Title: Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4
Abstract: Article8 March 2007free access Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4 Ruth Scherz-Shouval Ruth Scherz-Shouval Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Elena Shvets Elena Shvets Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Ephraim Fass Ephraim Fass Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Hagai Shorer Hagai Shorer Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Lidor Gil Lidor Gil Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Zvulun Elazar Corresponding Author Zvulun Elazar Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Ruth Scherz-Shouval Ruth Scherz-Shouval Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Elena Shvets Elena Shvets Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Ephraim Fass Ephraim Fass Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Hagai Shorer Hagai Shorer Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Lidor Gil Lidor Gil Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Zvulun Elazar Corresponding Author Zvulun Elazar Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Author Information Ruth Scherz-Shouval1, Elena Shvets1, Ephraim Fass1, Hagai Shorer1, Lidor Gil1 and Zvulun Elazar 1 1Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel *Corresponding author. Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, 76100 Israel. Tel.: +972 8 9343682; Fax: +972 8 9344112; E-mail: [email protected] The EMBO Journal (2007)26:1749-1760https://doi.org/10.1038/sj.emboj.7601623 Correction(s) for this article Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg415 May 2019 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Autophagy is a major catabolic pathway by which eukaryotic cells degrade and recycle macromolecules and organelles. This pathway is activated under environmental stress conditions, during development and in various pathological situations. In this study, we describe the role of reactive oxygen species (ROS) as signaling molecules in starvation-induced autophagy. We show that starvation stimulates formation of ROS, specifically H2O2. These oxidative conditions are essential for autophagy, as treatment with antioxidative agents abolished the formation of autophagosomes and the consequent degradation of proteins. Furthermore, we identify the cysteine protease HsAtg4 as a direct target for oxidation by H2O2, and specify a cysteine residue located near the HsAtg4 catalytic site as a critical for this regulation. Expression of this regulatory mutant prevented the formation of autophagosomes in cells, thus providing a molecular mechanism for redox regulation of the autophagic process. Introduction Autophagy is a major pathway for delivery of proteins and organelles to lysosomes/the vacuole, where they are degraded and recycled. Autophagy plays an essential role in differentiation and development, as well as in cellular response to stress. It is activated during amino-acid deprivation and has been associated with neurodegenerative diseases, cancer, pathogen infections and myopathies (reviewed in Cuervo, 2004; Shintani and Klionsky, 2004). Autophagy is initiated by the surrounding of cytoplasmic constituents by the crescent-shaped isolation membrane/phagophore, which forms a closed double-membrane structure, called autophagosome. Finally, the autophagosome fuses with a lysosome to become an autolysosome, and its content is degraded by lysosomal hydrolases. The molecular mechanism underlying autophagy has been extensively researched in the past decade, and the genes participating in this process, denoted ATGs (autophagy-related; Klionsky et al, 2003), were found to be conserved from yeast to man (Ohsumi, 2001; Huang and Klionsky, 2002). Yet, the roles played by the different gene products and their modes of action are to be resolved. A hallmark event in the autophagic process is the reversible conjugation of the Atg8 family of proteins to the autophagosomal membrane (Ichimura et al, 2000). This ubiquitin-like (UBL) protein family, which has been implicated in a variety of cellular processes, includes Atg8p in yeast (Lang et al, 1998; Ichimura et al, 2000) and GATE-16 (Golgi-associated ATPase enhancer of 16 kDa; Sagiv et al, 2000), LC3 (light-chain 3; Mann and Hammarback, 1994) and GABARAP (GABA receptor-associated protein; Wang et al, 1999) in mammals. All Atg8 homologues (hereafter termed Atg8s) serve as substrates for the Atg4 family of cysteine proteases (Kirisako et al, 2000; Hemelaar et al, 2003; Marino et al, 2003; Scherz-Shouval et al, 2003). Atg4s cleave Atg8s near the C-terminus, downstream of a conserved glycine. This cleavage allows the conjugation of Atg8 to phosphatidylethanolamine (PE) through the exposed