Oxidative stress contributes to autophagy induction in response to endoplasmic reticulum stress in Chlamydomonas

! 1! Running&title:&ROS!trigger!autophagy!upon!ER!stress& & Corresponding&author:! José!L.!Crespo! Instituto!de!Bioquímica!Vegetal!y!Fotosíntesis,!Consejo!Superior!de!Investigaciones! Científicas!(CSIC)IUniversidad!de!Sevilla,!41092!Seville,!Spain! eImail:![email protected]! Tel:!+34!954489574! Fax:!+34!954460065! & Research&Area:&! Signaling!and!Response! ! 2! & Reactive& oxygen& species& mediate& autophagy& induction& in& response& to& endoplasmic&reticulum&stress&in&Chlamydomonas& & Marta!PérezIMartín1,!María!Esther!PérezIPérez2,3,!Stéphane!D.!Lemaire2,3!and!José! L.!Crespo1*! ! 1Instituto! de! Bioquímica! Vegetal! y! Fotosíntesis,! Consejo! Superior! de! Investigaciones!Científicas!(CSIC)IUniversidad!de!Sevilla,!41092!Seville,!Spain! 2Centre!National!de!la!Recherche!Scientifique,!UMR8226,!Laboratoire!de!Biologie! Moléculaire! et! Cellulaire! des! Eucaryotes,!Institut!de!Biologie!PhysicoIChimique,! 75005!Paris,!France! 3Sorbonne! Universités,! UPMC! Univ! Paris! 06,! UMR8226,! Laboratoire! de! Biologie! Moléculaire! et! Cellulaire! des! Eucaryotes,! Institut! de! Biologie! PhysicoIChimique,! 75005!Paris,!France! ! Summary:& Reactive!oxygen!species!generated!during!endoplasmic!reticulum!stress!contribute! to!autophagy!activation!in!Chlamydomonas.! ! ! 3! Footnotes:& This!work!was!supported!in!part!by!an!IEF!EU!Marie!Curie!Fellowship!(PIEFIGAI 2011I298652IREDOXDYNAMICS! to! MEPP),! by! LABEX! DYNAMO! ANRI11ILABXI 0011! (to! MEPP! and! SDL),! by! the! Spanish! Ministry! of! Economy! and! Competitiveness! (grants! BFUI2009I07368! and! BFU2012I35913! to! JLC),! and! by! Junta!de!Andalucía!(grant!CVII7336!to!JLC).! ! Corresponding&author:! José!L.!Crespo!([email protected])! ! ! ! 4! ABSTRACT! The! accumulation! of! unfolded/misfolded! proteins! in! the! endoplasmic! reticulum! (ER)! results! in! the! activation! of!stress!responses!such!as!the! unfolded! protein! response! or! the! catabolic! process! of! autophagy! to! ultimately! recover! cellular! homeostasis.! ER! stress! also! promotes! the! production! of! reactive! oxygen! species! (ROS),!which!play!an!important!role!in!autophagy!regulation.!However,!it!remains! unknown! whether! ROS! are! involved! in! ER! stressIinduced! autophagy.! In! the! present! study,! we! provide! evidence! connecting! ROS! generated! during! ER! stress! and! autophagy! activation! in! the! model! unicellular! green! alga! Chlamydomonas+ reinhardtii.!Treatment!of!Chlamydomonas!cells!with!the!ER!stressors!tunicamycin! or!dithiothreitol!resulted!in!upregulation!of!the!expression!of!genes!encoding!ER! resident! ERO1! oxidoreductase! and! protein! disulfide! isomerases.! ER! stress! also! triggered! autophagy! in! Chlamydomonas! based! on! the! protein! abundance,! lipidation,!cellular!distribution!and!mRNA!levels! of!the!autophagy! marker!ATG8.! Moreover,!an!increase!in!the!level!of!ROS,!in!the!oxidation!of!the!glutathione!pool! and! in! the! expression! of! oxidative! stressIrelated! genes! were! detected! in! tunicamycinItreated! cells.!Our! results! revealed! that! the! antioxidant! glutathione! partially! suppressed! ER! stressIinduced! autophagy! and! decreased! the! toxicity! of! tunicamycin,! indicating! that! oxidative! stress! participates! in! the! control! of! autophagy! in! response! to!ER!stress!in! Chlamydomonas.! In! close! agreement,! we! also!found!that!autophagy!activation!by!tunicamycin!was!more!pronounced!in!the! Chlamydomonas! sor1! mutant,! which! shows! increased!expression!of!oxidative! stressIrelated!genes.! ! 5! INTRODUCTION! All!living!organisms!have!evolved!sophisticated!mechanisms!to!efficiently!respond! and! adapt! their! growth! and! metabolism! to! different!types! of! stress.! A! wellI documented!example!of!such!stressIinduced!responses!is!the!process!of!autophagy! or! selfIdegradation,! which! is! structurally! and! functionally! conserved! in! all! eukaryotes.!During! autophagy! (also! known! as! macroautophagy),! cytoplasmic! components! including! proteins,! membranes! and! even! organelles! are! nonI selectively!enclosed!within!a!doubleImembrane!vesicle!known!as!autophagosome! and! delivered! to! the! vacuole/lysosome! for! degradation! of! toxic! or! damaged! components! and! recycling! of! needed! nutrients!(Xie! and! Klionsky,! 2007;! Nakatogawa!et!al.,!2009;!Li!and!Vierstra,!2012;!Liu!and!Bassham,!2012).!! Autophagy! is! mediated! by! a! set!of!proteins!coded!by!ATG! (autophagyIrelated)! genes!that!are!widely!conserved!from!yeasts!to!humans.!Homologs!of!ATG!genes! have!been!reported!in!plant!and!algal!genomes,!indicating!that!autophagy!is!also! conserved!in!photosynthetic!organisms!(Thompson!and!Vierstra,!2005;!Bassham! et! al.,! 2006;! DiazITroya! et! al.,! 2008;! AvinIWittenberg! et! al.,! 2012).! Some! ATG! proteins!play!a!structural!role!in!autophagy!and!are!essential!for!the!formation!of! the!autophagosome.!For!instance,!the!ATG8!protein!anchors!to!the!autophagosome! membrane! through! its! covalent! binding! to! phosphatidylethanolamine! (PE),! and! the! ATG8IPE! conjugate! is! essential! for! the! formation! and! completion! of! the! autophagosome! (Ichimura! et! al.,! 2000).! ATG!genes!are!conserved!in! Chlamydomonas!