Control of Autophagy in Chlamydomonas Is …...Control of Autophagy inChlamydomonas Is Mediated through Redox-Dependent Inactivation of the ATG4 Protease1 María Esther Pérez-Pérez*,

Control of Autophagy in Chlamydomonas Is Mediatedthrough Redox-Dependent Inactivation of theATG4 Protease1

María Esther Pérez-Pérez*, Stéphane D. Lemaire, and José L. Crespo

Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas-Universidad deSevilla, Sevilla, Spain (M.E.P.-P., J.L.C.); and Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR8226,Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique,75005 Paris, France (S.D.L.)

ORCID IDs: 0000-0003-0779-6665 (M.E.P.-P.); 0000-0003-3514-1025 (J.L.C.).

Autophagy is a major catabolic pathway by which eukaryotic cells deliver unnecessary or damaged cytoplasmic material to thevacuole for its degradation and recycling in order to maintain cellular homeostasis. Control of autophagy has been associated withthe production of reactive oxygen species in several organisms, including plants and algae, but the precise regulatory molecularmechanisms remain unclear. Here, we show that the ATG4 protease, an essential protein for autophagosome biogenesis, plays acentral role for the redox regulation of autophagy in the model green alga Chlamydomonas reinhardtii. Our results indicate that theactivity of C. reinhardtii ATG4 is regulated by the formation of a single disulfide bond with a low redox potential that can beefficiently reduced by the NADPH/thioredoxin system. Moreover, we found that treatment of C. reinhardtii cells with norflurazon,an inhibitor of carotenoid biosynthesis that generates reactive oxygen species and triggers autophagy in this alga, promotes theoxidation and aggregation of ATG4. We propose that the activity of the ATG4 protease is finely regulated by the intracellular redoxstate, and it is inhibited under stress conditions to ensure lipidation of ATG8 and thus autophagy progression in C. reinhardtii.

Macroautophagy (hereafter termed autophagy) is acatabolic process by which eukaryotic cells degradeand recycle intracellular material, including proteins,membranes, ribosomes, and entire organelles, in re-sponse to stress or for proper differentiation and de-velopment. During autophagy, cytosolic componentsare engulfed in bulk within a double-membrane vesicleknown as the autophagosome and delivered to thevacuole/lysosome for degradation to recycle needednutrients or degrade toxic or damaged components (Heand Klionsky, 2009; Mizushima et al., 2011; Liu andBassham, 2012). Therefore, autophagy plays a funda-mental role in cellular homeostasis and the response tostress. The degradation of intracellular components viaautophagy can be selective or nonselective dependingon the nature of cargo that is targeted to the vacuole,and several types of selective autophagy have been

reported such as mitophagy, pexophagy, or chloro-phagy (Floyd et al., 2012; Schreiber and Peter, 2014;Oku and Sakai, 2016; Young and Bartel, 2016).

Autophagy is highly conserved through evolution.The autophagy process is mediated by autophagy-related (ATG) genes, which were originally identified inyeasts (Tsukada and Ohsumi, 1993) and subsequentlyin other eukaryotes, including animals, plants, and al-gae (Bassham et al., 2006; Díaz-Troya et al., 2008b;Avin-Wittenberg et al., 2012; Shemi et al., 2015). Amongthese ATG genes, a subgroup of around 18 genes isrequired for autophagosome formation and constitutesthe autophagy core machinery in most eukaryotes.Core ATG proteins include the ATG8 and ATG12ubiquitin-like conjugation systems that catalyze thecovalent binding of the ubiquitin-like protein ATG8 tothe phospholipid phosphatidylethanolamine (PE), anessential event in the formation of the autophagosome(Mizushima et al., 2011; Feng et al., 2014). The conju-gation of ATG8 to PE is catalyzed by the sequentialaction of ATG4 (a Cys protease), ATG7 (E1-activatingenzyme), ATG3 (E2-activating enzyme), and the ATG12-ATG5-ATG16 complex (E3-like ligase). First, the ATG4protease cleaves nascent ATG8 at a conserved Gly residuelocated at theC terminus of the protein. ProcessedATG8 isthen activated by the E1-like enzyme ATG7, transferred tothe E2-like enzyme ATG3, and finally conjugated to PE atthe exposedGly in a reaction that requires the participationof the E3-like ligase ATG12-ATG5-ATG16 (Mizushimaet al., 2011; Feng et al., 2014). ATG8 is also recycled from

1 This work was supported in part by Ministerio de Economía yCompetitividad grants BFU2012-35913 and BFU2015-68216-P andJunta de Andalucía CVI-7336 (to J.L.C.), and Agencie Nationale dela Recherche grants 12-BSV5-0019 REDPRO2 and LABEX DYNAMOANR-11-LABX-0011 (to S.D.L.).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:María Esther Pérez-Pérez ([email protected]).

M.E.P.-P., S.D.L., and J.L.C. designed research, analyzed data, andwrote the manuscript; M.E.P.-P. performed the experiments.

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the autophagosome membrane by the delipidatingactivity of ATG4 that cleaves the amide bond betweenATG8 and PE (Kirisako et al., 2000). Hence, ATG4 canact both as a conjugating and deconjugating enzyme,and the fine regulation of these two activities is es-sential for the normal function of autophagy (Nairet al., 2012; Nakatogawa et al., 2012; Yu et al., 2012).

The autophagy machinery is well conserved in theplant kingdom, although some differences have beenreported between plants and algae. Higher plants suchasArabidopsis (Arabidopsis thaliana),maize (Zeamays), orrice (Oryza sativa) evolved either small or large familiesfor many of the core ATG genes (Bassham et al., 2006;Chung et al., 2009; Avin-Wittenberg et al., 2012) likelyallowing specialized functions. However, all predictedATG genes appear to be in single copy in the genome ofseveral algal species, including the model green algaChlamydomonas reinhardtii (Díaz-Troya et al., 2008b;Avin-Wittenberg et al., 2012; Pérez-Pérez and Crespo,2014; Shemi et al., 2015). The availability of plant mu-tant collections and established techniques for gene si-lencing has been fundamental to understand the role ofautophagy in the plant response to stress. The analysis ofArabidopsis mutants defective in core autophagy genessuch as ATG3, ATG4, ATG5, ATG7, or ATG10 revealedthat autophagy plays a central role in the plant responseto nutrient (nitrogen or carbon) starvation, different abi-otic stresses, and pathogen infection (Li and Vierstra,2012; Liu and Bassham, 2012; Avila-Ospina et al., 2014;Minina et al., 2014; Michaeli et al., 2016). Moreover, thiscatabolic process has been involved in the selective deg-radation of chloroplasts andperoxisomes in plants (Ishidaet al., 2008; Farmer et al., 2013; Kim et al., 2013; Shibataet al., 2013). In algae, it has been reported that autophagyis activated in response to nitrogen or carbon limitation,oxidative stress, metal toxicity, endoplasmic reticulum(ER) stress, or virus infection (Pérez-Pérez et al., 2010,2012; Davey et al., 2014; Goodenough et al., 2014; Pérez-Martin et al., 2014, 2015; Schatz et al., 2014).

Control of autophagy has been associated with theproduction of reactive oxygen species (ROS) in severalsystems. Upon starvation, human cells generateROS, specifically H2O2, that seem to be essential forautophagosome biogenesis and autophagic degradation(Scherz-Shouval et al., 2007). In the model yeast Saccha-romyces cerevisiae, autophagy has also been linked withthe cellular redox state. Incubation of yeast cells with theROS inducer H2O2 triggers autophagy, whereas activa-tion of autophagy by the TOR inhibitor rapamycin resultsin a pronounced increase of the total glutathione (reducedglutathione [GSH] + oxidized glutathione [GSSG]) pool, amarker of redox unbalance (Pérez-Pérez et al., 2014).In Arabidopsis, it has been demonstrated that oxida-tive damage caused by the ROS generators methylviologen and H2O2 triggers autophagy, and accord-ingly plants defective in autophagy show enhancedsensitivity to oxidative stress (Xiong et al., 2007). In thesame line, a strong correlation between the activationof autophagy and the accumulation of ROS in carotenoid-depleted cells or in response to methyl viologen or H2O2

treatment has been shown in C. reinhardtii (Pérez-Pérezet al., 2010; Perez-Perez et al., 2012).

Despite the extensive experimental evidence con-necting autophagy and redox signaling, the precisemolecular mechanisms by which ROS control this cat-abolic process are largely unknown. At present, ATG4is the only ATG protein whose activity has been pro-posed to be redox regulated. The proteolytic activity ofhuman ATG4A and ATG4B is reversibly inhibited byoxidation in a process that involves Cys-81, which mayinterfere with the activity of the nearby catalytic Cys-77(Scherz-Shouval et al., 2007). The activity of the yeastATG4 protease is also subject to redox regulation, al-though the underlying molecular mechanism differsfrom the one described in humans, because Cys-81 isnot conserved in yeasts or other eukaryotes such asplants and algae (Pérez-Pérez et al., 2014). Indeed, wehave recently demonstrated that yeast ATG4 is regu-lated by oxidoreduction of a single disulfide bond be-tween Cys-338 and Cys-394. Mutation of any of thesetwo cysteines resulted in loss of redox regulation ofATG4, and the mutant proteins displayed constitutiveactivation (Pérez-Pérez et al., 2014). Moreover, thesmall oxidoreductase thioredoxin (Trx) was identifiedin this study as a regulator of ATG4 activity based on itsability to efficiently reduce the regulatory disulfidebond in this protease (Pérez-Pérez et al., 2014). Inplants, it has been shown that the activity of Arabi-dopsis ATG4a and ATG4b is reversibly inhibited byoxidation (Woo et al., 2014), although the underlyingmolecular mechanism has not been reported.

In this study, we unraveled the molecular mechanismthat mediates the redox regulation of C. reinhardtiiATG4.This regulation controls oxidative inactivation of ATG4under conditions that promote autophagy inC. reinhardtii.