glycine, a process mediated through a ubiquitination-like mechanism. Atg8-PE serves as another substrate for Atg4, which cleaves Atg8 and releases it from the membrane. Notably, this conjugation process must be preceded by another ubiquitination-like process, conjugating Atg12 to Atg5 (Tanida et al, 2004b). In mammalian cells, amino-acid deprivation induces lipidation of LC3 (Kabeya et al, 2000) as well as of GATE-16 and GABARAP (Kabeya et al, 2004). Lipidated Atg8s associate with phagophaores and autophagosomes and remain there until fusion with lysosomes, at which point intra-autophagosomal Atg8 is probably degraded (Kabeya et al, 2000, 2004). At least four Atg4 mammalian homologues have been reported based on sequence homology to the yeast Saccharomyces cerevisiae (Sc)Atg4. Two of the homologues, HsAtg4A and HsAtg4B, were shown to cleave the three mammalian Atg8s with different efficiencies: HsAtg4A cleaves mainly GATE-16, whereas HsAtg4B cleaves all three homologues (GATE-16, GABARAP and LC3), with the highest efficiency for LC3 (Kabeya et al, 2004). Atg4s act both as conjugating and deconjugating enzymes and therefore their activity is expected to be tightly regulated. Hence, in the process of autophagy, following the initial cleavage of Atg8-like proteins, Atg4 must become inactive so as to ensure the conjugation of Atg8 to the autophagosomal membrane. Later on, as the autophagosome fuses with the lysosome, Atg4 might be locally re-activated in order to delipidate and recycle Atg8. How is this regulation achieved? We have previously shown that recombinant HsAtg4A is active as a cysteine protease of GATE-16 only in the presence of the reducing agent DTT (Scherz-Shouval et al, 2003). This could implicate that, in vivo, the delipidating activity of Atg4 is regulated through changes in redox potentials that take place under different conditions and at specific subcellular microenvironments. Such regulation could be inflicted, among other factors, by reactive oxygen species (ROS). At high levels, ROS are deleterious to cells, leading to programmed cell death (PCD) (Jabs, 1999; Lee et al, 2003; Macip et al, 2003). At low levels, however, ROS can serve as signaling molecules, by oxidizing factors in a variety of pathways that lead to growth and survival. Here, we report for the first time the involvement of ROS as signaling molecules in nutrient starvation-induced autophagy, which is essentially a survival pathway. We show that under starvation conditions, cells form ROS, specifically H2O2, which is essential for autophagosome formation and autophagic degradation. The oxidative signal is partially PI3K dependent and leads to inhibition of Atg4. Using an in vitro assay, we could demonstrate that H2O2 directly regulates HsAtg4A. Moreover, we identified Cys81, situated four amino acids downstream of the active site, as an essential residue for the redox regulation of HsAtg4A. Expression of HsAtg4A mutated in Cys81, or the corresponding mutant of HsAtg4B, inhibited the formation of GATE-16- or LC3-labeled autophagosomes in cells. Results Starvation induces formation of ROS ROS serve as signaling molecules in a variety of cellular processes, including growth, differentiation, adhesion and PCD. To test whether ROS play a role in autophagy, we measured ROS production under starvation using a fluorescent probe, dihydroethidum (DHE). DHE reacts with peroxides to form a compound that upon binding to DNA fluoresces and can be visualized by confocal fluorescent microscopy (Vanden Hoek et al, 1997). Indeed, upon nutrient starvation, DHE staining was evident, indicating that cells accumulate peroxides under these conditions (Figure 1A). We then used another probe, 2′,7′-dichlorofluorescin diacetate (DCF-DA), which reacts mainly with H2O2 to form a fluorescent compound at the point of interaction (Cathcart et al, 1983; Vanden Hoek et al, 1997). DCF staining was already evident 15 min after the induction of starvation (Figure 1Bd and Supplementary Figure 1A, see text below). The pattern of DCF staining was punctated as well as cytosolic, and it was also apparent following overnight starvation (Figure 1Bh). These results indicate that H2O2 is most likely the oxidative agent accumulating in starved cells. To rule out the possibility that the oxidative signal associated with starvation is leading to PCD, we tested the viability of cells grown under nutrient starvation conditions. As shown in Supplementary Figure 1B, 5 h of nutrient starvation did not affect the viability of the cells, and the percent of cell death was equally low in control and starved cells. Taken together with the finding that the oxidative signal appears minutes after induction of starvation, these results suggest that H2O2 serves as a