(DiazITroya!et!al.,!2008;!PerezIPerez!et!al.,!2010),!and!autophagy! has! been! investigated! in! this! model! system! by! monitoring! the! abundance,! lipidation!state!and!cellular!distribution!of!the!ATG8!protein!under!several!growth! ! 6! and!stress!conditions.!As!reported!for!other!systems,!nitrogen!or!carbon!depletion! triggers!autophagy!in!Chlamydomonas!(PerezIPerez!et!al.,!2010).!Moreover,!entry! of! Chlamydomonas! cells! into! stationary! growth! phase! activates! autophagy! in! a! reversible!manner!since!the!process!is!quickly!downregulated!when!cells!return!to! exponential! growth!(PerezIPerez! et! al.,! 2010).! Autophagy! is! also! induced! in! Chlamydomonas!cells!treated!with!H2O2!or!methyl!viologen!(MV),!indicating!that! oxidative!stress!triggers!this!process!in!algae!(PerezIPerez!et!al.,!2010;!PerezIPerez! et!al.,!2012a).!Reactive!oxygen!species!(ROS)!are!potent!inducers!of!autophagy!in! Chlamydomonas! and! plants! (Liu! and! Bassham,! 2012;! PerezIPerez! et! al.,! 2012b).! Indeed,!a!link!between!photoIoxidative!damage,!ROS!accumulation!and!autophagy! activation!has!been! shown!in!Chlamydomonas!cells! with!a!decreased! carotenoid! content! caused! by! either! the! mutation! of! phytoene! synthase! or! the! inhibition! of! phytoene! desaturase! by! the! herbicide! norflurazon!(PerezIPerez! et! al.,! 2012a).! Moreover,!ROS!generated!in!the!chloroplast!of!carotenoidIdeficient!cells!or!in!the! chloroplast! of! wildItype! cells! subjected! to! high! light! stress! activate! autophagy! (PerezIPerez!et!al.,!2012a).! The!accumulation!of!un/misfolded!proteins!in!the!endoplasmic!reticulum!(ER)!is! known!to!trigger!autophagy!in!yeasts!(Bernales!et!al.,!2006;!Yorimitsu!et!al.,!2006)! and! mammals! (Ogata! et! al.,! 2006).! More! recently,! induction! of! autophagy! by! ER! stress!has!also!been!reported!in!land!plants!and!algae!(PerezIPerez!et!al.,!2010;!Liu! et! al.,! 2012),! indicating! that! the! signaling! pathways! controlling! autophagy! activation! in! response! to! this! intracellular! stress! might! be! conserved!in! photosynthetic! organisms.! In! Chlamydomonas,! tunicamycin,! which! induces! ER! stress! by! inhibition! of! NIlinked! glycosylation,! strongly! increases! the! abundance! and! lipidation! of! ATG8! and! modifies! its! cellular! distribution!(PerezIPerez! et! al.,! ! 7! 2010).!The!accumulation!of!misfolded!proteins!in!the!ER!is!a!potent!stress!signal! that! induces! the! expression! of! chaperones! and! other! proteins! required! for! the! reestablishment! of! cell! homeostasis,! a! signaling! process! known! as! the! unfolded! protein!response!(UPR)!(Walter!and!Ron,!2011).!ER!stress!is!perceived!in!the!cell! by! key! signaling! proteins! such! as! the! highly! conserved! IRE1! kinase,! which! transduces! stress! signals! to! the! nucleus! by! promoting! the! splicing! of! bZIPIlike! transcription! factors!(Walter! and! Ron,! 2011).! Eukaryotic! cells! employ! a! quality! control!mechanism!that!recognizes!aberrantly!folded!proteins!in!the!ER!for!their! degradation! via! the! proteasome,! a! process! that! is! known! as! ERIassociated! degradation! (ERAD)! (Walter! and! Ron,! 2011).! Prolonged! ER! stress! also! triggers! autophagy! to! remove! unfolded! proteins! and! to! counterbalance! ER! expansion! caused!by!UPR!(Bernales!et!al.,!2006).!! Unlike! other! cellular! compartments,! the! ER! provides,! through! ERIresident! oxidoreductases,! an! oxidative! environment! that! facilitates! the! oxidation! of! cysteines!and!thereby!the!formation!of!disulfide!bonds!(Tu!and!Weissman,!2004).! In!yeasts!and!mammals,!it!has!been!shown!that!the!ER!oxidoreductin!1!(ERO1)!is!a! major! source! of! ROS! in! the! ER! and! in! the! cell! (Haynes! et! al.,! 2004;! Tu! and! Weissman,!2004).!Oxidative!protein!folding!in!the!ER!occurs!in!part!through!the! formation!of!disulfide!bonds!via!protein!disulfide!isomerases!(PDIs).!To!introduce! disulfides! into! client! proteins,! PDI! must! be! maintained! in! an! oxidized! state,! and! ERO1!is!the!main!responsible!for!keeping!PDI!oxidized!and!active!in!the!ER!(Frand! and!Kaiser,!1999).!ERO1!utilizes!molecular!oxygen!as!final!electron!acceptor!and! hence! forms! one! molecule! of! H2O2!for!every!disulfide!it!introduces!(Tu! and! Weissman,! 2004).! Several! studies! have! established! an! association! between! ER! stress!and!ROS!generation!(Malhotra!and!Kaufman,!2007;!Rutkowski!and!Kaufman,! ! 8! 2007;! Ozgur! et! al.,! 2014),! and! the! molecular! mechanisms! by! which! ROS! are! produced!during!UPR!have!been!thoroughly!reviewed!(Santos!et!al.,!2009).!! ROS! have! been! proposed! to! play! an! important! role! in! the! mechanisms! of! autophagy!induction!in!response!to!various!stress!conditions!in!mammals,!plants! and!algae!(Huang!et!al.,!2011;!ScherzIShouval!and!Elazar,!2011;!Szumiel,!2011;!Li! et! al.,! 2012;! PerezIPerez! et! al.,! 2012b).! Although! ER! stress! promotes! ROS! production!and!induces!autophagy,!it!remains!unknown!whether!ROS!are!involved! in! ER! stressIinduced! autophagy.!In! the! present!study,!we!provide! evidence! connecting!autophagy!activation!in!ERIstressed!cells!with!ROS!generated!from!the! UPRIregulated!oxidative!folding!machinery!in!Chlamydomonas,!and!propose!that! oxidative!stress!contributes!to!the!induction!of!autophagy!by!ER!stress.! ! 9! RESULTS! Tunicamycin&triggers&ER&stress&and&autophagy&in&Chlamydomonas& In! a! previous! study,! we! showed! that! tunicamycin!triggers! autophagy! in! Chlamydomonas! (PerezIPerez! et! al.,! 