC- yeetooain n the and electroporation, by II(hroScientific), (Thermo dIII m m itmn)or histamine) M /lampicillin) g/ml m M 1 Journal of Cell Science oa,L,Yn,W,Femn .R,Sen .adFamnat R. Flaumenhaft, and H. Steen, R., M. Freeman, W., Yang, L., Dowal, eo,F,Fsat .adCee,E. Chevet, and D. Fessart, F., Delom, eBio .M n crao L. Scorrano, and M. O. Brito, de o,H,Bdr,K,Gonny,J,Jn,J n ihlk M. Csorda Michalak, and J. Jung, J., Groenendyk, K., Bedard, H., Coe, M. Michalak, and H. Coe, rne,Z,Seka,M,Knrte,M n eekee,G Z. G. Lederkremer, and M. Kondratyev, M., F. Shenkman, Grassi, Z., and Frenkel, S. Porcellini, M., Panigada, D., Ferrera, upeetr aeilaalbeoln at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.125856/-/DC1 online available material was Supplementary 1070/10]. Alberta number is L.G.B. [grant the G.Z.L. Foundation Science and 24425]; Health 99085]; Israel number 81248] the of [grant by MOP NHG (ACRI) supported Institutes number Institute Research number [grant Canadian Cancer CIHR [grant by by supported supported (CIHR) is Research and Chemistry hvt . og .N,Gre,D,Cce,C,Fzl . aeo,P . Gushue, H., P. Cameron, A., Fazel, C., Cochet, D., Gerber, N., H. 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