RESULTS

Identification of ATG4 in C. reinhardtii

We have previously reported that a single ATG4 geneis conserved in the nuclear genome of C. reinhardtii andthat anATG8-proteolytic activity can be detected in totalextracts from C. reinhardtii (Pérez-Pérez et al., 2010).According to the most up-to-date annotated version ofthe C. reinhardtii genome at the Joint Genome Institute(genemodel version 5.5 at the Phytozome 10.3 database),the C. reinhardtii ATG4 gene (Cre12.g510100.t1.1) codesfor a protein of 640 amino acids that contains two pep-tidase domains highly conserved among ATG4 proteins(Sugawara et al., 2005; Fig. 1A). The predicted size of C.reinhardtii ATG4 (approximately 65 kD) is significantlyhigher compared toATG4proteins fromother organisms,including algae, plants, yeasts, or mammals. Amino acidsequence comparison of ATG4 proteins revealed a lowidentity in this family of proteases, with Volvox carteribeing the closest ortholog to C. reinhardtii ATG4(Supplemental Fig. S1). Phylogenetic analysis of ATG4proteins also showed that ATG4 homologs comprisethree main branches: fungi, metazoans, and the green

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lineage, which includes green algae in a separate cladefrom land plants (Supplemental Fig. S2).To characterize the ATG4 protein from C. reinhardtii,

an in vitro assay was established using recombinantpurified ATG4 and the ATG8 protein from C. reinhardtiias substrate. The endoproteolytic activity of ATG4 canbe monitored by SDS-PAGE based on the different mo-bility of processed and unprocessed ATG8, sinceC. reinhardtiiATG8 contains a 15-amino acid extension atthe C terminus of the protein that is cleaved by ATG4(Kirisako et al., 2000; Pérez-Pérez et al., 2010). Our resultsindicated that ATG4 efficiently processes the C terminusof ATG8within 15 to 30min using a low concentration ofthe protease in the assay (1 mM; Fig. 1, B–E). To confirmthatATG8processing occurs at the highly conservedGlyresidue that is recognized by ATG4 (Kirisako et al.,2000), we performed a proteolytic assay with a mutant

version of ATG8 where Gly-120 has been replaced byAla (ATG8G120A) or a truncated form lacking the last15 amino acids (ATG8G120). As predicted, C. reinhardtiiATG4 cleaves ATG8 at the conserved Gly-120 (Fig. 1F).

C. reinhardtii ATG4 Activity Is Redox Regulated andDepends on a Single Disulfide Bond with a Very LowRedox Potential

It has been shown that the proteolytic activity ofhuman and yeast ATG4 proteins is regulated by thereduction of a regulatory Cys adjacent to the catalyticCys or the formation of a disulfide bond, respectively(Scherz-Shouval et al., 2007; Pérez-Pérez et al., 2014). Toinvestigate whether the activity ofC. reinhardtiiATG4 issubject to redox regulation, we performed an in-depthanalysis of this protein under different redox conditions.

Figure 1. C. reinhardtii ATG4 cleaves ATG8 at the conserved Gly Gly-120. A, Domain structure of the ATG4 protease fromC. reinhardtii. Twopeptidase domains comprising residues frompositions 49-182 and 437-590, respectively,were identified using theConserved Domains search tool at the NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). All cysteines present inC. reinhardtii ATG4 are indicated as gray balls, including the catalytic (red) and two putative regulatory (green) cysteines. B,C. reinhardtii ATG4 (1 mM) activity was monitored by following the cleavage of recombinant C. reinhardtii ATG8 (5 mM) from theunprocessed (ATG8) to the processed (pATG8) form (indicated by arrowheads) along time and subsequent SDS-PAGE and CoomassieBrilliant Blue staining. C, Quantification of ATG4 activity from B. The reference sample for quantification was the one at 60 min and itwas considered as activity = 1 (in arbitrary units). D, Different concentrations of ATG4 were used to analyze C-terminal processing ofATG8 (5 mM) to pATG8 after 30 min. E, Quantification of ATG4 activity from D. The reference sample for quantification was the onewith the maximum ATG4 concentration (9 mM was considered as activity = 1, in arbitrary units). F, C. reinhardtii wild-type ATG8(ATG8WT) and themutant formsATG8G120A (Gly-120 is replacedbyAla) andATG8G120 (a truncated protein atGly-120 corresponding toa processed-like formandused as a control)were incubated in an assaymixture in the absenceor presence of theATG4protease for 1 h.

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Redox Control of C. reinhardtii ATG4



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First, ATG4 was incubated with increasing concentra-tions of reducing (dithiothreitol [DTT]) or oxidant (H2O2)agents and processing of ATG8 was analyzed. ATG4activity was enhanced with increasing concentrations ofDTT (Fig. 2, A and B), indicating that the protein is ac-tivated by reduction. By contrast, the activity of ATG4prereduced with a low concentration of DTT (100 mM)was inhibited by H2O2 in a concentration-dependentmanner (Fig. 2, C and D). Moreover, we found thatboth reduction and oxidation of C. reinhardtii ATG4 arefully reversible (Fig. 2E), strongly suggesting that theregulation of ATG4 activity is controlled by redoxposttranslational modifications. ATG4 proteins are Cysproteases whose activity is strictly dependent on a cat-alytic Cys that can be irreversibly blocked by alkylatingagents such as iodoacetamide (IAM).C. reinhardtiiATG4previously reduced and activated with low DTT wasirreversibly inactivated by IAM, demonstrating that thisprotein is a Cys protease (Fig. 2F). Next, to determine ifC. reinhardtii ATG4 activity depends on the redox po-tential, we analyzed ATG8 processing in the presence ofdifferent ratios of reduced and oxidized DTT (DTTred andDTTox, respectively; Fig. 2G). It was found that ATG4 isactivated at a DTTred/DTTox ratio between 1021 and 1022,suggesting that ATG4 activation requires a very lowredox potential. In addition, we observed that C. rein-hardtii ATG4 is activated by reduction with 1 mM DTT(Em,7 = 2330 mV) but not with GSH (Em,7 = 2240 mV)even at 5 mM (Fig. 2H), indicating again that a very lowredox potential is required for ATG4 activation (Em,7:midpoint redox potential at pH 7.0). Finally, we per-formed a complete DTT redox titration by varying theambient redox potential (Eh at pH 7.0) between 2232and 2350 mV. ATG4 was incubated at each potential,and the range of ATG4 activation was determined withthe ATG8 processing assay (Fig. 2I). Our data indicatedthat there is a strong correlation between ATG4 acti-vation and the redox potential. No significant activitywas detected when the Eh was higher than 2262 mV.The experimental data of this in-gel redox titration gavegood fits to the Nernst equation for the reduction of asingle 2-electron component, with a midpoint redoxpotential (Em), at pH 7.0, of 2278 mV (Fig. 2J). Thus,taken together, our data indicated that C. reinhardtiiATG4 activity is redox regulated and that themolecularmechanism of this redox regulation involves the oxi-doreduction of a single regulatory disulfide bondwith avery low midpoint redox potential.

The NADPH/NTR/Trx System Regulates C. reinhardtiiATG4 Activity

Trx are small oxidoreductases that are able to reducedisulfide bonds with very low redox potentials (Buchananand Balmer, 2005). To reduce a disulfide bond in a targetprotein, Trx has to be reduced first by the NADPH-dependent thioredoxin reductase (NTR), which takeselectrons from NAPDH (Jacquot et al., 1994). Trx canalso receive electrons from DTT, a classical electron

donor widely used in biochemical assays to test theability of Trx to reduce a target protein, since DTT isable to efficiently reduce Trx in vitro but reacts veryslowly with the disulfide bond of the target protein. Totest the ability of Trx to reduce and activate C. rein-hardtii ATG4, TRXh1 from C. reinhardtii, the majorcytosolic Trx (Lemaire and Miginiac-Maslow, 2004),was used in the ATG8 proteolytic assay. Our resultsindicated that TRXh1 efficiently activated the proteo-lytic activity of ATG4 in the presence of 10 mM DTT,whereas no ATG8 processing was detected in thepresence of DTT only or when TRXh1 was replaced bya mutant form of this protein where the catalytic Cysis replaced by Ser (TRXh1C39S; Fig. 3A). To furthercharacterize the role of Trx in ATG4 reduction, weanalyzed the time-course of ATG8 cleavage in thepresence of TRXh1 and a very low concentration ofDTT (10 mM). ATG4 was efficiently reduced and acti-vated by TRXh1 but not with DTT alone or in thepresence of TRXh1C39S (Fig. 3B). Nevertheless, thephysiological electron donor to TRXh1 is NTR ratherthan DTT, and therefore to investigate the regulationof C. reinhardtii ATG4 by TRXh1 we reconstituted thecomplete NADPH/NTR/Trx system using NTRBfrom Arabidopsis as electron donor for TRXh1. Weobserved that in the presence of the complete system,ATG4 is able to efficiently process ATG8, whereas nocleavage occurred when the reducing system was notcomplete or the mutant TRXh1C39S was used (Fig. 3C).Therefore, our results strongly suggest a role ofthe NADPH/NTR/Trx system in the regulation ofC. reinhardtii ATG4 by the oxidoreduction of a regula-tory disulfide bond.

