Glutathione Participates in the Regulation of Mitophagy in Yeast
2009; Elsevier BV; Volume: 284; Issue: 22 Linguagem: Inglês
10.1074/jbc.m109.005181
ISSN1083-351X
AutoresMaïka S. Deffieu, Ingrid Bhatia-Kiššová, Bénédicte Salin, Anne Galinier, Stéphen Manon, Nadine Camougrand,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoThe antioxidant N-acetyl-l-cysteine prevented the autophagy-dependent delivery of mitochondria to the vacuoles, as examined by fluorescence microscopy of mitochondria-targeted green fluorescent protein, transmission electron microscopy, and Western blot analysis of mitochondrial proteins. The effect of N-acetyl-l-cysteine was specific to mitochondrial autophagy (mitophagy). Indeed, autophagy-dependent activation of alkaline phosphatase and the presence of hallmarks of non-selective microautophagy were not altered by N-acetyl-l-cysteine. The effect of N-acetyl-l-cysteine was not related to its scavenging properties, but rather to its fueling effect of the glutathione pool. As a matter of fact, the decrease of the glutathione pool induced by chemical or genetical manipulation did stimulate mitophagy but not general autophagy. Conversely, the addition of a cell-permeable form of glutathione inhibited mitophagy. Inhibition of glutathione synthesis had no effect in the strain Δuth1, which is deficient in selective mitochondrial degradation. These data show that mitophagy can be regulated independently of general autophagy, and that its implementation may depend on the cellular redox status. The antioxidant N-acetyl-l-cysteine prevented the autophagy-dependent delivery of mitochondria to the vacuoles, as examined by fluorescence microscopy of mitochondria-targeted green fluorescent protein, transmission electron microscopy, and Western blot analysis of mitochondrial proteins. The effect of N-acetyl-l-cysteine was specific to mitochondrial autophagy (mitophagy). Indeed, autophagy-dependent activation of alkaline phosphatase and the presence of hallmarks of non-selective microautophagy were not altered by N-acetyl-l-cysteine. The effect of N-acetyl-l-cysteine was not related to its scavenging properties, but rather to its fueling effect of the glutathione pool. As a matter of fact, the decrease of the glutathione pool induced by chemical or genetical manipulation did stimulate mitophagy but not general autophagy. Conversely, the addition of a cell-permeable form of glutathione inhibited mitophagy. Inhibition of glutathione synthesis had no effect in the strain Δuth1, which is deficient in selective mitochondrial degradation. These data show that mitophagy can be regulated independently of general autophagy, and that its implementation may depend on the cellular redox status. Autophagy is a major pathway for the lysosomal/vacuolar delivery of long-lived proteins and organelles, where they are degraded and recycled. Autophagy plays a crucial role in differentiation and cellular response to stress and is conserved in eukaryotic cells from yeast to mammals (1Huang W.P. Klionsky D.J. Cell Struct. Funct. 2002; 27: 409-420Crossref PubMed Scopus (162) Google Scholar, 2Reggiori F. Klionsky D.J. Curr. Opin. Cell Biol. 2005; 17: 415-422Crossref PubMed Scopus (237) Google Scholar). The main form of autophagy, macroautophagy, involves the non-selective sequestration of large portions of the cytoplasm into double-membrane structures termed autophagosomes, and their delivery to the vacuole/lysosome for degradation. Another process, microautophagy, involves the direct sequestration of parts of the cytoplasm by vacuole/lysosomes. The two processes coexist in yeast cells but their extent may depend on different factors including metabolic state: for example, we have observed that nitrogen-starved lactate-grown yeast cells develop microautophagy, whereas nitrogen-starved glucose-grown cells preferentially develop macroautophagy (3Kissová I. Salin B. Schaeffer J. Bhatia S. Manon S. Camougrand N. Autophagy. 