signaling molecule in the autophagic pathway, and not as part of an oxidative stress leading to PCD. Figure 1.Cells accumulate ROS under starvation conditions. (A) CHO cells were grown in a control medium (see Materials and methods) or starved for 3 or 13 h, after which they were incubated in 50 μM DHE and visualized as detailed in Materials and methods. (B) CHO cells were grown as in (A), treated with 5 nM MitoTracker Red for 30 min at 37°C, washed, treated with 30 μM DCFDA and visualized as detailed in Materials and methods. (C) HeLa cells grown in 96-well plates were deprived of serum (αMEM), completely starved (EBSS) or maintained in a control medium (α-MEM, 10% FCS) for 2 h, after which they were treated with DCFDA as in (B) and subsequently analyzed in a fluorimeter, as explained in Materials and methods. (D) Data collected from the fluorometric measurements were analyzed as detailed in Materials and methods. Download figure Download PowerPoint The pattern of DCF staining resembled mitochondrial staining, and therefore we double stained the cells with DCF and MitoTracker Red, a marker for functional mitochondria. We found significant overlap between the two markers (Figure 1Bf and j; enlargements in g and k, respectively). All the DCF puncta either colocalized with or were in close vicinity to MitoTracker-stained structures, suggesting mitochondria as the source of ROS generated during starvation. This finding coincides with our observation that phagophores, identified by a double staining with Atg5 and Atg16 (Mizushima et al, 2003), tend to colocalize with MitoTracker Red-stained mitochondria (Supplementary Figure 1C), suggesting a functional link between ROS generated in the mitochondria during starvation and autophagosome formation. This result is consistent with recently reported findings (Kissova et al, 2006). In order to assess the rise in ROS in a more accurate manner, we measured the increase in DCF fluorescence using a fluorimeter set at 485 nm excitation and 535 nm emission in two different cell lines (CHO and HeLa). The cells were either deprived of serum, completely starved (deprived of serum and nutrients) or grown in control medium for 2 h, after which they were treated with DCF and the fluorescent signal of the whole population (3 × 104 cells) was monitored. As shown in Figure 1C, complete starvation of the cells resulted in a dramatic increase in the fluorescent signal over time, as compared to the control, whereas serum starvation had only a mild effect on the fluorescent signal, indicating a slight increase in ROS production. These measurements were further processed and summarized as the increase in DCF fluorescence over 20 min of incubation with the reagent (Figure 1D). Class III PI3K is involved in starvation-dependent ROS formation In the attempt to understand where ROS production fits in the autophagic signaling pathway, we monitored the effect of two known autophagy inhibitors, wortmannin and 3-methyladenine (3MA), on starvation-dependent DCF staining (Petiot et al, 2000). Starvation of cells for a 2-h period in the presence of either drug reduced the level of DCF fluoresence by 25–30% (Figure 2A, left panel). As expected, the autophagic activity in the treated cells, tested by the rate of degradation of long-lived proteins (known to increase during autophagy, see Mizushima et al, 2001), was significantly reduced (Figure 2A, right panel). Wortmannin and 3MA are phosphatidyl inositol 3 kinase (PI3K) inhibitors. Class III PI3K plays a critical role in the early stages of autophagosome formation in mammals through formation of an essential complex with Beclin 1 and, hence, inhibition of its activity inhibits the autophagic process (Petiot et al, 2000; Tassa et al, 2003). This finding, therefore, indicates that ROS production is partially PI3K dependent. Indeed, partial silencing of Beclin 1 (previously shown to cause a partial autophagic defect; Reef et al, 2006) or the PI3K hVps34 resulted in 15 and 20% (respectively) reduction in starvation-induced ROS production (Supplementary Figure 2A). Notably, partial silencing of hVps34 caused 30% reduction in the number of autophagosomes per cell (Supplementary Figure 2B, lower right panel). Figure 2.ROS formation occurs partially downstream of class III PI3K activation. (A) Left panel: HeLa cells were starved for 2 h in the presence or absence of either 100 nM wortmannin or 10 mM 3MA, after which they were treated with DCFDA and analyzed by a fluorimeter as described above. Right panel: the rate of degradation of long-lived proteins was measured in cells incubated in either α-MEM medium or EBSS medium in the absence or presence of 100 nM wortmannin or 10 mM 3MA. Values are represented as the means±s.d. of three separate