Cys-400 Plays a Key Role in the Redox Control of C.reinhardtii ATG4

All our results indicate that a single disulfide bondmay play a key role for the redox regulation of C.reinhardtii ATG4, as recently described for yeast ATG4(Pérez-Pérez et al., 2014). Cys-338 and Cys-394 havebeen shown to establish a regulatory disulfide bond inyeast ATG4, and mutation of any of these residues re-sults in constitutive activation of the protein (Pérez-Pérez et al., 2014). Cys-338 is widely conserved amongATG4 proteins, whereas Cys-394 seems to be restrictedto some ascomycete species (Pérez-Pérez et al., 2014).Multiple sequence alignments revealed that the tworegulatory cysteines identified in yeast ATG4 might beconserved in C. reinhardtii as Cys-400 and Cys-473, re-spectively (Supplemental Fig. S3). To test this hypoth-esis, Cys-400 and Cys-473 from C. reinhardtii ATG4were mutated to Ser, and the corresponding recombi-nant proteins were produced and purified. We alsoidentified Cys-84 as the catalytic Cys of C. reinhardtiiATG4. This Cys is the only one strictly conserved in allorganisms (Supplemental Fig. S3). As expected, a mu-tant having this Cys replaced by Ser was constitutivelyinactive and was used as a negative control in these








Figure 2. The protease activity of C. reinhardtii ATG4 is redox regulated. A, Effect of DTTon ATG4 activity. ATG4 was incubatedwith ATG8 for 1 h in the presence of DTT concentrations ranging from 25mM to 1mM. Control (lanes 1 and 2): incubation with noaddition. Lane 1 does not contain ATG4. B, Quantification of ATG4 activity from A. The highest DTT concentration sample wasused as reference sample for quantification (activity = 1, in arbitrary units). C, Effect of H2O2 on the activity of prereduced ATG4.After pretreatment with 100 mM DTT for 30 min, ATG4 was treated with increasing H2O2 concentrations for 20 min and thenincubated with ATG8 for 1 h. Control (lanes 1 and 2): incubation with no addition. Lane 1 does not contain ATG4. D, Quanti-fication of ATG4 activity from C. The prereduced but not treated with H2O2 sample was used as reference for quantification(activity = 1, in arbitrary units). E, Inhibition of ATG4 activity by H2O2 is reversed by DTT. ATG4 protein was pretreated with100 mM DTT for 30 min (lane 2); then, ATG4 was incubated with 500 mM H2O2 for 20 min (lane 3); finally, ATG4 was newlytreated with 1 mM DTT for 30 min (lane 4). Control (lane 1): incubation with no addition. For all lanes, ATG8 was added to thereactionmixture for 1 h after each specific treatment to assess ATG4 activity. F, ATG4 is irreversibly inactivated by the alkylatingagent IAM. ATG4 was pretreated with 100 mM DTT for 30 min (lane 2), then incubated with 20 mM IAM for 30 min (lane 3);next, ATG4 was again incubated with 50 mM DTT for 30 min (lane 4). Control (lane 1): incubation with no addition. For alllanes, ATG8 was added to the reaction mixture for 1 h after each specific treatment to study ATG4 activity. G, ATG4 activity is






experiments (Fig. 4). The ATG8 cleavage activity of allATG4 variants was analyzed in the presence of a lowconcentration of DTT with or without TRXh1. Asmentioned above, wild-type ATG4 was activated byreduction with TRXh1 but not by low DTT. As ex-pected, the mutant lacking the catalytic Cys (ATG4C84S)was inactive in all tested conditions (Fig. 4). In contrast,the ATG4C400S mutant was readily active even in the

absence of TRXh1 (Fig. 4), indicating that Cys-400 isrequired for the redox control of C. reinhardtii ATG4.However, mutation of Cys-473 had no effect on theproteolytic activity of the protein, strongly suggestingthat this Cys does not participate in the redox regula-tion of C. reinhardtii ATG4 (Fig. 4). Nevertheless, thebiochemical study performed on C. reinhardtii ATG4demonstrated that the activity of this protein is regulated

Figure 2. (Continued.)dependent on the DTTred/DTTox ratio. ATG4 processing activity wasmonitored after incubation during 2 h in the presence of variousDTTred/DTTox ratios (1/1, 1/10, 1/100, 10/1, 100/1 in mM). H, ATG4 is not activated by GSH. ATG4 activity was determined afterincubation for 1 h in the presence of 0.5, 1, or 5 mM GSH (lanes 2, 3, and 4, respectively), 1 mM DTT (lane 5), or in the absence ofreducing agent (lane 1). I, Redox titration of ATG4 activity. ATG4 activitywas analyzed after incubation for 2 h at indicated Eh poisedby 20 mM DTT in various dithiol/disulfide ratios. ATG8 was added to the reaction mixture for 15 min after DTT treatment in allsamples. The -∞ sample was used as reference for quantification. J, ATG4 activities monitored as in I were interpolated by nonlinearregression of the data using a Nernst equation for 2 electrons exchanged (n = 2) and one redox component. The average midpointredox potential (Em,7) of three independent experiments is reported in the figure as mean 6 SD.

Figure 3. C. reinhardtii ATG4 is activated by the NADPH/NTR/TRX system. A, ATG4 activity was measured after incubation for30 min in the presence (+) or absence (2) of 10 mM DTT, 5 mM TRXh1, and 5 mM TRXh1C39S as indicated. Proteins were visualizedby Coomassie Brilliant Blue staining. pATG8, processed ATG8. B, Kinetics of ATG8 processing by ATG4 in the presence of 10 mM

DTT, 5mM TRXh1, or 5mM TRXh1C39S. ATG4 activity was assayed as described in A. C, ATG4 activity was determined as describedin (A) but using NADPH and NTR as the physiological electron donor for TRX. ATG4 activity was analyzed after incubation for30 min in the presence (+) or absence (2) of 2 mM NADPH, 1 mM Arabidopsis NTRB, 5 mM TRXh1, and 5 mM TRXh1C39S as in-dicated. The ATG8 and pATG8 forms NTRB and TRXh1 are marked with arrowheads. D, Schematic representation of the pro-posed mechanism of activation of C. reinhardtii ATG4 through reduction by the NADPH/NTR/Trx system.






through the formation of a disulfide bond efficientlyreduced by thioredoxin, where Cys-400 plays an im-portant role.

Oligomerization of C. reinhardtii ATG4

In the course of the biochemical analysis ofC. reinhardtiiATG4,we observed that this protein is able to oligomerizein the absence of 2-mercaptoethanol, an efficient reducerof disulfide bonds (Fig. 5A). Moreover, we found that theoligomerization of ATG4 is also redox regulated, sinceincubation of this protein with increasing DTT concen-trations resulted in the monomerization of ATG4 (Fig.5B), indicating that DTT prevents the aggregation ofATG4. However, treatment of ATG4 with H2O2 stronglystimulated the oligomerization of the protein in a re-versible manner (Fig. 5, B and C). To further characterizethe oligomerization of C. reinhardtiiATG4, we performeda DTT redox titration by incubating the protein with dif-ferent concentrations of oxidized and reduced DTT. Ourresults revealed that there is a strong correlation betweenthe oligomerization of C. reinhardtii ATG4 and the redoxpotential (Fig. 5D). Next, we investigated whether the

oligomerization of C. reinhardtii ATG4 might be con-served in other ATG4 proteins. We recently demon-strated that the proteolytic activity of yeast ATG4 isalso controlled by the redox potential (Pérez-Pérezet al., 2014), although the study of the aggregation ofthis protein was not addressed. A detailed analysis ofyeast ATG4 revealed that, interestingly, this proteinshows an oligomerization pattern similar to the onedescribed in C. reinhardtii ATG4, including the redoxcontrol of this process (Supplemental Fig. S4). Thus,taken together, these findings indicate that the redoxregulation of ATG4 proteins may also involve theformation of aggregates in vitro.

Stress Conditions Promote the Oxidation and Aggregationof ATG4 in C. reinhardtii

To study the regulation of ATG4 in C. reinhardtii cells,we used an antibody raised against the recombinantprotein. This antibody was able to recognize lowamounts of purified C. reinhardtii ATG4 (Fig. 6A). Wealso tested the ability of this antibody to detect ATG4 intotal extracts from C. reinhardtii cells. A band with a sizecomparable to C. reinhardtii ATG4 was observed in totalextracts, although additional bands of lower and highersizes could also be detected (Supplemental Fig. S3). Thebands of lower size may likely correspond to degrada-tion products of ATG4, whereas those of higher sizemight be due to posttranslational modifications or ag-gregation of this protein in vivo as observed with puri-fiedATG4. To explore this hypothesis,we analyzed if theC. reinhardtiiATG4 antibody is able to detect monomericand oligomeric ATG4. Our results revealed that indeedthe antibody reacted with both forms of purified ATG4(Fig. 6A).

Next, we investigated whether the detection of olig-omeric ATG4 in C. reinhardtii total extracts may be re-lated with the function of this protein in vivo. To thisaim, ATG4was analyzed in C. reinhardtii cells subjectedto different stress conditions that activate autophagy inthis organism. First, ATG4 was examined in stationaryphase cells, since autophagy is up-regulated whenC. reinhardtii cells reach stationary growth (Pérez-Pérezet al., 2010). We found that the abundance of ATG4, inparticular oligomeric forms, increased in stationarycells (Fig. 6B). As previously reported (Pérez-Pérezet al., 2010), lipidated ATG8 could also be detected instationary cells (Fig. 6B). Second, we investigated theeffect of tunicamycin on ATG4, since this drug triggersER stress and autophagy in C. reinhardtii (Pérez-Pérezet al., 2010). Our results indicated that monomericand oligomeric ATG4 forms increased in tunicamycin-treated cells (Fig. 6C). Third, autophagy was alsoinduced in C. reinhardtii by treating cells with nor-flurazon, an inhibitor of the carotene biosynthesispathway (Sandmann and Albrecht, 1990) that activatesthis degradative process in C. reinhardtii as a result ofROS production and photo-oxidative damage of thecell (Pérez-Pérez et al., 2012). ATG8 lipidation was

Figure 4. TheCys-to-Sermutant protein ATG4C400S is not redox-regulated.ATG4 activity was monitored as described in Figure 3. A, The activity ofATG4WT, ATG4C84S, ATG4C400S, or ATG4C473S was determined afterincubation for 30 min in the presence (+) or absence (2) of 10 mM DTTand 5 mM TRXh1 as indicated. B, Quantification of ATG4 activity fromA. The reference sample for quantification was ATG4WT incubatedwith DTT and TRXh1 (activity = 1, in arbitrary units). The data arerepresented as mean 6 SD (n = 3). **, Differences were significant atP, 0.01 according to the Student’s t test between the ATG4WTand theindicated mutant. The ATG8 and pATG8 forms and TRXh1 are markedwith arrowheads.








detected in C. reinhardtii cells treatedwith norflurazon,as previously described (Pérez-Pérez et al., 2012; Fig.6D). Monomeric and oligomeric forms of ATG4 weredetected in cells with basal autophagy (Fig. 6D).However, activation of autophagy by norflurazonresulted in a pronounced detection of oligomericATG4 forms as well as an increase in the overallsignal of ATG4 (Fig. 6D). Up-regulation of autoph-agy in stationary cells also increased the abundanceof oligomeric ATG4 forms, although to a minor ex-tent compared to cells treated with norflurazon for48 h (Fig. 6D).