2010).! To! further! characterize! the! effect! of! tunicamycin!on!autophagy,! we! analyzed! the! expression! of! the! ATG8!gene!by! quantitative! realItime! PCR! (qPCR).! Tunicamycin! treatment! resulted! in! a! progressive! increase! of! ATG8! transcript! levels! (Fig.! 1A).! In! close! agreement,! an! increase!in!ATG8!protein!abundance!and!the!appearance!of!modified!ATG8!forms! were!observed!in!ERIstressed!cells!(Fig.!1B).!Transcription!of!ATG3,!encoding!an! E2Ilike! enzyme! involved! in! ATG8! lipidation! (Ichimura! et! al.,! 2000),! was! also! analyzed!in!tunicamycinItreated!cells!to!investigate!the!participation!of!other!ATG! genes!in!ER!stress.!qPCR!analysis!revealed!that!similar!to!ATG8,!ATG3!expression! was!upregulated!with!tunicamycin!and!also!with!other!stressors!previously!shown! to!induce!autophagy!such!as!H2O2,!methyl!viologen!or!norflurazon!(PerezIPerez!et! al.,! 2010;! PerezIPerez! et! al.,! 2012a)!(SFig.!1).! As! expected,! tunicamycin! had! no! effect!on!the!expression!of!FKBP12,!an!ER!stress!unrelated!gene!(SFig!2).! The!cellular!response!to!ER!stress!in!Chlamydomonas!is!still!poorly!characterized! and! no! markers! have! been! established! to! investigate! this! process! in! this! model! alga.!In!Arabidopsis,!it!has!been!reported!that!expression!of!some!UPR!genes!such! as!the!ERIresident!chaperone!CalreticulinI2!(At1g09210)!increases!in!response!to! tunicamycin! (Martinez! and! Chrispeels,! 2003).! We! identified! a! member! of! the! calreticulin!family!in!Chlamydomonas!nuclear!genome!(SFig.!3),!which!we!denoted! as! CAL2.! To! investigate! whether! the! expression! of! CAL2!is!subject! to! UPR! regulation!in!Chlamydomonas,!transcription!of!this!gene!was!determined!by!qPCR! ! 16! Protein!glycosylation!in!the!ER!is!essential!for!cell!growth!and!thus!its!inhibition!by! tunicamycin!is!toxic!for!the! cell.!Indeed,!we!found!that!tunicamycin! reduced!cell! viability! in! Chlamydomonas! after! 24! hours! of! treatment! (Fig.! 6A).! Our! results! indicated! that! the! presence! of! exogenous! GSH! in! the! medium! decreased!the! toxicity!of!tunicamycin!as!revealed!by!Evans!blue!staining!(Fig.!6A)!or!serial!spot! dilution!assays!(Fig.!6B).!Interestingly,! cells! were! still! fully! viable! at! the! time! of! autophagy! activation! by! tunicamycin! (6I8! hours;! Fig.! 6A),! supporting! a!proI survival!role!of!this!catabolic!process!in!response!to!ER!stress.!In!relation!to!the! effect! of! tunicamycin! on! cell! viability,! we! analyzed! the! expression! of! the! DAD1! gene,!a!homologue!of!defender!against!apoptotic!cell!death!1!that!is!conserved!in! many!eukaryotes!including!Chlamydomonas!(Kelleher!and!Gilmore,!1994;!Gallois! et!al.,!1997;!Kelleher!and!Gilmore,!1997;!Moharikar!et!al.,!2007).!The!DAD1!protein! is!part!of!the!oligosaccharyltransferase!complex!and!appears!to!play!an!important! role! in! NIlinked! glycosylation! in! the! ER! (Kelleher! and! Gilmore,! 1994),! a! process! specifically! inhibited! by! tunicamycin.! Our! results! revealed! that! DAD1!expression! was! upregulated! upon! prolonged! exposure! (48! hours)! to! tunicamycin! (Fig.! 6C),! when! cell! viability! is! significantly! compromised! (Fig.! 6A).! No! induction! of! DAD1! was!observed!in!cells!treated!with!tunicamycin!for!6!hours!(SFig.!8),!indicating!that! DAD1! expression! is! associated! to! tunicamycinIinduced! loss! of! cell! viability.! In! agreement!with!the!protective!role!of!GSH!on!cell!growth!in!ERIstressed!cells,!the! presence! of! this! antioxidant! decreased! tunicamycinImediated! DAD1!expression! (Fig.!6C).! ! The&sor1&mutant&displays&increased&autophagy&in&response&to&ER&stress& ! 17! To! further! investigate! the! redox! regulation! of!ER!stressIinduced! autophagy! in! Chlamydomonas! we! analyzed! the! activation! of! this! degradative! pathway! by! tunicamycin!in!the!sor1!mutant.!This!strain!lacks!the!SOR1!protein,!a!putative!bZIP! transcription! factor!that! controls! the! expression! of! a! large! number! of! oxidative! stress! response! and! detoxification! genes! including! GSTS1!(Fischer! et! al.,! 2012).! Treatment!of!cw15!and!sor1!cells!with!tunicamycin!revealed!a!more!pronounced! accumulation!of!the!ATG8!protein!in!the!sor1!mutant!(Fig.!7A),!suggesting!a!higher! induction!of!autophagy!in!this!mutant!in!response!to!ER!stress.!Expression!analysis! of!the!ATG8!gene!also!showed!a!higher!response!to!tunicamycin!in!sor1!compared! to!cw15!cells!(Fig.!7B).!As!previously!reported!(Fischer!et!al.,!2012),!we!found!that! sor1! mutant! cells! show! a! constitutively! higher! expression! of! GSTS1!(Fig.!7B).! Interestingly,! despite! this! elevated! expression,! tunicamycin! further! increased! GSTS1!mRNA!abundance!in!the!sor1!mutant!(Fig.!7B).!Taken!together,!these!results! strongly!suggested!that!autophagic!activity!triggered!by!ER!stress!is!higher!in!cells! with!anomalous!expression!of!oxidative!stressIrelated!genes.! ! ! 18! DISCUSSION& In! a! previous! report! we! demonstrated! that! ER! stress! induces! autophagy! in! Chlamydomonas! (PerezIPerez! et! al.,! 2010).! In! this! study! we! show! that! the! accumulation!of!misfolded!proteins!in!the!ER!under!tunicamycin!or!DTT!treatment! strongly! increased! the! expression! of! the! ATG8!gene,! in! agreement! with! the! pronounced!accumulation!of!the!ATG8!protein!and!its!modified!forms!detected!in! ER! stressed! cells! (Figs.! 1! and! 3).! Induction! of! autophagy! by! ER! stress! is! evolutionarily! conserved! and! has! been! reported! in! yeasts! (Bernales! et! al.,! 