2007; 3: 329-336Crossref PubMed Scopus (176) Google Scholar). Both macroautophagy and microautophagy are essentially non-selective, in the way that autophagosomes and vacuole invaginations do not appear to discriminate the sequestered material. However, selective forms of autophagy have been observed (4Nair U. Klionsky D.J. J. Biol. Chem. 2005; 280: 41785-41788Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) that target namely peroxisomes (5Sakai Y. Koller A. Rangell L.K. Keller G.A. Subramani S. J. Cell Biol. 1998; 141: 625-636Crossref PubMed Scopus (197) Google Scholar, 6Kiel J.A. Komduur J.A. Van der Klei I.J. Veenhuis M. FEBS Lett. 2003; 549: 1-6Crossref PubMed Scopus (41) Google Scholar), chromatin (7Pan X. Roberts P. Chen Y. Kvam E. Shulga N. Huang K. Lemmon S. Goldfarb D.S. Mol. Biol. Cell. 2000; 11: 2445-2457Crossref PubMed Scopus (235) Google Scholar, 8Roberts P. Moshitch-Moshkovitz S. Kvam E. O'Toole E. Winey M. Goldfarb D.S. Mol. Biol. 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Although non-selective autophagy plays an essential role in survival by nitrogen starvation, by providing amino acids to the cell, selective autophagy is more likely to have a function in the maintenance of cellular structures, both under normal conditions as a “housecleaning” process, and under stress conditions by eliminating altered organelles and macromolecular structures (14Tolkovsky A.M. Xue L. Fletcher G.C. Borutaite V.M. Biochimie. 2002; 84: 233-240Crossref PubMed Scopus (190) Google Scholar, 15Lemasters J.J. Rejuvenation Res. 2005; 8: 3-5Crossref PubMed Scopus (954) Google Scholar, 16Kundu M. Thompson C.B. Cell Death Differ. 2005; 12: 1484-1489Crossref PubMed Scopus (97) Google Scholar). Selective autophagy targeting mitochondria, termed mitophagy, may be particularly relevant to stress conditions. The mitochondrial respiratory chain is both the main site and target of ROS 4The abbreviations used are: ROS, reactive oxygen species; EA, ethacrynic acid; GSH, reduced glutathione; GSSG, glutathione disulfide; GFP, green fluorescent protein; mtGFP, mitochondria-targeted green fluorescent protein; NAC, N-acetyl-l-cysteine; PMSF, phenylmethanesulfonyl fluoride. production (17Turrens J.F. J. Physiol. 2003; 552: 335-344Crossref PubMed Scopus (3657) Google Scholar). Consequently, the maintenance of a pool of healthy mitochondria is a crucial challenge for the cells. The progressive accumulation of altered mitochondria (18Lenaz G. Bovina C. D'Aurelio M. Fato R. Formiggini G. Genova M.L. Giuliano G. Merlo Pich M. Paolucci U. Parenti Castelli G. Ventura B. Ann. N.Y. Acad. 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Med. 2006; 41: 1655-1661Crossref PubMed Scopus (62) Google Scholar). Furthermore, resveratrol treatment impaired autophagic degradation of both cytosolic and mitochondrial proteins and delayed rapamycin-induced cell death, suggesting that mitochondrial oxidation events may play a crucial role in the regulation of autophagy. This existence of regulation of autophagy by ROS has received molecular support in HeLa cells (30Scherz-Shouval R. Shvets E. Fass E. Shorer H. Gil L. Elazar Z. EMBO J. 2007; 26: 1749-1760Crossref PubMed Scopus (1674) Google Scholar): these authors showed that starvation stimulated ROS production, namely H2O2, which was essential for autophagy. Furthermore, they identified the cysteine protease hsAtg4 as a direct target for oxidation by H2O2. This provided a possible connection between the mitochondrial status and regulation of autophagy. Investigations of mitochondrial autophagy in nitrogen-starved lactate-grown yeast cells have established the existence of two distinct processes: the first one occurring very early, is selective for mitochondria and is dependent on the presence of the mitochondrial protein Uth1p; the second one occurring later, is not selective for mitochondria, is not dependent on Uth1p, and is a form of bulk microautophagy (3Kissová I. Salin B. Schaeffer J. Bhatia S. Manon S. Camougrand N. Autophagy. 2007; 3: 329-336Crossref PubMed Scopus (176) Google Scholar). The absence of the selective process in the Δuth1 mutant strongly delays and decreases mitochondrial protein degradation (3Kissová I. Salin B. Schaeffer J. Bhatia S. Manon S. Camougrand N. Autophagy. 2007; 3: 329-336Crossref PubMed Scopus (176) Google Scholar, 12Kissová I. Deffieu M. Manon S. Camougrand N. J. Biol. Chem. 2004; 279: 39068-39074Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). The putative protein phosphatase Aup1p has been also shown to be essential in inducing mitophagy (31Tal R. Winter G. Ecker N. Klionsky D.J. Abeliovich H. J. Biol. Chem. 2007; 282: 5617-5624Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Additionally several Atg proteins were shown to be involved in vacuolar sequestration of mitochondrial GFP (3Kissová I. Salin B. Schaeffer J. Bhatia S. Manon S. Camougrand N. Autophagy. 