Finally, we also investigated the formation ofATG4 oligomers in the C. reinhardtii mutant lts1-204,which lacks phytoene synthase (McCarthy et al.,2004), one of the first enzymes in the carotenoid bio-synthetic pathway. Carotenoid-less lts1-204 cells grow inthe dark on acetate-containing medium and are un-able to survive in the light even under very low lightconditions (McCarthy et al., 2004). We have previ-ously shown that shifting lts1-204 cells from dark tolight results in a strong production of ROS and theactivation of autophagy (Pérez-Pérez et al., 2012).Thus, we analyzed the effect of light on ATG4 a-bundance and oligomerization in the lts1-204mutantstrain. In close agreement with the data obtained innorflurazon (NF)-treated cells, we found that bothATG4 abundance and oligomerization significantlyincreased in mutant cells exposed to light (Fig. 6E).As previously shown (Pérez-Pérez et al., 2012),ATG8 was also up-regulated in light-stressed lts1-204 cells (Fig. 6E). Taken together, our results indi-cate that stress conditions that generate ROS and

activate autophagy promote the oxidation and aggre-gation of ATG4 in C. reinhardtii.

ATG4 Activity Depends on ATG4 Reductionand Monomerization

We reasoned that the oxidation and aggregation ofATG4 observed in C. reinhardtii cells treated with NFmight be due to the high production of ROS that takesplace in these cells (Pérez-Pérez et al., 2012). To explorethis hypothesis, we treatedC. reinhardtii cells withNF inthe presence or absence of the ROS scavenger N-acetylCys (NAC). We observed that NAC largely preventedthe effect of NF on ATG4 (Fig. 7A). Remarkably, thepresence of NAC also decreased the lipidation of ATG8in cells treated with NF (Fig. 7A), again supporting therole of ROS in the activation of autophagy in these cells(Pérez-Pérez et al., 2012) through inhibition of ATG4.

To further investigate the formation of oligomericATG4, total extracts from stationary cells were incu-bated with DTT, and the aggregation of ATG4 wasanalyzed by western blot. Our results revealed thatoligomeric ATG4was converted into monomeric ATG4by reduction with DTT and demonstrated that oligo-merization of ATG4 is redox regulated in C. reinhardtii(Fig. 7B, top panel). Next, we investigated whetheraggregated ATG4may represent an inactive state of theprotein. To this aim, we assayed the ATG4 proteolyticactivity present in total extracts from C. reinhardtii cellsusing recombinant 6His-ATG8 as substrate as previ-ously described (Pérez-Pérez et al., 2010). Our resultsindicated that DTT stimulated the proteolytic activity of

Figure 5. Redox control of C. reinhardtii ATG4 oligomerization. A, C. reinhardtii recombinant ATG4 (5 mM) was subjectedto 12% SDS-PAGE in the presence (+) or absence (2) of b-mercaptoethanol (bME) and stained with Coomassie BrilliantBlue. mATG4, monomeric ATG4; oATG4, oligomeric ATG4. B, ATG4 (5 mM) was incubated with different DTT concen-trations ranging from 25 to 1,000 mM for 1 h and then resolved by SDS-PAGE. C, ATG4 (5 mM) was preincubated with 100 mM

DTT for 1 h (lane 2); then, ATG4 was treated with 500 mM H2O2 for 20 min (lane 3); and finally, ATG4 was incubated with1 mM DTT for 30 min (lane 4). Control (lane 1): incubation with no addition. D, Redox titration of ATG4 oligomerization/monomerization. ATG4 monomerization was analyzed after incubation for 2 h at indicated Eh poised by 20 mM DTT invarious dithiol/disulfide ratios. The oligomeric (oATG4) and monomeric (mATG4) ATG4 forms are marked by arrowheads.bME-free buffer was used in B to D.






ATG4 (Fig. 7B, bottom panel). Furthermore, we foundthat incubation with IAM resulted in ATG4 inactiva-tion and had no effect on the conformation of theprotein (Fig. 7, B and C), indicating that aggregated,

oxidized ATG4 is inactive. This finding was alsoconfirmed in vitro using purified ATG4 and ATG8proteins. DTT efficiently reduced and activated re-combinant ATG4, and a tight correlation between

Figure 6. Autophagy-activating conditions lead to the oligomerization and inactivation of ATG4 in C. reinhardtii cells. A, C.reinhardtii recombinant ATG4 was subjected to 12% SDS-PAGE in the presence (+) or absence (2) of b-mercaptoethanol(bME) in the loading buffer and/or treated with 20 mMDTT for 1 h and then stained with Coomassie Brilliant Blue (5mM ATG4)or subjected to western-blot analysis with anti-ATG4 antibodies (0.25 mM ATG4). B,Western-blot analysis of ATG4 and ATG8during the growth phase in C. reinhardtii. Cells were grown in TAP from 1 3 106 cells/mL to 83 106 cells/mL, and the ATG4and ATG8 protein profiles were analyzed. C, Effect of ER stress on ATG4 and ATG8 in C. reinhardtii. Wild-type C. reinhardtiicells growing at exponential phase were treated with 5 mg/mL tunicamycin (tun) as previously described (Perez-Perez et al.,2010), and samples were taken at different times for its analysis. Untreated cells were used as control. D, Inhibition of thecarotenoid biosynthetic pathway leads to ATG8 lipidation and ATG4 oligomerization in C. reinhardtii. Wild-type C. rein-hardtii cells in exponential growth phase were treated with 20 mM NF during 48 h as previously described (Perez-Perez et al.,2012). Samples of nontreated cells were taken at the same time and used as control. E, Western-blot analysis of ATG4 andATG8 proteins on dark-to-light transition in the lts1-204 mutant. C. reinhardtii lts1-204 cells growing exponentially in TAPunder dark conditions were transferred to standard light illumination (25 mE) for 6 h. Cells maintained in dark conditionswere used as control. Then 15 mg of total extracts were resolved by 12% (for ATG4) or 15% (for ATG8 and FKBP12) SDS-PAGE followed by western blotting with anti-ATG4, anti-ATG8, and anti-FKBP12 antibodies. The oligomeric (oATG4) andmonomeric (mATG4) ATG4 forms are marked on the right. Molecular mass markers (kD) are indicated on the left.b-Mercaptoethanol-free buffer was used in B to E.






ATG4 reduction/monomerization and ATG8 pro-cessing was detected with increasing concentrationsof DTT (Fig. 7, D and E).

Aggregated ATG4 Localizes to Punctate Structures in C.reinhardtii Cells

We analyzed the cellular localization of ATG4 by im-munofluorescencemicroscopy inC. reinhardtii cells treatedor not with NF. In control untreated cells, the ATG4 signalwas weak and restricted to some spots localized to theapical end of the cell as revealed by basal body stainingwith an antiacetylated tubulin antibody (Díaz-Troya et al.,2008a; Fig. 8). Progression into stationary phase of

untreated cells resulted in a moderate increase of ATG4signal and the detection of intense spots in other parts ofthe cell in addition to the peri-basal body region (Fig. 8).Remarkably, NF treatment caused a pronounced increaseof ATG4 signal and more intense dots were readily ob-served (Fig. 8). Based on these results and the finding thatATG4 aggregates in response to NF treatment, the punc-tate structures observed in treated cells may likely containaggregated ATG4. To strengthen this hypothesis, we alsoinvestigated the localization of ATG4 in lts1-204 mutantcells shifted from dark to light, since this stress stronglypromotes ATG4 oligomerization (Fig. 6E). In agreementwith the punctate pattern observed in wild-type cellstreated with NF (Fig. 8), light stress in carotenoid-mutant

Figure 7. ATG4 activity depends on ATG4 reduction and monomerization. A, The ROS scavenger NAC decreased ATG4oxidation and aggregation in C. reinhardtii. Wild-type C. reinhardtii cells in exponential growth phase were treated with 20 mM

NF in the presence (+) or absence (2) of 10 mMNAC during 48 h. Samples of nontreated cells were taken before the treatment(s)and used as control (first lane). B, ATG4 protease activity in cell-free extracts of C. reinhardtii. Ten nanograms of recombinantHis-tagged ATG8 protein (6H-ATG8) were incubated with 50 mg of cell-free extracts (SE) of C. reinhardtii for 30 min at 25˚C.When required, cell-free extracts were previously incubated with a reducing (25 mM DTT) or alkylating (20 mM IAM) agent for1 h on ice. The reaction was stopped by the addition of loading buffer and boiling at 100˚C. Aliquots of the reaction mixtureswere resolved by 15% SDS-PAGE and analyzed by western blotting with the anti-ATG4 (top panel) and anti-ATG8 (bottompanel) antibodies. Exogenous, recombinant ATG8 protein containing the His-6 tag (6H-ATG8) could be distinguished fromendogenous C. reinhardtii ATG8 by their different size (Perez-Perez et al., 2010). The oligomeric (oATG4) and monomeric(mATG4) ATG4 forms, the unprocessed (6H-ATG8) and processed His-tagged (p6H-ATG8) ATG8 forms, and the endogenousATG8 protein (ATG8) are marked with arrowheads. C, Quantification of ATG4 activity from B. Three independent experimentswere used for quantification and they are shown as mean 6 SD. D, Effect of DTT on both ATG4 oligomerization and ATG4activity. Purified ATG4 (2.5mM) was incubatedwith ATG8 (5mM) for 30min in the presence of DTT concentrations ranging from10 to 1 mM. Control (lane 1): incubation with no addition. The oligomeric (oATG4) and monomeric (mATG4) ATG4 forms, andthe unprocessed (ATG8) and processed ATG8 (pATG8) forms are marked by arrowheads. Molecular mass markers (kD) areshown on the left. A b-mercaptoethanol-free buffer was used. E, Quantification of ATG4 oligomerization (black circles) andATG4 activity (dark-gray circles) from D. The DTT-lacking sample and the highest DTT concentration-sample were used asreference sample for quantification of oligomerization (oligomerization = 1, in arbitrary units) and quantification of activity(activity = 1, in arbitrary units), respectively. Three independent experiments were used for quantification.






cells resulted in a sharp localization of ATG4 in acentral region of the cell (Fig. 9). Altogether, these re-sults indicate that ROS-generating stress leads to thedetection of ATG4 as intense spots likely containingoligomeric forms of this protein.