determinations. (B) WT MEFs or Atg5 (−/−) MEFs from two separate clones were starved for 2 h before treatment with DCFDA and visualization or fluorometric analysis. Download figure Download PowerPoint Next, we assessed the level of DCF staining in cell lines deleted for the Atg5 gene, whose product is essential for autophagosome formation, acting downstream of the class III PI3K (Suzuki et al, 2001; Tanida et al, 2004b). DCF staining in the mutant cells did not decrease, as compared with control cells (Figure 2B), but rather increased significantly (40–50%), indicating that ROS production takes place upstream of the Atg5-dependent step of autophagosome formation, and suggesting that in the absence of a functional autophagic pathway, cells accumulate ROS. ROS formation is essential for autophagy The results described above suggest that starvation induces ROS formation. But are ROS essential for autophagy? To address this question, we tested the effect of N-acetyl-L-cysteine (NAC), a general antioxidant, and catalase, which specifically decomposes H2O2, on the formation of autophagosomes using GATE-16 and LC3 as markers. LC3 and GATE-16 were shown to label autophagosomes under starvation conditions (Kabeya et al, 2004; Supplementary Figure 3A), and as shown in Supplementary Figure 3B, the two proteins exhibit significant colocalization under these conditions. Addition of either NAC or catalase to the growth medium (Sakurai and Cederbaum, 1998; Preston et al, 2001; Xu et al, 2003) of the cells for 10 min before a 2-h starvation period resulted in reduced lipidation of GATE-16 and LC3 and abolished the formation of GATE-16- and LC3-labeled autophagosomes (Figure 3A and Supplementary Figure 3C). As expected, the same concentration of antioxidative reagents significantly reduced the level of ROS in the treated cells (Figure 3B). These results were further validated by testing the effect of antioxidants on starvation-induced protein degradation (Figure 3C). Treatment with NAC caused 60% inhibition in starvation-induced degradation, and catalase caused 25% inhibition in starvation-induced degradation. Taken together, we conclude that starvation conditions induce formation of ROS, which are required for induction of autophagy. Notably, treatment of cells with H2O2 alone did not result in formation of autophagosomes (data not shown). Figure 3.ROS accumulation is essential for autophagy. (A) Upper panel: CHO cells stably transfected with GFP-GATE-16 were preincubated in the presence or absence of 10 mM NAC or 1000 μ/ml catalase for 10 min before starvation for 2 h in the presence or absence of these drugs, or grown in a control medium containing the drugs for 2 h. The cells were then fixed, permeabilized and stained with anti-GFP monoclonal antibodies. Representative images are shown. Lower panel: HEK 293 cells were transfected with GFP-GATE-16. At 24 h post-transfection, the cells were treated with NAC or catalase and starved as explained above, lysed in Ripa buffer and 100 μg of each lysate was separated on 10% SDS–PAGE and subsequently analyzed with anti-GFP antibodies to detect the transfected GATE-16 and anti-tubulin antibodies as control. The data were quantified using NIH image program and are depicted as the percentage of lipidated protein from the total GATE-16. (*) indicates non-lipidated and (**) indicates lipidated GFP-GATE-16. (B) CHO cells treated with NAC or catalase and starved as detailed in (A) were incubated with DCFDA and visualized by a confocal microscope or analyzed by a fluorimeter as in Figure 1. (C) The rate of degradation of long-lived proteins was measured in CHO cells incubated in either α-MEM medium or EBSS medium, or following pretreatment with 10 mM NAC for 10 min or with 1000 μ/ml catalase overnight. A representative experiment is shown. Download figure Download PowerPoint Atg4 is attenuated in response to starvation in a redox-dependent manner Next, we searched for a cellular target of the starvation-induced oxidative signal. The amount of the lipidated form of Atg8s increases significantly during starvation (Kabeya et al, 2004). As shown in Figure 4A, this elevation, in mammalian Atg8s, does not result from increased transcription, as previously shown for ScAtg8 (Kirisako et al, 1999). Consistently, we could not observe changes in the protein levels of mammalian Atg8s under these conditions (Figure 4B and data not shown). We therefore deduce that the increase in lipidated mammalian Atg8 proteins in response to nutrient starvation is due to post-translational regulation of the processing enzymes. To test this hypothesis, cells were starved for 3 or 13 h, after which they were lysed and the lysates were tested for their ability to cleave GATE-16. In this assay, recombinant