2007; 3: 329-336Crossref PubMed Scopus (176) Google Scholar, 12Kissová I. Deffieu M. Manon S. Camougrand N. J. Biol. Chem. 2004; 279: 39068-39074Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 32Rosado C.J. Mijaljica D. Hatzinisiriou I. Prescott M. Devenish R.J. Autophagy. 2008; 4: 205-213Crossref PubMed Scopus (95) Google Scholar, 33Kanki T. Klionsky D.J. J. Biol. Chem. 2008; 283: 32386-32393Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). Recently, the protein Atg11p, which had been already identified as an essential protein for selective autophagy has also been reported as being essential for mitophagy (33Kanki T. Klionsky D.J. J. Biol. Chem. 2008; 283: 32386-32393Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). The question remains as to identify of the signals that trigger selective mitophagy. It is particularly intriguing that selective mitophagy is activated very early after the shift to a nitrogen-deprived medium (3Kissová I. Salin B. Schaeffer J. Bhatia S. Manon S. Camougrand N. Autophagy. 2007; 3: 329-336Crossref PubMed Scopus (176) Google Scholar). Furthermore, selective mitophagy is very active on lactate-grown cells (with fully differentiated mitochondria) but is nearly absent in glucose-grown cells (3Kissová I. Salin B. Schaeffer J. Bhatia S. Manon S. Camougrand N. Autophagy. 2007; 3: 329-336Crossref PubMed Scopus (176) Google Scholar). In the present paper, we investigated the relationships between the redox status of the cells and selective mitophagy, namely by manipulating glutathione. Our results support the view that redox imbalance is a trigger for the selective elimination of mitochondria. Yeast Strains, Plasmids, and Growth Conditions—W303-1B (MAT α, ade2, his3, leu2, trp1, ura3) was used as the parental strain of the mutant Δuth1 (MAT α, ade2, his3, leu2, trp1, ura3, uth1::TRP1) (34Camougrand N. Grelaud-Coq A. Marza E. Priault M. Bessoule J.J. Manon S. Mol. Microbiol. 2003; 47: 495-506Crossref PubMed Scopus (68) Google Scholar). For measurement of alkaline phosphatase activity, the PHO8 locus was replaced with PHO8Δ60 by transformation with an HindIII fragment of plasmid pTN9 (a gift from Y. Ohsumi, NIBB Okazaki, Japan) bearing PHO8Δ60 as described previously (35Noda T. Matsuura A. Wada Y. Ohsumi Y. Biochem. Biophys. Res. Commun. 1995; 210: 126-132Crossref PubMed Scopus (295) Google Scholar). For fluorescent microscopy experiments, cells were transformed with plasmid pGAL-CLbGFP containing the presequence of the mitochondrial citrate synthase fused to GFP, under the control of a GAL1/10 promoter (36Okamoto K. Perlman P.S. Butow R.A. J. Cell Biol. 1998; 142: 613-623Crossref PubMed Scopus (122) Google Scholar). The strain Δgsh2 carrying the deletion gsh2::kanMX4 and its parent BY4742 (MATα, his3, leu2, lys2, ura3) were from the Euroscarf collection. Yeast cells were grown aerobically at 28 °C in a minimal medium containing 0.175% YNB (yeast nitrogen base without amino acids and ammonium sulfate), 0.5% ammonium sulfate, 0.1% potassium phosphate, 0.2% Drop-Mix, 0.01% auxotrophic requirements (pH 5.5) supplemented with 2% dl-lactate as a carbon source. Nitrogen starvation medium (SD-N) contained 0.175% YNB without amino acids and ammonium sulfate, supplemented with 2% lactate as a carbon source. Western Blot Analyses—Protein sample preparations, SDS-PAGE, and Western blots were previously described (34Camougrand N. Grelaud-Coq A. Marza E. Priault M. Bessoule J.J. Manon S. Mol. Microbiol. 2003; 47: 495-506Crossref PubMed Scopus (68) Google Scholar). Primary antibodies were rabbit polyclonal anti-yeast Atp6p (1/10,000), mouse monoclonal anti-yeast porin (Molecular Probes; 1/5,000), mouse monoclonal anti-yeast phosphoglycerate kinase (Molecular Probes, 1/5,000), and mouse monoclonal anti-GFP (Roche, 1/5,000). Peroxidase-coupled secondary anti-mouse and anti-rabbit antibodies (Jackson Laboratories) were used at 1/5,000. ECL+ (GE Healthcare) was used for detection. Scanning of non-saturated Western blots were quantified with ImageJ software. Alkaline Phosphatase Assay—The alkaline phosphatase activity assay using α-naphtyl phosphate as a substrate was performed as described previously (12Kissová