2006;! Yorimitsu!et!al.,!2006),!mammals!(Ogata!et!al.,!2006)!and!more!recently!in!plants! (Liu! et! al.,! 2012).! Studies! performed! in! yeasts! strongly! suggest! that! autophagy! serves! to! counterbalance! ER! expansion! that! occurs! as! a! consequence! of! UPR! signaling! (Bernales! et! al.,! 2006).! Degradation! of! ER! membranes! via! autophagy! during! ER! stress! is! conserved! in! plants! since! this! type! of! membrane! has! been! detected!inside!autophagic!bodies!in!Arabidopsis!plants!treated!with!tunicamycin! (Liu!et!al.,!2012).!! The!present!study!also!demonstrated!that!the!accumulation!of!unfolded!proteins!in! the!ER!triggers!oxidative!stress!in!Chlamydomonas.!Formation!of!disulfide!bonds! in!the!ER!and!associated!oxidative!protein!folding!are!linked!to!the!generation!of! H2O2!by!the!activity!of!the!ERO1!oxidoreductase!that!catalyzes!the!reIoxidation!and! activation!of!PDI!(Tu!and!Weissman,!2004).!Originally!identified!in!yeasts!(Frand! and!Kaiser,!1998;!Pollard!et!al.,!1998),!ERO1!is!widely!conserved!in!all!eukaryotes! including! plants! (Aller! and! Meyer,! 2012).! In! this! study,! we! found! that! ERO1! is! conserved! in! Chlamydomonas! and! its! expression! is! highly! induced! by! the! ERI stressors! tunicamycin! and! DTT! (Figs.! 1! and! 3).! ERO1! being! an! H2O2Igenerating! ! 19! enzyme,!its!elevated!expression!may!lead!to!increased!ROS!production!under!ER! stress.!Indeed,!our!results!indicated!that!ROS!accumulate!in!ERIstressed!cells!(Fig.! 2A).! Moreover,! Chlamydomonas! cells! subjected! to! ER! stress! displayed! elevated! expression!of!oxidative!stressIrelated!genes!and!higher!levels!of!the!intracellular! pool!of!GSSG!(Fig.!2B,!2C!and!3E).!Thus,!different!lines!of!evidence!indicate!that!ER! stress!triggers!oxidative!stress!in!Chlamydomonas.!In!yeasts,!ROS!such!as!H2O2!are! generated! during! ER! stress! and! overexpression!of!ERO1!causes!a!significant! increase! in! ROS! and! GSSG! pool,! suggesting! that! upregulation! of! the! oxidative! protein!folding!machinery!by!the!UPR!contributes!to!ROS!accumulation!(Haynes!et! al.,!2004).!In!a!recent!report,!it!has!also!been!shown!that!ER!stress!triggers!ROS! signaling! in! plants! (Ozgur! et! al.,! 2014),! indicating! that! ROS! production! upon!ER! stress!is!widely!conserved.! In! addition! to! ERO1,! we! identified! other! genes! subjected! to! UPR! regulation! in! Chlamydomonas.!The!expression!levels!of!two!PDI!genes,!Rb60!and!PDI6,!and!the! CAL2!chaperone!were!increased!in!response!to!ER!stress!(Figs.!2!and!4).!Rb60!is!a! canonical!PDI! with!an! atypical!dual! localization! in!the! ER!and!in! the!chloroplast! (Levitan! et! al.,! 2005)! where! it! regulates! psbA! translation! by! light! (Danon! and! Mayfield,!1991).!The!function!of!Rb60!in!the!ER!is!unclear!although!based!on!its! high!identity!to!classical!PDI!and!its!localization,!it!likely!participates!in!oxidative! protein!folding!in!the!ER.!PDI6!encodes!a!PDIIlike!protein!containing!an!NIterminal! DnaJ!domain!that!is!conserved!among!ER!coIchaperones!(Schroda,!2004)!and!a!CI terminal! arginineIrich! domain! involved! in! ER! stress! quality! control! in! mammals! (Mizobuchi! et! al.,! 2007).! The! domain! architecture! of! PDI6! appears! to! be! well! conserved!in!microalgae!and!less!represented!in!plants,!where!the!DnaJ!domainI containing! protein! ATERDJ3A! from! Arabidopsis+ thaliana! seems! to! be!the!closest! ! 20! homologue! (SFig.! 3).! No! clear! homologues! of! this! protein! have! been! found! in! yeasts! or! humans.! Our! results! revealed! that! PDI6! mRNA! levels! are! particularly! sensitive!to!ER!stress!and! a!massive!increase!in!the!expression!of!this!gene!was! observed!with!tunicamycin!or!DTT!(Fig.!1,!3!and!4).!Expression!of!the!calreticulin! CAL2!gene!was!also!found!to!be!upregulated!by!the!UPR!in!Chlamydomonas!(Fig.!1,! 3!and!4).!Similar!to!ERO1,!CAL2!is!conserved!in!plants!and!other!systems!(SFig.!3),! and!microarray!analysis!of!Arabidopsis!seedlings!treated!with!tunicamycin!or!DTT! suggested!that!this!gene!is!upregulated!under!ER!stress!conditions!(Martinez!and! Chrispeels,!2003).!Our!results!indicate!that!the!abundance!of!ERO1,!Rb60,!PDI6!or! CAL2!mRNAs!can!be!used!as!markers!of!the!UPR!in!Chlamydomonas!and!strongly! suggest! that! the! mechanisms! of! oxidative! protein! folding! described! in! other! eukaryotes!are!conserved!in!green!algae.! Given!that!autophagy!is!regulated!by!ROS!(PerezIPerez!et!al.,!2012b)!and!ER!stress! leads!to!ROS! production!in!Chlamydomonas!(Fig.!2),! ER!stressIderived!ROS!may! likely!contribute!to!autophagy!regulation!in!response!to!this!stress!(Fig.!8).!Indeed,! the!finding!that!the!antioxidant!GSH!downregulates!autophagy!in!ERIstressed!cells! (Figs.!4!and!5)!strongly!supports!the!implication!of!ROS!in!the!control!of!ER!stressI induced!autophagy.!Glutathione!is!the!main!free!soluble!thiol!in!the!cell!and!acts!as! a! redox! buffer! by! maintaining! the! intracellular! environment! in! a! reduced! state! (Foyer! and! Noctor,! 2011).! Prolonged! UPR! activation! in! a! yeast! strain! that! is! genetically!predisposed!to!sustained!ER! stress!causes!oxidative!stress!ultimately! leading!to!cell!death!