DISCUSSION

Recent genomic analyses revealed that core autoph-agy genes are highly conserved among plants and algae

(Díaz-Troya et al., 2008b; Avin-Wittenberg et al., 2012;Pérez-Pérez and Crespo, 2014; Shemi et al., 2015).However, our current knowledge about the molecularmechanisms that regulate most of the ATG proteins inthe plant kingdom is limited compared to yeasts ormammals. In this study, we unraveled the molecularmechanism underlying the redox regulation of theATG4 protease from the model green alga C. reinhardtii.A singleATG4 gene has been previously identified in C.reinhardtii (Díaz-Troya et al., 2008b) as well as in othergreen algae (Pérez-Pérez and Crespo, 2014; Shemi et al.,

Figure 8. ATG4 localizes to punctate structures in C. reinhardtii cells in response to autophagy activation. A, Immunolocalization ofATG4 in wild-type C. reinhardtii cells treated with NF. C. reinhardtii cells growing exponentially were treated with 20 mM NF for 48 h.Nontreated cells at 0 and 48 h were used as control. Cells were collected and processed for immunofluorescence microscopy analysiswith anti-ATG4 and antiacetylated tubulin antibodies. Scale bar = 8 mm. A zoom of some of the cells is also shown (right panels for thecontrol cells and lower panels for NF-treated cells). B, Quantification of the immunofluorescence signal detected in individual cellsfrom the experiment described in A. A minimum of 200 cells was analyzed for each condition using ImageJ software. ** Differenceswere significant at P , 0.0001 according to Student’s t test. AU, arbitrary units.






2015), whereas plants such as Arabidopsis, maize, orrice have two ATG4 genes (Yoshimoto et al., 2004;Chung et al., 2009; Avin-Wittenberg et al., 2012).Amino acid sequence comparison of ATG4 proteinsfrom different organisms showed a low identityeven in the green lineage (Supplemental Figs. S1 andS2). The ATG4 protein from C. reinhardtii is about200 to 300 amino acids larger than its orthologs inyeasts, plants, or mammals, which might indicatethat this protein has acquired additional domains inC. reinhardtii. In this regard, an unusually large(about 3,800 amino acids) ATG4 protein with ATG8-endoproteolytic activity has been reported in theparasitic protist Toxoplasma gondii (Kong-Hap et al.,2013). The low identity found among ATG4 prote-ases together with their variable size suggests thatthis key autophagy protein may have developedspecific features and/or functions in evolutionarilydistant eukaryotes.

Our results demonstrated that the activity of C.reinhardtiiATG4 is subjected to redox regulation, sincethe protein is active in the reduced form and inactive inthe oxidized form. Moreover, we found that this reg-ulation is reversible, suggesting the involvement ofredox posttranslational modifications. A similar redoxregulation has been reported for yeast, human, andArabidopsis ATG4 (Scherz-Shouval et al., 2007; Pérez-Pérez et al., 2014; Woo et al., 2014), indicating that theredox control of ATG4 activity is broadly conserved ineukaryotes despite the low identity observed amongATG4 proteins. The mechanism of action by whichredox signals control ATG4 activity has been shown inonly mammals and yeasts. In human ATG4A andATG4B, this redox regulation involves Cys-81, whichinterferes with the activity of the adjacent catalytic

Cys-77 (Scherz-Shouval et al., 2007). Regulatory Cys-81 from human ATG4s is not conserved in yeasts, andconsequently the molecular mechanism that mediatesthe redox control of ATG4 in this unicellular modelsystem is different. A detailed biochemical analysisdemonstrated that Atg4 activity is controlled in yeaststhrough dithiol/disulfide exchange that involves thetransfer of two electrons and the oxidation/reductionof a single disulfide bond (Pérez-Pérez et al., 2014).Our studies on the ATG4 protein fromC. reinhardtii arein close agreement with these findings, since the redoxcharacterization of this protein indicated that the ac-tivity of C. reinhardtii ATG4 is regulated by the re-duction of a single disulfide bond with a very lowredox potential. In the cell, these regulatory disulfideswith very low redox potential are typically reduced bysmall oxidoreductases such as thioredoxins (Buchananand Balmer, 2005). Indeed, it has been shown thatyeast ATG4 can be efficiently reduced and activatedby the cytoplasmic thioredoxin Trx1 (Pérez-Pérezet al., 2014). We found that C. reinhardtiiATG4 also canbe readily reduced by the cytoplasmic thioredoxinTRXh1 from this alga, suggesting that control of ATG4activity by thioredoxins might be evolutionarily con-served. In close agreement, thioredoxin and thioredoxinreductase have been identified as autophagosome-associated proteins in a quantitative proteomic study(Dengjel et al., 2012). Moreover, we were able to recon-stitute in vitro the whole ATG4 redox regulation processby the NADPH/NTR/thioredoxin system using NTRBfrom Arabidopsis as electron donor for C. reinhardtiiTRXh1. The finding that NADPH can act as the electrondonor of C. reinhardtii ATG4 suggests that this reducingmolecule may link the energetic and redox state of thecell with the regulation of the autophagy processthrough the control of ATG4 activity.

Site-directed mutagenesis established that the regu-latory disulfide bond in yeast Atg4 is formed betweenCys-338 and Cys-394 (Pérez-Pérez et al., 2014). Cys-338appears to be conserved in yeasts, plants, and mam-mals, whereas Cys-394 is restricted to some ascomycetespecies (Pérez-Pérez et al., 2014). Our results demon-strated that Cys-400 (the equivalent to Cys-338 in yeastAtg4) is required for the redox regulation of C. rein-hardtii ATG4 activity and strengthen the hypothesisthat the key role of this residue in the control of ATG4is conserved through evolution. However, we foundthat Cys-473 (the counterpart to Cys-394 in yeast Atg4)does not participate in the redox regulation of ATG4activity, since point mutation of this residue had nosignificant effect on the redox control of this protein.This observation is consistent with the poor conser-vation of Cys-394 from yeast ATG4 in other organisms(Pérez-Pérez et al., 2014). Nevertheless, the findingthat C. reinhardtii ATG4 activity is regulated throughthe reduction of a single disulfide bond by thioredoxinand that one of the regulatory cysteines is conservedstrongly suggests that the mechanism of ATG4 regu-lation is evolutionarily conserved in unicellular eu-karyotes like yeasts and algae.

Figure 9. Localization of ATG4 inC. reinhardtii carotenoidmutant cellsexposed to light. lts1-204 mutant cells growing exponentially in TAPmedium in dark conditions were exposed to low light (20 mE m22 s21)for 6 h or maintained in dark conditions as control. Cells were collectedand processed for immunofluorescence microscopy analysis with anti-ATG4 antibodies. Scale bar = 8 mm.








The formation of permanent or transient protein-protein complexes is essential for protein functionalityand regulation. Some proteins such as the antioxidantenzyme peroxiredoxins undergo major redox-dependentconformational changes that are pivotal for its physio-logical function and regulation (Dietz, 2011). In this study,we report the formation of oligomers or aggregates of theC. reinhardtii ATG4 protein both in vitro and in vivo. Ourresults revealed that C. reinhardtiiATG4 aggregates uponoxidation, whereas the reduction of the protein preventsits aggregation, indicating that ATG4 oligomerization istightly connected to the redox state of the protein. Re-markably, we found that yeast ATG4 also undergoesredox-dependent oligomerization, which may suggestthis is a conserved feature among ATG4 proteins. Wepropose that C. reinhardtii ATG4 can be found in threedifferent states that are modulated by the intracellularredox potential (Fig. 10). Under low redox potential, thethioredoxin system keeps ATG4 in a reduced and activeform (State 1). The presence of ROS promotes the oxida-tion and consequent inactivation of ATG4 (State 2) thatcan be further oxidized as the redox potential increases,leading to the aggregation of the protein (State 3). Oxi-dation and aggregation of ATG4 can be reversed by re-ducing agents, which maintain the equilibrium for theproper redox regulation of ATG4 activity.The proteolytic activity of ATG4 is essential for the

conjugation of ATG8 to PE, a key step for autophago-some biogenesis, but this enzyme is also crucial during

the final steps of autophagosome formation, sinceATG4 can delipidate ATG8-PE to release it from itslipid anchor, a fundamental step for normal autophagicactivity (Kirisako et al., 2000). Thus, ATG4 activitymustbe finely regulated to ensure autophagy progression.C-terminal maturation of ATG8 might be constitutive,since ATG4 cleaves newly synthesized ATG8 in yeasts(Kirisako et al., 2000). In close agreement, unprocessedATG8 has not been detected in C. reinhardtii (Pérez-Pérez et al., 2010), suggesting that cleavage of ATG8 atthe C terminus must be highly efficient. These findingssuggest that regulation of ATG8 delipidation by ATG4may play a central role in the control of the autophagyprocess. Mounting evidence indicates that ATG8 deli-pidation must indeed be tightly regulated. In humancells, it has been proposed that nutrient starvationgenerates ROS, which in turn inactivates ATG4 at thesite of autophagosome formation to promote lipidationof ATG8 (Scherz-Shouval et al., 2007). According to thismodel, the accumulation of lipidated Atg8s detected inhuman cells under starvation might result from inhi-bition of Atg4 delipidating activity (Scherz-Shouvalet al., 2007). In the same line, it has been shown thatdeconjugation of ATG8-PE is required for efficientautophagosome biogenesis in yeasts (Nair et al., 2012;Nakatogawa et al., 2012; Yu et al., 2012). Our results inC. reinhardtii strengthen the hypothesis that ATG4 isinactivated under stress conditions to prevent delipi-dation of ATG8 at the site of autophagosome formation.