I. Deffieu M. Manon S. Camougrand N. J. Biol. Chem. 2004; 279: 39068-39074Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). Fluorescence intensity was measured at 472 nm (excitation at 345 nm) in a Safas Xenius spectrofluorimeter. Protein concentration was measured with the Lowry method. Activities were expressed as arbitrary fluorescence units per minute per mg of proteins. Epifluorescence Microscopy—To induce mtGFP expression, cells carrying the pGAL-CLbGFP plasmid were grown overnight in the appropriate medium supplemented with 0.5% galactose. GFP was visualized on an epifluorescence microscope (Leica Microsystems DM-LB). The images were acquired with a SIS camera and processed with Corel Draw 9.0 suite software. Transmission Electron Microscopy—For electron microscopy experiments, cells were grown and starved in the presence of 1 mm PMSF to prevent the rapid proteolytic degradation of autophagic bodies. Harvested cells were placed on the surface of Formvar-coated copper grids (400 mesh). Each loop was quickly submersed in liquid propane (–180 °C) and then transferred to a precooled solution of 4% osmium tetroxide in dry acetone at –82 °C for 48 h for substitution/fixation. Samples were gradually warmed to room temperature, and washed in dry acetone. Specimens were stained for 1 h with 1% uranyl acetate in acetone at 4 °C, rinsed, and infiltrated with araldite (epoxy resin, Fluka). Ultrathin sections were stained with lead citrate. Observations were performed on a Philips Tecnai 12 Biotwin (120 kV) electron microscope. Glutathione Measurement—6 × 107 cells were harvested and the pellet was resuspended in 350 μl of 3.4% metaphosphoric acid. Glass beads (0.4 mm diameter) were added and cells were vortexed (4 × 30 s) and then centrifuged for 2 min at 10,000 × g. Supernatants were used to measure reduced glutathione (GSH) and glutathione disulfide (GSSG) by reversephase high pressure liquid chromatography with electrochemical detection (37Melnyk S. Pogribna M. Pogribny I. Hine R.J. James S.J. J. Nutr. Biochem. 1999; 10: 490-497Crossref PubMed Scopus (199) Google Scholar). Cell Respiration—Oxygen consumption by growing or nitrogen-starved cells was measured polarographically at 28 °C using a Clark oxygen electrode in a 1-ml thermostatically controlled chamber. Respiratory rates (JO2) were determined from the slope of a plot of O2 concentration versus time. NAD(P)H and NAD(P)+ Measurements (38Klingenberg M. Methods of Enzymatic Analysis VII. VCH, Weinheim1985: 251-271Google Scholar)—NAD(P)H was measured in neutralized methanolic KOH extracts. NAD(P)H disappearance was fluorometrically monitored after the addition of dihydroxyacetone phosphate and glycerol-3-phosphate dehydrogenease (EC 1.1.1.8) for NADH or α-cetoglutarate and glutamate dehydrogenase (EC 1.4.1.3) for NADPH. NAD(P)+ was measured in neutralized HClO4 extracts. NAD+ and NADP+ were fluorometrically monitored as the formation of NADH or NADPH following the addition of ethanol and alcohol dehydrogenase (EC 1.1.1.1) for NAD+ or glucose 6-phosphate and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) for NADP+. All the measurements were done on a Safas Xenius fluorimeter (excitation at 365 nm, emission at 460 nm). ROS Measurements—Growing or nitrogen-starved cells were diluted in a fresh medium (2 × 106 cells/ml) and incubated for 15 min in the presence of 20 μm dihydroethidium, in the dark, under agitation. The fluorescence of ethidium resulting from dihydroethidium oxidation was measured in a Partec Galaxy flow cytometer (FL3 channel). N-Acetyl-l-cysteine (NAC) Inhibits Nitrogen Starvation-induced Mitophagy—We have previously reported that hydrophobic antioxidants resveratrol and α-tocopherol inhibited pharmacologically induced mitochondrial autophagy by the TOR (target of rapamycin) kinase inhibitor rapamycin (29Kissová I. Deffieu M. Samokhvalov V. Velours G. Bessoule J.J. Manon S. Camougrand N. Free Radic. Biol. Med. 2006; 41: 1655-1661Crossref PubMed Scopus (62) Google Scholar). To investigate the possible role of oxidative events in the regulation of mitochondrial autophagy under physiological conditions, lactate-grown cells were submitted to nitrogen starvation, in the presence of various known antioxidants, namely NAC, Tiron, l-ascorbic acid, and resveratrol. Mitochondrial morphology and vacuolar delivery were followed