GATE-16, tagged at both termini (His6-GATE-16-HA), is used so that cleaved GATE-16 can be detected by its faster mobility in SDS–PAGE. As shown in Figure 4C, incubation of GATE-16 in a lysate prepared from cells that were starved for a short period of 3 h resulted in a decrease of up to 40% in the cleavage activity as compared with control cells. Starvation of cells for longer periods of 13 h (during which the cells are still viable) resulted in further inhibition in cleavage activity. However, addition of 1 mM DTT to the lysates brought about recovery of the cleavage activity, suggesting that the inhibition of Atg4 was caused by oxidation of the protein. Consistently, treatment of cells with 1 mM H2O2 for 1 h before lysis resulted in strong inhibition of the cleavage activity of Atg4 as compared with control cells. Addition of DTT to the lysate restored most of the lost activity, confirming that the inhibition by oxidation is reversible. Notably, the protein levels of the GATE-16-specific protease, HsAtg4A, did not change under any of the tested conditions (Supplementary Figure 4A), supporting the idea that the regulation of Atg4 activity is post-translational. Specific anti-HsAtg4A antibodies were utilized to confirm that the cleavage activity shown in Figure 4C is mediated by HsAtg4A (for details see Supplementary Figure 4B), indicating that Atg4 is a target for redox regulation under starvation, and suggesting that accumulation of lipidated Atg8s under starvation might result from inhibition of Atg4 activity. Indeed, we found that treatment with 1 mM H2O2 resulted in accumulation of lipidated LC3 (Supplementary Figure 4C). Figure 4.The activity of endogenous Atg4 is inhibited under nutrient starvation. (A) CHO cells (for mammalian Atg8s) or cells of S. cerevisiae (for ScAtg8) were incubated in starvation medium (EBSS or SD-N, respectively) for different time periods as indicated before isolation of RNA by the Tri-reagent as explained in Materials and methods. For each gene, data obtained from non-starved cells were set to an arbitrary value of 1, and results from the corresponding starved cells were normalized accordingly. Results represent the means±s.d. of three separate experiments. (B) HEK 293 cells were grown in control medium or starved for 2 h in EBSS in the absence or presence of 100 nM bafilomycin A1. Lysates (100 μg) obtained in Ripa buffer were run on 12% SDS–PAGE and analyzed by Western blot, using anti-GATE-16 or anti-tubulin antibodies. (C) CHO cells were grown in a control medium in the presence or absence of 1 mM H2O2 for 1 h, or starved for 3 or 13 h. Lysates (10 μg) obtained in Ripa buffer were incubated with recombinant His6-GATE-16-HA (0.3 μg) in 50 KT reaction buffer (25 mM Tris, pH 7.4, 50 mM KCl) at 30°C for 45 min, in the presence or absence of 1 mM DTT. The reaction was stopped by addition of sample buffer and boiling, after which the samples were resolved on 15% SDS–PAGE and subsequently analyzed by Western blot, using anti-His monoclonal antibodies. The experiment was repeated six times; a representative blot is shown. (D) Left panel: HeLa cells transfected with Myc-GATE-16-HA or with an empty vector as control were labeled with [35S]methionine for 10 min and lysed immediately or chased for 1 h before lysis in Ripa buffer. Lysates were immunoprecipitated using anti-Myc antibodies and the immunoprecipitates were resolved on 15% SDS–PAGE. Middle panel: HeLa cells transfected with Myc-GATE-16-HA were kept in control medium or starved for 30 min or 13 h in EBSS before labeling with [35S]methionine for 10 min, immediate lysis, analysis by SDS–PAGE and quantification (right panel) using NIH image program. Values are presented as the average percentage of unprimed form out of the total GATE-16. (*) in all sections of this figure indicates non-cleaved His6-GATE-16-HA or Myc-GATE-16-HA and (**) indicates cleaved His6-GATE-16 or Myc-GATE-16. Download figure Download PowerPoint Atg4 acts both in the initial processing of Atg8s (priming) and in the delipidation step. The priming step was previously shown to occur immediately after translation under nutrient-rich conditions (Kabeya et al, 2000). To test whether this step is also inhibited during starvation, we transfected cells with GATE-16 tagged with Myc at its N-terminus and HA at the C-terminus, starved them for 30 min (a time period in which autophagosomes are expected to form; Fass et al, 2006) or 13 h and subjected them to a [35S]methionine pulse–chase experiment as detailed in Materials and methods (Figure 4D). The control experiment shows that two bands at approximately 19 kDa are detected immediately after pulse labeling in the transfected cells (Figure 4D, left panel). A 1 h chase resulted in disappearance of the