(Haynes!et!al.,!2004).!In!this!strain,!the!presence!of!GSH!in!the! culture! medium! relieved! oxidative! stress! and! prevented! cell! death! without! diminishing!UPR!activation,!indicating!that!ROS!accumulate!during!ER!stress!and! are!toxic!for!the!cell!(Haynes!et!al.,!2004).!The!effect!of!GSH!in!alleviating!oxidative! ! 21! stress! and! promoting! growth! of! ERIstressed! cells! might! be! conserved! in! Chlamydomonas! since! our! results! showed! that! GSH! decreases! the! toxicity! of! tunicamycin! (Fig.! 6).!The!protective!role!of!GSH!was! emphasized!by!the!finding! that! this! antioxidant! decreased! the! expression! of! the! DAD1!gene,!which!is! specifically!upregulated! when!cell!viability!is!compromised!due!to! prolonged!ER! stress! (Fig.! 6C).! DAD1!is!a!putative!antiIapoptotic! gene! widely! conserved! in! eukaryotes! including! humans,! plants,! yeasts! and! algae! (Nakashima! et! al.,! 1993;! Kelleher! and! Gilmore,! 1994;! Gallois! et! al.,! 1997;! Kelleher! and! Gilmore,! 1997;! Moharikar!et!al.,!2007).!The!DAD1!protein!is!localized!in!the!ER!and!forms!part!of! the! oligosaccharyltransferase! complex,! which! catalyzes! NIlinked! glycosylation! (Kelleher!and!Gilmore,!1994).!Although!the!precise!function!of!this!protein!is!not! clear,!DAD1!appears!to!be!involved!in!preventing!cell!death!and!in!regulating!NI linked!glycosylation.!Our!results!are!in!agreement!with!these!putative!roles!since! expression! of! the! Chlamydomonas! DAD1! gene! is! induced! by! tunicamycin! treatment,! which! is! toxic! for! the! cell! and! specifically! inhibits! NIlinked! glycosylation.! The!hypothesis!that!ER!stressIinduced!autophagy!and!redox!signaling!are!linked! was! strengthened! by! the! finding! that! tunicamycin! triggers! autophagy! more! potently!in!sor1!mutant!cells!(Fig.!7).!The!sor1!strain!was!isolated!in!a!screen!for! mutants! with! increased! tolerance! to! singlet! oxygen! and! shows! a! constitutively! higher! expression! of! a! large! number! of! oxidative! stress! response! and! detoxification!genes!(Fischer!et!al.,!2012).!Mapping!of!the!sor1!mutation!identified! a!putative!bZIP!transcription!factor!denoted!as!SOR1!that!controls!among!others! the!expression!of!GSTS1!(Fischer!et!al.,!2012),!which!is!upregulated!in!ERIstressed! cells!(Fig.!2B).!The!high!autophagic!activity!observed!in!sor1!mutant!cells!treated! ! 22! with!tunicamycin!might!be!related!to!the!increased!expression!of!GSTS1!and!other! SOR1Icontrolled! genes! in! this! mutant,! emphasizing! again! the! link! between! ER! stressIinduced!autophagy!and!redox!signaling.! Overall,!our!results!indicate!that!ER!stress!triggers!ROS!production!and!autophagy! in!Chlamydomonas.!Under!ER!stress!triggered!by!tunicamycin,!GSH!was!found!to! decrease! autophagy! induction! while! UPR! markers! remained! unaffected.! This! uncoupling!of!UPR!and!autophagy!in!the!presence!of!a!ROS!scavenging!molecule! strongly!suggests!that,!under!ER!stress,!ROS!production!plays!a!prominent!role!in! the! induction! of! autophagy! in! Chlamydomonas!(Fig.!8).! Considering! the! high! conservation! of! the! mechanisms! of! ER! stress! and! autophagy! in! eukaryotes,! it! is! possible!that!ROS!may!also!be!involved!in!autophagy!induction!in!other!organisms.! Further!studies!will!be!required!to!unravel!how!ROS!control!autophagy!induction! and!to!examine!the!interplay!between!ROS!and!other!ER!stress!signaling!pathways! such!as!those!involving!the!IRE1!kinase.! ! 23! MATERIALS&AND&METHODS& Strains&and&growth&conditions& Chlamydomonas+reinhardtii!cw15!4B+!and!sor1!mutant!strains!were!obtained!from! the!Chlamydomonas!Culture!Collection.!Chlamydomonas!cells!were!grown!under! continuous! illumination! at! 25°C! in! TrisIacetate! phosphate! (TAP)! medium! as! described!(Harris,!1989).!When!required,!cells!in!exponential!growth!phase!(106! cells/ml)!were!treated!with!5!µg/ml!tunicamycin!(Calbiochem,!654380)!from!a!5! mg/ml!stock!in!dimethylformamide,!2,5!mM!DTT!(Applichem,!A2948),!1!mM!H2O2! (Sigma,! H1009),! 1! µM! methyl! viologen! (SigmaIAldrich,! 85617I7)! or! 20! µM! norflurazon!(SigmaIAldrich,!PS1044).! ! Gene&accession&numbers& Chlamydomonas! genes! analyzed! in! this! study! were! identified! at! the! Phytozome! website! (http://www.phytozome.net/cgiIbin/gbrowse/chlamy/)! under! the! following! accession! numbers:! ATG3,! Cre02.g102350.t1.2;! ATG8,! Cre16.g689650.t1.2;! CAL2,! Cre01.g038400.t1.2;! ERO1,! Cre17.g723150.t1.3;! PDI6,! Cre12.g518200.t1.3;!Rb60,!Cre02.g088200.t1.2;!GPXH,!Cre10.g458450.t1.3;!GSTS1,! Cre16.g688550.t1.2;!DAD1,!Cre02.g108400.t1.2.! ! RNA&isolation&and&quantification& Chlamydomonas! total! RNA! was! isolated! from! frozen! cell! pellets! as! previously! described!(Crespo!et!al.,!2005).!FirstIstrand!cDNA!was!produced!using!2!µg!total! ! 24! RNA,! oligo(dT)! primer,! and! 100! units! of! SuperScript! II! RNase! HI!reverse! transcriptase! (Invitrogen,! 18064I014)! in!a!50! µl! reaction.!Reverse! transcriptase! quantitative!PCR!was!performed!with!a!StepOne!RealITime!PCR!System!(Applied! Biosystems).!PCR!reactions,!in!a!final!volume!of!20!µl,!contained!10!µl!of!FastStart! Universal!SYBR!Green!Master!(Roche,!04913850001),!1!