Figure 10. Putative model for the redox regulation of ATG4 in C. reinhardtii. C. reinhardtii ATG4 can be found in at least threeconformational states: an active and monomeric form (state 1), an inactive and monomeric form (state 2), and an inactive andoligomeric form (state 3). Taken together, our data strongly suggest that the presence of ATG4 in these three states depends on theintracellular redox potential.We propose amodel in which two different oxidation levels result in the inactivation of the protease.A first level of oxidation (disulfide bond formation, state 2) results in the inactivation of ATG4,which can be either further oxidizedto an oligomeric and inactive form (state 3) or newly reduced to a monomeric and active form (state 1). The presence of oxidants,ROS, or a positive redox potential may induce a high rate of state 2 and state 3 forms of ATG4. In contrast, the presence of reducingagents, the action of reducing proteins such as the Trx system, or a negative redox potential may lead to the reduction and ac-tivation of ATG4 (state 1). Thus, the activity of the ATG4 proteasemay be finelymodulated depending on the redox conditions andthe intracellular redox state in C. reinhardtii cells.






We found that ATG4 is oxidized and forms oligomersin C. reinhardtii cells treated with NF, an inhibitor of thecarotenoid biosynthesis pathway that generates highlevels of ROS and activates autophagy in this green alga(Pérez-Pérez et al., 2012). ATG4 aggregates were alsoobserved in the carotenoid-lessmutant lts1-204 exposedto light, a stress condition that produces ROS and in-duces autophagy (Pérez-Pérez et al., 2012), or whenwild-type cells reached stationary growth, a physiolog-ical condition that triggers autophagy in C. reinhardtii(Pérez-Pérez et al., 2010). Concomitant with ATG4 ag-gregation, we also detected an accumulation of lipidatedATG8 under all these stress conditions, strongly sug-gesting that aggregated ATG4 is inactive. Together, ourresults provide the molecular basis for the redox controlof autophagy in a photosynthetic organism through theoxidation and inactivation of the ATG4 protease understress conditions.

MATERIALS AND METHODS

Strains, Media, and Growth Conditions

Chlamydomonas reinhardtiiwild-type 4A+ (CC-4051) and the lts1-204mutant(CC-4199) strains were obtained from the Chlamydomonas Culture Collection. C.reinhardtii cells were grown under continuous illumination at 25°C in Tris-acetate phosphate (TAP) medium as described (Harris, 1989). When required,cells in exponential growth phase (106 cells/mL) were treated with NF (Sigma-Aldrich; 34364). The lts1-204 mutant strain was maintained in complete dark-ness as previously described (McCarthy et al., 2004).

Gene Cloning and Protein Purification

The DNA coding sequence of the ATG4 gene from C. reinhardtii was syn-thesized and cloned into pBluescript SK (+) vector by GeneCust Europe(http://www.genecust.com). The ATG4 coding region was cloned into thepET28a (+) (Novagen; 69864-3) plasmid at NdeI and XhoI sites for expression ofthe corresponding His-tagged protein. The different ATG4 mutants (ATG4C84S,ATG4C400S, and ATG4C473S) were synthesized by GeneCust Europe and clonedinto pBluescript SK (+) vector. The different ATG4 mutants were also clonedinto the pET28a (+) plasmid at NdeI and XhoI sites. All clones were verified byDNA sequencing and used to transform Escherichia coli BL21 (DE3) strain. Re-combinant proteins were expressed by induction at exponential growth phase(OD600� 0.5) with 0.5mM isopropyl-b-D-thiogalactopyranoside (Sigma-Aldrich;I6758) for 2.5 h at 37°C. The recombinant proteins ATG4WT, ATG4C84S, ATG4C400S

and ATG4C473S were purified by affinity chromatography on a His-SelectNickel Affinity Gel (Sigma-Aldrich; P6611) following the manufacturer’sinstructions. Prior to purification, wild-type ATG4 and the Cys variants werefirst solubilized with 4 M urea (Sigma-Aldrich; 51456) due to the presence of therecombinant proteins in inclusion bodies and subsequently purified from thesoluble fraction by affinity chromatography as described above. The ATG8protein from C. reinhardtii and the ATG4 protein from Saccharomyces cerevisiaewere purified as previously described (Pérez-Pérez et al., 2010, 2014), respec-tively. The TRXh1WT and TRXh1C39S proteins fromC. reinhardtiiwere purified asdescribed in Goyer et al. (1999). The NTRB protein from Arabidopsis (Arabi-dopsis thaliana) was purified as described in Jacquot et al. (1994). All proteinsused in this study are listed in Supplemental Table S1.

In Vitro ATG4 Cleavage Assay

A typical reaction mixture included 1 mM ATG4, 5 mM ATG8, 1 mM 1,4-DTT(Sigma-Aldrich; 43815), and 1mMEDTA (Sigma-Aldrich; E6511) in 50mMTrizmabase, 138 mM NaCl, and 27 mM KCl, pH 8. When indicated, ATG4 was pre-incubated in the presence of reducing [DTT (Sigma-Aldrich; 43815) or GSH(Sigma-Aldrich; G4251)], oxidizing [DTTox (Sigma-Aldrich; D3511) or H2O2(Sigma-Aldrich; H1009)], or alkylating [IAM (Sigma-Aldrich; I1149)] compoundsalone or in combination at the indicated times and concentrations. For the analysis

of TRX activity, 10mMDTT and 5mM TRXh1 or TRXh1C39S fromC. reinhardtiiwereused at the indicated incubation time. The reaction mixtures were incubated at25°C for the indicated time and stopped by addition of b-mercaptoethanol-freeLaemmli sample buffer followed by 5 min of boiling. Proteins were resolved on15% SDS-PAGE gels and stained with Coomassie Brilliant Blue (Sigma-Aldrich;27816). For ATG4 activity quantification, gels were scanned with Odyssey (LI-COR, Biosciences) and the signals corresponding to unprocessed (ATG8) andprocessed (pATG8) ATG8 were quantified with the software Image Studio (LI-COR, Biosciences). The ATG4 activity was considered as the relation betweenpATG8/(ATG8+pATG8), and it was normalized between 0 and 1, with 0 corre-sponding to totally oxidized and inactive ATG4 and 1 corresponding to totallyreduced and active ATG4. The ATG4 activities (in arbitrary units) were plottedusing the software OriginPro 8 (OriginLab).

Protein Preparation and Immunoblot Analysis

C. reinhardtii cells from liquid cultures were collected by centrifugation(4,000g for 5 min), washed in 50 mM Tris-HCl (pH 7.5) buffer, and resuspendedin a minimal volume of the same solution. Cells were lysed by two cycles ofslow freezing to –80°C followed by thawing at room temperature. The solublecell extract was separated from the insoluble fraction by centrifugation (15,000gfor 20 min) in a microcentrifuge at 4°C. For immunoblot analyses, total proteinextracts (15 mg) were subjected to 12% or 15% SDS-PAGE and then transferredto nitrocellulose membranes (Bio-Rad; 162-0115). Anti-CrATG4 (obtained incollaborationwithAgrisera Antibodies), anti-CrATG8 (Pérez-Pérez et al., 2010),anti-CrFKBP12 (Crespo et al., 2005), and secondary antibodies were diluted1:5,000, 1:3,000, 1:5,000, and 1:10,000, respectively, in PBS containing 0.1% (v/v)Tween 20 (Applichem; A4974) and 5% (w/v) milk powder. The Luminata Cre-scendoMillipore immunoblotting detection system (Millipore;WBLUR0500) wasused to detect the proteins with horseradish peroxidase-conjugated antirabbitsecondary antibodies (Sigma-Aldrich; A6154). Proteins were quantified with theCoomassie dye binding method (Bio-Rad; 500-0006).

In Vivo ATG4 Cleavage Assays

A typical reaction mixture contained 50 mg total extract from C. reinhardtiiand 10 ng of 6His-tagged ATG8 (denoted as 6H-ATG8). When indicated, re-ducing agent (25 mM DTT) or alkylating agent (20 mM IAM) was added. Thereaction mixture was incubated at 25°C for the indicated time and stopped byaddition of b-mercaptoethanol-free Laemmli sample buffer followed by 5 minof boiling. The samples were analyzed by western-blot analysis with anti-CrATG8 or anti-CrATG4 antibodies as described above.

Immunofluorescence Microscopy

C. reinhardtii-untreated (control 0 h and control 48 h) or NF-treated (NF 48 h)cells were fixed and stained for immunofluorescence microscopy as previouslydescribed (Crespo et al., 2005). The primary antibodies used were anti-CrATG4(1:250) and antiacetylated Tubulin (Sigma-Aldrich; T7451; 1:500 dilution). Forsignal detection, fluorescein isothiocyanate-labeled goat anti-rabbit antibody(Sigma-Aldrich; F4890; 1:500 dilution) and Alexa Fluor 594 donkey anti-mouse(Jackson ImmunoResearch; 715-515-150; 1:5,000 dilution) were used. Preparationswere photographed on a DM6000B microscope (Leica) with an ORCA-ER camera(Hamamatsu) and processed with the Leica Application Suite Advanced Fluores-cence softwarepackage (Leica). For comparative analysis, the same acquisition timewas fixed for the fluorescein isothiocyanate or Alexa Flour 594 signals.

Statistical Methods

All experimentswereperformedat least in triplicateanddataarepresentedasmean6 SD. The statistical significance of datawas verified by the Student’s t testwhenever required.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Table showing the identity (%) of ATG4 proteinsfrom different species.

Supplemental Figure S2. Unrooted maximum likelihood phylogenetic treeof ATG4 proteins.




http://www.genecust.com






Supplemental Figure S3. Multiple sequence alignment of ATG4 from di-verse organisms.

Supplemental Figure S4. Redox control of yeast ATG4 oligomerization.

Supplemental Figure S5.Detection of ATG4 in total extracts from C. reinhardtii.

Supplemental Table S1. Proteins used in this study.