by fluorescence microscopy of mtGFP (Fig. 1). Under growth conditions, lactate-grown cells exhibited a well differentiated mitochondrial network. Under conditions of physiologically induced autophagy by nitrogen starvation, the appearance of patches of mtGFP at the periphery of the cells, and the simultaneous disappearance of the mitochondrial network were observed. These changes in mitochondrial morphology were followed by extensive delivery of mtGFP into vacuoles: pictures taken after 24 h nitrogen starvation revealed that about 95% of cells had mtGFP in the vacuoles. Tiron, l-ascorbic acid, and resveratrol had no effect on this process. On the contrary, in the presence of NAC, the mitochondrial network was still disrupted but only 4% of cells had mtGFP in the vacuoles. No increase in vacuolar delivery of mtGFP was observed even after 72 h of nitrogen starvation (data not shown). To examine the possibility that NAC could be a source of nitrogen for starved cells, each of 20 amino acids was individually assayed for a possible effect on vacuolar delivery of mitochondria induced by nitrogen starvation. Results showed that cysteine had the same effect as NAC, whereas none of the other amino acids had any effect, as exemplified in Fig. 2 for glutamine, asparagine (which are efficient sources of nitrogen for yeast), and proline (which is poorly utilized as source of nitrogen) (39Watson T.G. J. Gen. Microbiol. 1976; 96: 263-268Crossref PubMed Scopus (68) Google Scholar). This showed that the protective effects of NAC and cysteine on mitophagy are not related to nitrogen supply. Furthermore, to confirm that NAC actually impaired mitophagy, the hallmarks of this process were investigated. The amounts of two mitochondrial proteins localized in the outer membrane (porin, Por1p) and inner membrane (subunit 6 of ATP synthase, Atp6p) were followed by Western blot analysis, in comparison to a cytosolic protein, phosphoglycerate kinase (Pgk1p) (Fig. 3, A and B). After 24 h of nitrogen starvation, the content of both mitochondrial proteins was depressed, whereas the amount the cytosolic protein was only marginally affected. The degradation of the two mitochondrial proteins was largely impaired when cells were starved in the presence of NAC. In the presence of the hydrophobic antioxidant resveratrol, mitochondrial proteins were still degraded, in accordance with results from fluorescence microscopy (Fig. 1). We also followed the degradation of the cit1-GFP fusion protein by Western blot during nitrogen starvation. GFP is resistant to proteolysis and the vacuolar delivery of fusion proteins with GFP was shown to be associated to a size reduction corresponding to the degradation of the fused fragment (33Kanki T. Klionsky D.J. J. Biol. Chem. 2008; 283: 32386-32393Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). We observed cleavage of fusion protein cit1-GFP to GFP during nitrogen starvation, which was inhibited both by PMSF (inhibitor of vacuolar proteases) and NAC (Fig. 3C). This further confirmed the protective effect of NAC on autophagic mitochondrial proteins degradation. Electron microscopy of nitrogen-starved lactate-grown cells has previously revealed the existence of two distinct processes for mitochondrial autophagy (3Kissová I. Salin B. Schaeffer J. Bhatia S. Manon S. Camougrand N. Autophagy. 2007; 3: 329-336Crossref PubMed Scopus (176) Google Scholar). The first process was characterized by the early and transient appearance of vacuoles/mitochondria contacts and the later appearance of mitochondria-containing vesicles inside the vacuoles. The second process involved a late and non-exclusive engulfment of mitochondria with a significant proportion of surrounding cytosol (non-selective microautophagy). After 2 h of nitrogen starvation under lactate as the carbon source, a maximum of 85% of cells displayed vacuole/mitochondria contacts (Figs. 4 and supplemental S1). As previously reported (3Kissová I. Salin B. Schaeffer J. Bhatia S. Manon S. Camougrand N. Autophagy. 