μL!of!cDNA!dilution,!250! nM! of! each! primer,! and! distilled! water! up! to! 20! µl.! Conditions! used! for! amplification!in!the!thermocycler!were:!preIincubation!at!95°C!for!10!min!followed! by!40!cycles!of!denaturation!at!95°C!for!15!s,!annealing!and!elongation!at!55I58°C! depending!on!the!gene!analyzed!for!60!s.!All!reactions!were!performed!in!triplicate! with!2I4!biological!replicates.!The!CBLP!gene!was!used!as!a!control!constitutively! expressed!gene!(Pootakham!et!al.,!2010).!The!primer!pairs!used!for!qPCR!were:!5’I CTTCTCGCCCATGACCACI3’! and! 5’ICCCACCAGGTTGTTCTTCAGI3’! for! CBLP;! 5’I CGAGTTCAAGGTCGAGCAGTI3’!and!5’ICCACCCACAGACATGGTGTAI3’!for!ATG3;!5’I TCCCCGATATCGACAAGAAGI3’!and!5’ITGCGGATGACGTACACAAATI3’!for!ATG8;!5’I ACCCTGACTACGTCCACGACI3’! and! 5’IGTCCTCAGCGAACTTCTTGGI3’! for! CAL2;! 5’I TGTCAACCTGCTCATCAACCI3’! and! 5’ICTGCTGCTGCTACTGCTGTCI3’! for! ERO1;! 5’I GGTGTGGCTGGTTGAGTTCTI3’! and! 5’ICTCTTTGGCGTCCTCACAGTI3’! for! PDI6;! 5’I CCAAGCGCTTTAAGAAGGTGI3’!and!5’IGTAGGGAAGCCCTTGACCTCI3’!for!Rb60;!5’I AGGTTCTGGATGCGTTCCTAI3’!and!5’IACACAGTCAGGGCGAAGAAGI3’!for!DAD1;!5’I GCGGTCGCCAATAACCAATI3’!and!5’IAAGGGCTGTCCCGAAAGCI3’!for!GPXH+(Fischer! et!al.,!2009);!5’ICTGACCATCAGCCACGACTI3’!and!5’IACATCGAACACCAGGGTAGCI3’! for!FKBP12;!5’I!CAGAGGTGAAAGGCGGATACI3’!and!5’IGTGTTGCAATGGACTTCAGCI 3’!for!GSTS1!(Fischer!et!al.,!2012).! ! ! 25! Protein&preparation&and&immunoblot&analysis& Chlamydomonas! cells! from! liquid! cultures! were! collected! by! centrifugation! (4,000g,!5!min),!washed!once!in!50!mM!TrisIHCl!(pH!7.5)!buffer,!and!resuspended! in!a!minimal!volume!of!the!same!solution.!Cells!were!lysed!by!two!cycles!of!slow! freezing! to! I80°C! followed! by! thawing! at! room! temperature.! The! soluble! cell! extract!was!separated!from!the!insoluble!fraction!by!centrifugation!(15,000g,!15! min)!in!a!microcentrifuge!at!4°C.!For!immunoblot!analyses,!total!protein!extracts! (30!µg)!were!subjected!to!15%!SDSIPAGE,!and!then!transferred!to!nitrocellulose! membranes! (BioIRad,! 162I0115).! AntiICrATG8! (PerezIPerez! et! al.,! 2010)!and! secondary! antibodies! were! diluted! 1:2,500! and! 1:10,000,! respectively,! in! phosphateIbuffered!saline!containing!0.1%!Tween!20!(Applichem,!A4974)!and!5%! milk! powder.! The! ECLIPlus! immunoblotting! detection! system! (GE! Healthcare,! RPN2132)!was!used!to!detect!the!proteins!with!horseradish!peroxidaseI conjugated!antiIrabbit!secondary!antibodies!(SigmaIAldrich,!A6154).!AntiIFKBP12! antibody!was!diluted!1:3,000!and!used!as!loading!control!as!described!previously! (Crespo!et!al.,!2005).!Proteins!were!quantified!with!the!Coomassie!Brilliant!Blue! dyeIbinding!method!as!described!by!the!manufacturer!(BioIRad,!500I0006).! ! ROS&determination& The! analysis! of! ROS! was! performed! as! previously! described!(PerezIPerez! et! al.,! 2012a).!Total! protein!extracts! from! Chlamydomonas!cells! growing!exponentially! were!obtained!as!described!above.!200I400!µg!of!total!protein!were!used!for!ROS! quantification.! Each! measurement! was! performed! on! two! equal! aliquots,! one! of! ! 32! protein,!DAD1,!whose!defect!causes!apoptotic!cell!death!in!hamster!BHK21! cells.!Mol!Cell!Biol!13:!6367I6374! Nakatogawa&H,&Suzuki&K,&Kamada&Y,&Ohsumi&Y!(2009)!Dynamics!and!diversity! in! autophagy! mechanisms:! lessons! from! yeast.! Nat! Rev! Mol! Cell! Biol! 10:! 458I467! Ogata& M,& Hino& S,& Saito& A,& Morikawa& K,& Kondo& S,& Kanemoto& S,& Murakami& T,& Taniguchi&M,&Tanii&I,&Yoshinaga&K,&Shiosaka&S,&Hammarback&JA,&Urano& F,& Imaizumi& K! (2006)! Autophagy! is! activated! for! cell! survival! after! endoplasmic!reticulum!stress.!Mol!Cell!Biol!26:!9220I9231! Ozgur&R,&Turkan&I,&Uzilday&B,&Sekmen&AH!(2014)!Endoplasmic!reticulum!stress! triggers! ROS! signalling,! changes! the! redox! state,! and! regulates! the! antioxidant!defence!of!Arabidopsis!thaliana.!J!Exp!Bot!65:!1377I1390! PerezEPerez& ME,& Couso& I,& Crespo& JL! (2012a)! Carotenoid! deficiency! triggers! autophagy!in!the!model!green!alga!Chlamydomonas!reinhardtii.!Autophagy! 8:!376I388! PerezEPerez&ME,&Florencio&FJ,&Crespo&JL!(2010)!Inhibition!of!target!of!rapamycin! signaling! and! stress! activate! autophagy! in! Chlamydomonas! reinhardtii.! Plant!Physiol!152:!1874I1888! PerezEPerez& ME,& Lemaire& SD,& Crespo& JL!(2012b)!Reactive!oxygen!species!and! autophagy!in!plants!and!algae.!Plant!Physiol!160:!156I164! Pollard& MG,& Travers& KJ,& Weissman& JS!(1998)!Ero1p:!a!novel!and!ubiquitous! protein! with! an! essential! role! in! oxidative! protein! folding! in! the! endoplasmic!reticulum.!Mol!Cell!1:!171I182! Pootakham& W,& GonzalezEBallester& D,& Grossman& AR!(2010)!Identification!and! regulation! of! plasma! membrane! sulfate! transporters! in! Chlamydomonas.! Plant!Physiol!153:!1653I1668! Queval&G,&Noctor&G!(2007)!A!plate!reader!method!for!the!measurement!of!NAD,! NADP,! glutathione,! and! ascorbate! in! tissue! extracts:! Application! to! redox! profiling!during!Arabidopsis!rosette!development.!Anal!Biochem!363:!58I 69! Rhee& SG,& Chang& TS,& Jeong& W,& Kang& D!(2010)!Methods!for!detection!and! measurement! of! hydrogen! peroxide! inside! and! outside! of! cells.! Mol! Cells! 29:!539I549! Rutkowski& DT,& Kaufman& RJ! (2007)! That! which! does! not! kill! me! makes! me! stronger:!adapting!to!chronic!ER!stress.!Trends!Biochem!Sci!32:!469I476! Santos& CX,& Tanaka& LY,& Wosniak& J,& Laurindo& FR! (2009)! Mechanisms! and! implications! of! reactive! oxygen! species! generation! during! the! unfolded! protein! response:! roles! of! endoplasmic! reticulum! oxidoreductases,! mitochondrial! electron! transport,! and! NADPH! oxidase.! Antioxid! Redox! Signal!11:!2409I2427! ScherzEShouval&R,&Elazar&Z!