ACKNOWLEDGMENTS

We thank Christophe Marchand for sharing C. reinhardtii TRXh1 andTRXh1C39S proteins, Emmanuelle Issakidis-Bourguet for kindly providingArabidopsis NTRB, KrishnaNiyogi formaking carotenoidmutant strains avail-able to the research community, and Francisco J. Florencio for critical reading ofthe manuscript.

Received October 12, 2016; accepted October 15, 2016; published October 17,2016.

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1

Figure S1. Table showing the identity (%) of ATG4 proteins from different species

(Chlamydomonas reinhartdii; Volvox carteri; Coccomyxa subellipsoidea; Arabidopsis

thaliana; Oryza sativa; Homo sapiens; Saccharomyces cerevisiae;

Schizosaccharomyces pombe). The identity was obtained by alignment of the two

sequences using Pairwise Sequence Alignment EMBOSS Needle. The accession

numbers are indicated in Figure S2.

Figure S2. Unrooted Maximum likelihood phylogenetic tree of ATG4 proteins. Sub-families are indicated by color-coded circles. Species abbreviations and the

accession numbers are indicated. Cr: Chlamydomonas reinhardtii

(Cre12.g510100.t1.1), Vc: Volvox carteri (Vocar.0016s0213.1), Cs: Coccomyxa

subellipsoidea (EIE27123), At: Arabidopsis thaliana (AT2G44140.1 for A;

AT3G59950.1 for B), Al: Arabidopsis lyrata (935334 for A; 486460 for B), Zm: Zea

mays (GRMZM2G064212_T02 for A; GRMZM2G173682_T01 for B), Sl: Solanum

lycopersicum (Solyc01g006230.2.1), St: Solanum tuberosum

(PGSC0003DMT400081879), Gm: Glycine max (Glyma.18G248400.1 for A;

Glyma.09G244800.1 for B), Mt: Medicago truncatula (Medtr7g081230.1), Vv: Vitis

vinifera (GSVIVT01003913001), Os: Oryza sativa (LOC_Os03g27350.1 for A;

LOC_Os04g58560.1 for B), Sc: Saccharomyces cerevisiae (KZV08400), Sp: Schizosaccharomyces pombe (CAC00556), Ca: Candida albicans (XP_713234), Nc: Neurospora crassa (EAA30202), An: Aspergillus nidulans (XP_661074), Hs: Homo

sapiens (BAB83889 for A; BAB83890 for B), Mm: Mus musculus (NP_777364 for A;

NP_777363 for B), Zf: Zebrafish (AAH95617), Dm: Drosophila melanogaster

(NP_608563). ATG4 sequences were obtained from Phytozome

(https://phytozome.jgi.doe.gov/pz/portal.html) for algae and plants, and NCBI

(https://www.ncbi.nlm.nih.gov/pubmed) for fungi and animals.

Figure S3. Multiple sequence alignment of ATG4 from diverse organisms. (A) Alignment of the region containing the catalytic Cys84. (B) Alignment of the C-

terminus region containing putative regulatory Cys400 and Cys473. The proteins

were aligned with the ClustalW program using default parameters. Residues boxed in

red correspond to 70% identity. Residues boxed in blue correspond to 70%

conservation with the PAM120 matrix. The accession numbers are indicated in

Figure S2.

2

Figure S4. Redox control of yeast ATG4 oligomerization. (A) Yeast recombinant

ATG4 (5 µM) was subjected to 12 % SDS-PAGE in the presence (+) or absence (-)

of β-mercaptoethanol (βME) and stained with coomassie brilliant blue (CBB).

mATG4, monomeric ATG4; oATG4, oligomeric ATG4. (B) ATG4 (5 µM) was

preincubated with 100 µM DTT for 1 h (lane 2); then, ATG4 was treated with 500 µM

H2O2 for 20 min (lane 3); and finally, ATG4 was incubated with 1 mM DTT for 30 min

(lane 4). Control (lane 1): incubation with no addition. (C) Redox titration of ATG4

oligomerization/monomerization. ATG4 monomerization was analyzed after

incubation for 2 h at indicated Eh poised by 20 mM DTT in various dithiol/disulfide

ratios. Arrowheads indicate oligomeric (oATG4) and monomeric (mATG4) ATG4

forms. β-mercaptoethanol-free buffer was used in (B and C).

Figure S5. Detection of ATG4 in total extracts from Chlamydomonas. Different

amounts (2.5, 10, 20, 30 and 40 µg) of soluble extracts from stationary cells were

resolved by 12 % SDS-PAGE followed by Western-blot analysis with the anti-ATG4

antibody. The oligomeric (oATG4) and monomeric (mATG4) ATG4 forms are marked

on the right. Molecular mass markers (kDa) are indicated on the left. β-

mercaptoethanol-free buffer was used.

Chlamydomonas

Chl

amyd

omon

asVolvox

Vol

vox

Coccomyxa

Coc

com

yxa

Arabidopsis A

Ara

bido

psis

AArabidopsis B

Ara

bido

psis

BOryza A

Ory

za A

Oryza B

Ory

za B

Human AHuman B

Hum

an A

Hum

an B

100 46100

2827

100

2023

31

100

2021

29

73

100

2023

29

48

47

100

1923

31

50

50

82

100

2022

29

25

26

26

24

100

1921

31

26

25

25

21

53

100

Sac

char

omyc

es

Sch

izos

acch

arom

yces

SaccharomycesSchizosaccharomyces

17 16

10010020

18 17

18 25

18

25

25

23

21

19

21

26

21

20 26

27

Identity (%)

Figure Suppl 1

AtA

TG4A

AlAT

G4A

AtAT

G4B

AlATG4B

VvATG4 MtATG4

GmATG4A

GmATG4B

SlATG4 StATG

4

ZmAT

G4A

ZmAT

G4B

OsA

TG4A

OsATG4B

CsATG4

CrATG4

VcATG4

DmATG4A

HsATG4B

MmATG4B

ZfATG4 HsATG4A

Mm

ATG4A

NcAT

G4

AnAT

G4

SpAT

G4

ScAT

G4

CaATG

4

0.2

algae higher plants

metazoa

fungi

green lineage Figure Suppl 2

HsATG4B 136 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S I G QWY G P N T V AQ V L K K L A V F D T WS S L A V H I A - - - - 168

CrATG4 392 - - - - - - - - - - - - - - - - - S S T A A S S C CG - - - - - - - - E A E D A A - A HG R R C RG V A AG T - - - - - - C G RG - - - - - - - H S L L L L V P L T L GM D K - I N P V Y I P Q L QQ V 452ScATG4 330 - - - - - - - - - - - - - - - - - E D S F V E S C H - - - - - - - - - T S K F G K - - - - L Q L S E M D P S - - - - - - - - - - - - - - - - - - - - - - M L I G I L I K G E K DWQQWK L E V A E S A 378OtATG4 224 - - - - - - - - - - - - - - - - - - E L T V H V V S G R - - - - - - - E G E DGG - V P T V D E A E V R AK S - - - - - - A D VG - - - - - - - K A L L L F V P V V L G VG R T I N A R Y L S Q L R S M 285PtATG4 307 I F R V Y - V A P QG T V Y R - - D T I H A F M T T E AR - - - - - V R I E E K K K V K E Q T Q P Q AH P L D L E WE E E L M E S AN T V E WD T A L L L L V P L R L G L T S - L N E E Y VQ S L AH T 397AtATG4a 261 - - - - - N - - - - - - - - - Q T L P M A V H I V S G S - - - - - - E D G E RGG - A P I L C I E D A T K S - - - - C L E F S KGQ - - - S E WT P I I L L V P L V L G L D S - V N P R Y I P S L V A T 332AtATG4b 265 - - - - - H - - - - - - - - - K S F S M A V H I V S G S - - - - - - E D G E RGG - A P I L C I E D V T K T - - - - C L E F S E G E - - - T E WP P I L L L V P L V L G L D R - V N P R Y I P S L I A T 336HsATG4A 172 - - - - - - - - - - - - - - - M D N T V V I E D I K KM C R - V L P L S A D T AG D R P P D S L T A S NQ S K - - - - - G T S A Y C - - - S AWK P L L L I V P L R L G I N Q - I N P V Y V D A F K E C 247HsATG4B 168 - - - - - - - - - - - - - - - M D N T V VM E E I R R L C R T S V P C AG A T A F P AD S D R H C NG F P AG - - - - A E V T N R P - - - S P WR P L V L L I P L R L G L T D - I N E A Y V E T L K H C 245

CrATG4 453 L S WP Q S VG I VG G R P S A S - - L Y V CG VQ D A S F I Y L D P H E AQ L A V R T P A A T A A A T A A A A V A A P AGG A A A T V R A A AGGGG AG AGGQGGG A T AWP P T GWL A A P Q L 550ScATG4 379 I - - - - - I N V L A K RM D D F - - D V S C S M D D V E S V S S N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S M K K 409OtATG4 286 M A F KQ S VG I VG G R P N S S - - L Y L VG H S D D V F F Y L D P H T VQ V A S S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M V T M D F 332PtATG4 398 F S L P Q S VG V L GG R P RG ARWF Y G AQ K DG S K I F G L D P H T VQ T A P G RQ T AR V NG - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q A S S V V E L S D D Y 460AtATG4a 333 F T F P Q S VG I L G G K P G A S - - T Y I VG VQ E D KG F Y L D P H E VQQ V V T V N K E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T P D V D T 383AtATG4b 337 F T F P Q S L G I L G G K P G A S - - T Y I VG VQ E D KG F Y L D P H D VQQ V V T V K K E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N Q D V D T 387HsATG4A 248 F KM P Q S L G A L GG K P N N A - - Y Y F I G F L G D E L I F L D P H T T Q T F V D T E E N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - G T V N D 297HsATG4B 246 F MM P Q S L G V I G G K P N S A - - H Y F I G Y VG E E L I Y L D P H T T Q P A V E P T DG - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C F I P D 295