upper band, suggesting that this band corresponds to unprimed GATE-16 (Myc-GATE-16-HA), whereas the lower band corresponds to primed GATE-16 (Myc-GATE-16). Short starvation does not induce accumulation of the unprimed form (Figure 4D, middle panel), and this form accumulates only in cells that were starved for a long period of 13 h. These results indicate that HsAtg4A is redox regulated during starvation. As the priming activity is not affected by short starvation, we conclude that the delipidation activity is the main target of this regulation. To characterize the redox regulation of HsAtg4, we expressed HsAtg4A as a recombinant protein and tested its activity in the cell-free cleavage assay. As depicted in Figure 5A, the protease was gradually activated upon reduction of the reaction mixture and the activity saturated at 1 mM DTT. Next, we tested the inhibition of HsAtg4A activity by H2O2. The protein was preincubated in a low concentration of DTT for activation followed by addition of H2O2 for 5 min before incubation with GATE-16 for the cleavage assay. The activity of HsAtg4A decreased as the H2O2 concentration increased above 100 μM (Figure 5B). In some experiments, a slight increase in the protease activity was detected upon addition of 30 μM H2O2 to the reaction mixture. Finally, we tested the reversibility of HsAtg4 inhibition by H2O2. In this experiment, the protein was preincubated in a low concentration of DTT (Figure 5C, lane 2) followed by addition of H2O2 (lane 3) and then addition of a high concentration of DTT (lane 4; each incubated for 5 min before addition of the next reagent) before incubation with GATE-16. Under these conditions, HsAtg4A completely regained its activity. Taken together, these results show that reducing conditions activate HsAtg4A, whereas an oxidizing environment inhibits its activity. Moreover, these results indicate that H2O2 alone is sufficient to reversibly inhibit the activity of HsAtg4A in vitro, and therefore suggest that H2O2 might also act directly on the protease in vivo. Figure 5.H2O2 directly inhibits the activity of HsAtgA. (A) Cleavage activity was tested by incubation of recombinant His6-HsAtg4A (0.1 μg) and His6-GATE-16-HA (0.3 μg) in 50 KT reaction buffer at 30°C for 45 min in the presence of indicated concentrations of DTT followed by Western blot analysis, using anti-His monoclonal antibodies. The resulting bands from three separate experiments were quantified with a densitometer using the Bio-Rad Multi-Analyst program and are presented as the average percentage of cleaved form out of the total GATE-16. (*) indicates non-cleaved His6-GATE-16-HA and (**) indicates cleaved His6-GATE-16. (B) His6-HsAtg4A was incubated in the presence of 200 μM DTT at 4°C for 10 min. Reduced His6-HsAtg4A (0.1 μg) was then incubated in 50 KT (to obtain 15 μM DTT) with the indicated concentrations of H2O2 at 25°C for 5 min, after which recombinant His6-GATE-16-HA (0.3 μg) was added and incubation proceeded at 30°C for 45 min. Reaction mixtures from three separate experiments were analyzed and are presented as explained in (A). (C) His6-HsAtg4A (0.1 μg) was incubated with recombinant His6-GATE-16-HA (0.3 μg) after the following procedures: no treatment (lane 1); pretreatment with 200 μM DTT at 25°C for 5 min (lane 2); treatment with 200 μM DTT followed by treatment with 1 mM H2O2 at 25°C for 5 min (lane 3); and treatment with 200 μM DTT followed by treatment with 1 mM H2O2 for 5 min and then 2 mM DTT (lane 4). Reaction mixtures were analyzed by Western blot using anti-His monoclonal antibodies. Download figure Download PowerPoint Cys81 is a target for the redox regulation of HsAtg4A HsAtg4A contains 12 cysteines, seven of which are highly conserved among tetrapod homologues of Atg4A and Atg4B. The latter share an overall high conservation level with respect to the whole family (Figure 6). One of these conserved residues is the putative active residue of the protease Cys77 (Kirisako et al, 2000; Tanida et al, 2004a), the only cysteine conserved also in the yeast S. cerevisiae (Figure 6A, large arrow, numbering is according to HsAtg4B). Another conserved residue (Cys81 in HsAtg4A) is located four sites downstream of the active residue. We first sought to determine whether Cys77 is the catalytic residue, as suggested by homology to ScAtg4 and HsAtg4B. As shown in Figure 7A, recombinant HsAtg4A harboring a mutation of cysteine to alanine at position 77 (His6-HsHsAtg4AC77A) did not cleave GATE-16 in vitro, neither in the absence nor in the presence of DTT. Based on this result and the sequence homology described above, we conclude that Cys77 is part of the active site of HsAtg4A. Figure 6.Tetrapod homologues of Atg4 share several conserved cysteine residues. (A)