(2011)!Regulation!of!autophagy!by!ROS:!physiology! and!pathology.!Trends!Biochem!Sci!36:!30I38! Schroda& M! (2004)! The! Chlamydomonas! genome! reveals! its! secrets:! chaperone! genes! and! the! potential! roles! of! their! gene! products! in! the! chloroplast.! Photosynth!Res!82:!221I240! Szumiel&I!(2011)!Autophagy,!reactive!oxygen!species!and!the!fate!of!mammalian! cells.!Free!Radic!Res!45:!253I265! Thompson&AR,&Vierstra&RD!(2005)!Autophagic!recycling:!lessons!from!yeast!help! define!the!process!in!plants.!Curr!Opin!Plant!Biol!8:!165I173! ! 33! Tietze& F! (1969)! Enzymic! method! for! quantitative! determination! of! nanogram! amounts! of! total! and! oxidized! glutathione:! applications! to! mammalian! blood!and!other!tissues.!Anal!Biochem!27:!502I522! Tu&BP,&Weissman&JS!(2004)!Oxidative!protein!folding!in!eukaryotes:!mechanisms! and!consequences.!J!Cell!Biol!164:!341I346! Urano&F,&Wang&X,&Bertolotti&A,&Zhang&Y,&Chung&P,&Harding&HP,&Ron&D!(2000)! Coupling! of! stress! in! the! ER! to! activation! of! JNK! protein! kinases! by! transmembrane!protein!kinase!IRE1.!Science!287:!664I666! Walter&P,&Ron&D!(2011)!The!unfolded!protein!response:!from!stress!pathway!to! homeostatic!regulation.!Science!334:!1081I1086! Xie& Z,& Klionsky& DJ! (2007)! Autophagosome! formation:! core! machinery! and! adaptations.!Nat!Cell!Biol!9:!1102I1109! Yorimitsu&T,&Nair&U,&Yang&Z,&Klionsky&DJ!(2006)!Endoplasmic!reticulum!stress! triggers!autophagy.!J!Biol!Chem!281:!30299I30304! & ! 34! FIGURE&LEGENDS! Figure&1.!Tunicamycin!triggers!autophagy!and!ER!stress!in!Chlamydomonas.!Log! phase!cells!grown!in!TAP! medium!were!treated!with!5!µg/ml!tunicamycin!(tun)! and! samples! were! taken! at! the! indicated! times! and! processed! for! expression! analysis!of!ATG8+(A),!CAL2,!ERO1,!PDI6!and!Rb60!(C)!by!qPCR!or!western!blot!(B).! Thirty!micrograms!of!total!extracts!were!resolved!by!15%!SDSIPAGE!followed!by! western!blotting!with!antiIATG8!and!antiIFKBP12!antibodies.!Values!are!means!of! three!independent!experiments.! Figure&2.!Tunicamycin!triggers!oxidative!stress.!(A),!Log!phase!cells!grown!in!TAP! medium!were!treated!with!5!µg/ml!tunicamycin!(tun)!and!samples!were!taken!at! different! times! for! determination! of! ROS! levels! as! described! in! Materials! and! Methods.! (B),! Expression! analysis! of! GSTS1!and! GPXH!genes!by! qPCR! in! cells! treated! with! 5! µg/ml! tunicamycin! at! the! indicated! times.! (C),! The! ratio! of! intracellular! pools! of! reduced! (GSH)! and! oxidized! (GSSG)! glutathione! was! determined!in!cells!treated!with!5!µg/ml!tunicamycin.!Values!are!means!of!three! independent!experiments.!“*”,!Differences!were!significant!at!P<0.05!according!to! Student’s!tItest.! ! Figure& 3.!DTT! triggers! autophagy! and! ER! stress! in! Chlamydomonas.! Log! phase! cells! grown! in! TAP! medium! were! treated! with! 2.5! mM! DTT! and! samples! were! taken!at!the!indicated!times!for!qPCR!(A),!western!blot!(B)!or!immunofluorescence! (C)!analysis!of!ATG8.!Immunofluorescence!images!correspond!to!6!h!of!treatment.! In!addition!to!ATG8,!expression!of!the!UPRIregulated!genes!CAL2,!ERO1,!PDI6!and! ! 35! Rb60!(D)!or!the!oxidative! stressIrelated! genes! GSTS1!and!GPXH!genes!(E)! was! analyzed!by!qPCR.!Values!are!means!of!three!independent!experiments.! ! Figure& 4.! GSH! partially! prevents! ER! stressIinduced! autophagy.! Log! phase! cells! grown!in!TAP!medium!were!treated!with!2.5!mM!DTT!(A)!or!5!µg/ml!tunicamycin! (tun)!(B)!alone!or!in!combination!with!10!mM!GSH.!After!8!h!of!treatment,!cells! were!processed!for!expression!analysis!of!ATG8!and!PDI6!genes!by!qPCR.!Values! are!means!of!three!independent!experiments.!“*”,!Differences!were!significant!at! P<0.05! according! to! Student’s! tItest! between! DTT! and! DTT/GSH! or! tun! and! tun/GSH.!(C,!D),!Western!blot!analysis!of!ATG8!and!FKBP12!proteins!in!log!phase! cells! treated! with! 5! µg/ml! tunicamycin! (tun),! 5I10! mM! GSH! or! 2! mM! ascorbate! (asc)! or! combinations! of! these! compounds.! Thirty! micrograms! of! total! extracts! were!resolved!by!15%!SDSIPAGE!followed!by!western!blotting!with!antiICrATG8! and!antiICrFKBP12!antibodies.! ! Figure& 5.! GSH! decreases! tunicamycinIinduced! cellular! accumulation! of! ATG8! in! Chlamydomonas.! (A),! Immunolocalization! of! ATG8! in! Chlamydomonas! cells! treated!for!8!h!with!5!µg/ml!tunicamycin!(tun)!alone!or!in!combination!with!10! mM! GSH.! Scale! bar,! 5! µm.! (B),! Quantification! of! the! immunofluorescence! signal! detected! in! individual! cells! from! the! experiment! described! in! (A).! For! each! condition,!a!minimum!of!100!individual!cells!was!analyzed!using!ImageJ!software.! “**”!Differences!were! significant!at!P<0.01! according! to!Student’s! tItest!between! tun!and!tun/GSH.! ! 36! ! Figure& 6.! GSH! reduces! the! toxicity! of! tunicamycin! in! Chlamydomonas.! (A)! Log! phase!cells!grown!in!TAP!medium!were!treated!with!tunicamycin!(5!µg/ml;!tun),! GSH! (10! mM)! or! both! compounds! in! combination.! Untreated! cells! were! used! as! control.!Cell!viability!was!determined!by!Evans!blue!staining!at!the!indicated!times.! Results! are! means! of! four! independent! experiments.! (B)! Cells! were! subjected! to! 10Ifold! serial! dilutions! and! spotted! onto! TAP! plates! containing! the! indicated! concentrations! of! tunicamycin! (tun)! and! GSH.! Plates! were! grown! at! 25°C! under! continuous!illumination!for! 5!days.!