CrATG4 551 G T Y F C D - V V R V L P S AQ L D P S L A I G F V C T S S A E L E D L F A R L Q A L A T Q H S S A P L M T - - L T T G S G - - A A VG CG S D AD F T D - - - - - - - D V L E GG T GQQQ L D E WE 638ScATG4 410 D A S N N E - N L G V L E G - D Y V D I G A I F P H T T N T E D V D E Y D C F Q D I H C K KQ K I V VMG N - - T H T V N AN L T D Y E V E G V L V E K E - - - - - T VG I H S P I D E K C - - - - - - 494OtATG4 333 E S Y Y C P - T P L H V CGG D L D P T L A L G F Y C R DG D D V A S L L V D I E A L AR V N A T A P A L A - - I R P A D D R Y K S I S P R H A E R A S P - - - - - - - S V P P T D I R D D E F A DWT 422PtATG4 461 L R S C H T T C P E M F P F C KM D P S I A L G F Y C R T R AD L N H V L N S MG AWQ K E H S S I P E L F S V L D R A P D Y S AN V D D L L L GG D S S - - - - - MM E T S G F E D E A S D AD E Y V 555AtATG4a 384 S S Y H C N - V L R Y V P L E S L D P S L A L G F Y C R D K D D F D D F C L R A L K L A E E S NG A P L F T V T Q T H T - - - - - - A I N Q S N Y G F AD - - - - - - - D D S E - - - - D E R E D DWQ 465AtATG4b 388 S S Y H C N - T L R Y V P L E S L D P S L A L G F Y CQ H K D D F D D F C I R A T K L AG D S NG A P L F T V T Q S H R R N D CG I A E T S S S T E T S T - - - - - - - E I S G - - - - E E H E D DWQ 475HsATG4A 298 Q T F H C L Q S P Q RM N I L N L D P S V A L G F F C K E E K D F D NWC S L VQ K E I L K E N - L RM F E L VQ K H P S HWP P F V P P AK P E V T T T G A E F I D S T E Q L E E F D - - L E E D F E 394HsATG4B 296 E S F H CQ H P P C RM S I A N L D P S I A VG F F C K T E D D F N DWCQQ V K K L S L L GG A L P M F E L V E QQ P S H L - - - - - - A C P D V L N L S L D S S D - V E R L E R F F D S E D E D F E 388

CrATG4 639 L V - - 640ScATG4 494 - - - - 494OtATG4 423 F V - - 424PtATG4 556 M L - - 557AtATG4a 466 M L - - 467AtATG4b 476 L L - - 477HsATG4A 395 I L S V 398HsATG4B 389 I L S L 392

Cys400











Cys473

Cys84

CrATG4 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M S Q A L S L R K L S E L M HG T T S L S Q H - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 23OtATG4 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M T G T F AR V S R A A V AH H P R V R A L G R L V T R K T - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 30PtATG4 1 M S I V R N S T AD S S V V L V T A P GWS E R S R S DG L VQ V E A V R S E N V D I P K T N L I K H F V E S R F GG E T K DG H L T E M K E M P L K V T P S P NQQ T E AD AD F D ACQQ Y N C E T 100AtATG4a 1 - - - - - - - - - - - - M K A L C D R F V P QQ C S S S S K S D T H D K S P - - - L V S D S G P S D N K S K F T L WS N V F T S S S S V S Q P Y R E S S T S G H KQ V C T T R NGWT A F V K R V S M A 85AtATG4b 1 - - - - - - - - - - - - M K A I C D R F V P S K C S S S S T S E K R D I S S P T S L V S D S A S S D N K S N L T L C S D V V A S S S P V S Q L C R E A S T S G H N P V C T T H S S WT V I L K T A S M A 88ScATG4 1 - - - - - - - - - - - - - - - - - - MQ RWL Q L WKM D L VQ K V S HG V F E G S S E E P A A L M N H D Y I V L G E V Y P E R D - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 47HsATG4A 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M E S V L S K Y E DQ I T I F T D Y L E E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 21HsATG4B 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M D A A T L T Y D - - - T L R F A E F E D - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 18

CrATG4 23 - - - - - - - - - - - - - - - - - - - - - - - - T I C L L G K S Y G D AG T G DQ S K A L D A E A - - - - - - - - - - - - - - - L E AM L G D F R S RMWC T Y R K D F P A L G P S L - - - - - - - - - 75OtATG4 30 - - - - - - - - - - - - - - - L D D L P P D A S L VM F G V T HWD R E T S S G E R S N E VG R - - - - - - - - - - - - - - - - - R E WE R DWR S R CWM T Y R RG F E A L G R T K - - - - - - - - - 89PtATG4 101 S G E N E K E E A S D L NG P F G T M S A F A S A VM S S I L L DG N N S E S P Q S N P S V E S V S M S F V L G K T Y H P L H D Y S I R R D D E R S L F WF T Y R C D F P E I A P Y N - - - - - - - - - 191AtATG4a 86 S G A I R R F Q E R V L G P N R T G L P S T T S D VWL L G V C Y K I S A D E N S G E T D T G T V - - - - - - - - - - - - - - - L A A L Q L D F S S K I L M T Y R KG F E P F R D T T - - - - - - - - - 161AtATG4b 89 S G A I R R F Q D R V L G P S R T G I S S S T S E I WL L G V C Y K I S E G E S S E E AD AG R V - - - - - - - - - - - - - - - L A A F RQ D F S S L I L M T Y R RG F E P I G D T T - - - - - - - - - 164ScATG4 47 - - - - - - - - - - - - - - E E S G A E Q C E Q D C R Y RG E A V S DG F L S S L F G R E I S S Y T - - - - - - - - - - - - - - - K E F L L D VQ S R V N F T Y R T R F V P I A R A P DG P S P L S L N 118HsATG4A 21 - - - - - - - - - - - - - - - - - - Y P D T D E L VW I L G KQ H L L K T E K S K - - - - - - - - - - - - - - - - - - - - - - - - - - L L S D I S A R L WF T Y R R K F S P I G G T G - - - - - - - - - 68HsATG4B 18 - - - - - - - - - - - - - - - - - - F P E T S E P VW I L G R K Y S I F T E K D E - - - - - - - - - - - - - - - - - - - - - - - - - - I L S D V A S R L WF T Y R K N F P A I G G T G - - - - - - - - - 65

CrATG4 75 - - - - - - - - - - - - - - - - - - - - L T S D VGWG C T L R S GQM L L A E AMM R V A L G R DWQ R C S D N L E A V R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 117OtATG4 89 - - - - - - - - - - - - - - - - - - - - WC T D AGWG C T L R S AQMM L AN A L S I H S RG R HWR R E VQ L V A V H E N E T AD DG S K S P A V S F L S G V V N K L K I P Q S E R T R AG S D AQ 169PtATG4 191 - - - - - - - - - - - - - - - - - - - - I T S D AGWG CM L R S AQMM L GQ A L R L H F K S R DWR P P Q L L AR R RQ D - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S F I 237AtATG4a 161 - - - - - - - - - - - - - - - - - - - - Y T S D V NWG CM I R S S QM L F AQ A L L F H R L G R AWT K K - S E L P E Q E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Y 203AtATG4b 164 - - - - - - - - - - - - - - - - - - - - Y T S D V NWG CM L R S GQM L F AQ A L L F Q R L G R S WR K K D S E P AD E K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Y 207ScATG4 119 L L V R T N P I S T I E D Y I A N P D C F N T D I GWG CM I R T GQ S L L G N A L Q I L H L G R D F R V NG N E S L E R E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 180HsATG4A 68 - - - - - - - - - - - - - - - - - - - - P S S D AGWG CM L R CGQMM L AQ A L I C R H L G R DWS WE KQ K E Q P K E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Y 111HsATG4B 65 - - - - - - - - - - - - - - - - - - - - P T S D T GWG CM L R CGQM I F AQ A L V C R H L G R DWRWT Q R K RQ P D S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Y 108

CrATG4 117 - P V V A A L L D C - - - A E A P L S I H R I C D AG - G P AG I V P G RWL G P WM L C KG L E A L F S Q L AG AQQ E P L G L R L H V ACG AGGG AP E L HM D L I R A AM A P A AQ S A A T AG 212

A

B

Figure Suppl 3

C

BA

βmercaptoethanol+ -

10080

60

50

40

150

mATG4

oATG4

230

kDa

oATG4

mATG4

-311 -305-316 -299 -250-277-282-286-289-297 -273 -259-268 -264 -230-294 -292 -279 -271-infi +infiEm (mV)

1008060

50

40

150230

kDa

mATG4

oATG4

500 μM H2O2 100 μM DTT

1 mM DTT

---

+--

+++

++-

Figure Suppl 4

Figure Suppl 5

9864

250

---

Total extract (μg)

ATG4

kDa10 2.5 20 30 40

mATG4

oATG4

Table S1. Proteins used in this study Name Characteristics Reference

CrATG8WT His-‐tag CrATG8 1

CrATG4WT His-‐tag CrATG4 This study

CrATG4C84S His-‐tag CrATG4 where Cys84 was replaced by Ser This study



ScATG4WT His-‐tag ScATG4 2

CrTRXh1 His-‐tag ScTrx1 3

CrTRXh1C39S His-‐tag ScTrx1 where Cys33 was replaced by Ser 3

AtNTRB His-‐tag AtNTRB 4

Cr: Chlamydomonas reinhardtii; Sc: Saccharomyces cerevisiae; At: Arabidopsis thaliana. 1. Pérez-‐Pérez ME, Florencio FJ, Crespo JL (2010). Plant Physiol, 152:1874-‐1888. 2. Pérez-‐Pérez ME, Zaffagnini M, Marchand CH, Crespo JL, Lemaire SD (2014). Autophagy, 10(11):1953-‐1964. 3. Goyer A, Decottignies P, Lemaire S, Ruelland E, Issakidis-‐Bourguet E, Jacquot J-‐P, Miginiac-‐Maslow M (1999). FEBS Lett., 444(2-‐3):165-‐169. 4. Jacquot J-‐P, Rivera-‐Madrid R, Marinho P, Kollarova M, Le Maréchal P, Miginiac-‐Maslow M, Meyer Y (1994). J. Mol. Biol., 235(4):1357-‐1363.

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