(C)!Expression!analysis!of!the!DAD1!gene!by! qPCR.! Log! phase! cells! were! treated! with! 5! µg/ml! tunicamycin! (tun)! alone! or! in! combination!with!10!mM!GSH!for!48!hours!and!then!processed!for!RNA!isolation! and!analysis.!Values!are!means!of!three!independent!experiments.!“**”!Differences! were!significant!at!P<0.01!according!to!Student’s!tItest!between!tun!and!tun/GSH.! ! Figure&7.!The!sor1!mutant!displays!increased!autophagy!in!response!to!ER!stress.! (A)!Log!phase!cw15!and!sor1!cells!were!treated!with!tunicamycin!(5!µg/ml)!and!at! the!indicated!times!samples!were!taken!and!processed!for!western!blot!analysis.! (B)!Expression!analysis!of!ATG8!and!GSTS1!genes!by!qPCR.!Log!phase!cw15!and! sor1!cells!were!treated!with!tunicamycin!(5!µg/ml)!for!6!hours!and!subsequently! subjected! to! RNA! isolation! and! analysis.! Values! are! means! of! three!independent! experiments.! ! ! 37! Figure& 8.! Control! of! autophagy! by! ER! stress! and! ERIderived! ROS! in! Chlamydomonas.! Tunicamycin! (tun)! treatment! results! in! toxic! accumulation!of! unfolded! proteins! in! the! ER,! leading!to!autophagy!activation!as!a!defensive! mechanism.! ER! stress! also! increases!ROS!production!in!the!ER!via!de! oxidoreductase! ERO1,! which! contributes!to!the!upregulation! of! autophagy.! The! antioxidant! properties!of!GSH!counterbalance! ROS! signaling! to! the! autophagic! machinery! but! do! not! abrogate!ER!stress,! resulting! in! partial! inactivation! of! autophagy.! ! 0 2 4 6 8 10 12 14 16 control 02 46246 tun ATG8 012461246 control tun ATG8 FKBP12 1 2 3 4 5CAL2 2 4 6 8 10 12 14 ERO1 10 20 30 40 50 0 1 2 3 4 5 6 7 8 control 0246246 tun PDI6 Rb60 A B C Relative mRNA Relative mRNARelative mRNARelative mRNARelative mRNA time (h) time (h) time (h) Figure 1. Tunicamycin triggers autophagy and ER stress in Chlamydomonas. Log phase cells grown in TAP medium were treated with 5 μg/ml tunicamycin (tun) and samples were taken at the indicated times and processed for expression analysis of ATG8 (A), CAL2, ERO1, PDI6 and Rb60 (C) by qPCR or western blot (B). Thirty micrograms of total extracts were resolved by 15% SDSPAGE followed by western blotting with anti-ATG8 and anti-FKBP12 antibodies. Values are means of three independent experiments. control 0468 tun 0468 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 ROS (AU) control 02 46246 tun 2 4 6 8 10 12 Relative mRNA GSTS1 AB C time (h) time (h) 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 Relative mRNA GPXH 1 2 3 4 5 0control tun GSH/GSSG ratio 6* Figure 2. Tunicamycin triggers oxidative stress. (A), Log phase cells grown in TAP medium were treated with 5 μg/ml tunicamycin (tun) and samples were taken at different times for determination of ROS levels as described in Materials and Methods. (B), Expression analysis of GSTS1 and GPXH genes by qPCR in cells treated with 5 μg/ml tunicamycin at the indicated times. (C), The ratio of intracellular pools of reduced (GSH) and oxidized (GSSG) glutathione was determined in cells treated with 5 μg/ml tunicamycin. Values are means of three independent experiments. “*”, Differences were significant at P<0.05 according to Student’s t-test. 10 20 30 40 50 control 0246246 DTT ATG8 Relative mRNA 012461246 control DTT ATG8 FKBP12 time (h) control DTT tun ATG8 Nomarski 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 GSTS1 1 2 3 4 5 6 7GPXH control 0246246 DTT Relative mRNA Relative mRNA 0,5 1,0 1,5 2,0 2,5 3,0 3,5 CAL2 2 4 6 8 10 12 ERO1 10 20 30 40 50 PDI6 1 2 3 4 control 0246246 DTT Relative mRNARelative mRNARelative mRNARelative mRNA Rb60 AB C E D time (h) time (h) time (h) Figure 3. DTT triggers autophagy and ER stress in Chlamydomonas. Log phase cells grown in TAP medium were treated with 2.5 mM DTT and samples were taken at the indicated times for qPCR (A), western blot (B) or immunofluorescence (C) analysis of ATG8. Immunofluorescence images correspond to 6 h of treatment. In addition to ATG8, expression of the UPR-regulated genes CAL2, ERO1, PDI6 and Rb60 (D) or the oxidative stress-related genes GSTS1 and GPXH genes (E) was analyzed by qPCR. Values are means of three independent experiments. 5 10 15 20 25 30 35 40 2 4 6 8 10 12 14 16 18 20 control DTT DTT GSH GSH Relative mRNARelative mRNA PDI6 ATG8 2 4 6 8 10 12 14 16 18 20 5 10 15 20 25 control tun tun GSH GSH Relative mRNARelative mRNA PDI6 ATG8 C tun tun GSH tun asc GSH asc ATG8 FKBP12 tun GSH (mM) - - +++ -510 -- 510 ATG8 FKBP12 AB CD * * * Figure 4. GSH partially prevents ER stress-induced autophagy. Log phase cells grown in TAP medium were treated with 2.5 mM DTT (A) or 5 μg/ml tunicamycin (tun) (B) alone or in combination with 10 mM GSH. After 8 h of treatment, cells were processed for expression analysis of ATG8 and PDI6 genes by qPCR. Values are means of three independent experiments. “*”, Differences were significant at P<0.05 according to Student’s t-test between DTT and DTT/GSH or tun and tun/GSH. (C, D), Western blot analysis of ATG8 and FKBP12 proteins in log phase cells treated with 5 μg/ml tunicamycin (tun), 5-10 mM GSH or 2 mM ascorbate (asc) or combinations of these compounds. Thirty micrograms of total extracts were resolved by 15% SDS-PAGE followed by western blotting with anti-CrATG8 and anti- CrFKBP12 antibodies.