2007; 3: 329-336Crossref PubMed Scopus (176) Google Scholar), this proportion decreased to 35% after 3 h and completely disappeared after 4 h. These vacuole/mitochondria contacts were early and transient events. After 3 h of nitrogen starvation, the presence of mitochondria within vacuoles was visualized in 75% of the cells. However, in the presence of NAC, 13% of cells exhibited mitochondria/vacuoles contacts after 2 h of starvation and 13% of cells showed mitochondria sequestration in vacuoles after 3 h of starvation. These observations suggested that in the presence of NAC both processes (mitochondria/vacuoles contacts and mitochondria sequestration in vacuoles) were strongly impaired, although not completely abolished. Moreover, after 3 h of starvation, the average number of mitochondria sequestered in vacuoles was 1–2 units/slice in the presence of NAC compared with 5–7 units/slice in the absence of NAC (data not shown). These data confirmed that autophagic mitochondrial degradation was inhibited by NAC. NAC Does Not Inhibit Nonselective Autophagy—We investigated if the non-selective autophagic process was altered by NAC. A well established biochemical method to measure non-selective autophagy is to follow vacuolar delivery and the further activation of Pho8Δ60p, a cytosolic variant of alkaline phosphatase Pho8p. The full-length protein Pho8p is exported to the vacuoles via the normal secretory pathway, where proteinase B-dependent cleavage of the C-terminal propeptide leads to the activation of the alkaline phosphatase activity. The N-terminal truncated mutant Pho8Δ60p cannot be exported and remains inactive in the cytosol. Upon induction of autophagy, it is sequestered by autophagosomes (macroautophagy) or vacuole invaginations (microautophagy) and delivered to the vacuole where activation takes place. The level of alkaline phosphatase activity in yeast cells where Pho8Δ60p has been substituted for Pho8p is therefore a quantitative measurement of non-selective autophagy. The alkaline phosphatase activity was measured in lactate-grown Pho8Δ60p-bearing cells submitted to nitrogen starvation, in the absence or presence of NAC (Fig. 5A). Alkaline phosphatase activity increased, independently of the presence of NAC, showing that this molecule did not inhibit non-selective autophagy. To confirm this observation, electron micrographs were traced for the presence of hallmarks of non-selective autophagy. We have previously reported that macroautophagy is nearly absent from lactate-grown cells submitted to nitrogen starvation and that only hallmarks of microautophagy (direct sequestration of surrounding cytoplasm by the vacuole and appearance of autophagic vesicles) could be observed under these conditions (3Kissová I. Salin B. Schaeffer J. Bhatia S. Manon S. Camougrand N. Autophagy. 2007; 3: 329-336Crossref PubMed Scopus (176) Google Scholar). The characteristics of non-selective microautophagy have still been observed in the presence of NAC, further supporting the hypothesis that non-selective autophagy remained active in the presence of the molecule (Fig. 5B). The Role of Glutathione Metabolism—NAC has been shown to act as an antioxidant through at least two different processes: (i) it scavenges ROS through the reaction with its thiol group (40Aruoma O.I. Halliwell B. Hoey B.M. Butler J. Free Radic. Biol. Med. 1989; 6: 593-597Crossref PubMed Scopus (1627) Google Scholar) and (ii) it stimulates glutathione synthesis after being converted to cysteine. To discriminate between these two effects, different experiments were carried out. ROS production during nitrogen starvation was measured as the conversion of non-fluorescent dihydroethidium to fluorescent ethidium. Because cellular respiration strongly decreased when cells were submitted to starvation (Table 1, W303), ROS production was normalized to the rate of oxygen consumption (Fig. 6): this ratio markedly increased after 6 and 24 h of starvation. Moreover, NAC did not affect this increase. This result suggested that the effect of NAC on mitophagy did not occur through its ROS scavenging properties. This observation was confirmed by comparing the effects of the d- and l-stereoisomers of N-acetylcysteine. N-Acetyl-d-cysteine has the same scavenging properties as the N-acetyl-l-cysteine (NAC), but cannot enter the glutathione metabolic pathway (41Sjödin K. Nilsson E. Hallberg A. Tunek A. Biochem. Pharmacol. 1989; 38: 3981-3985Crossref PubMed Scopus (107) Google Scholar). Contrary to the l-stereoisomer, the d-stereoisomer was without effect on sequestration of the mitochondrial GFP in the vacuoles following nitrogen starvation, because 96% of cells had mtGFP in the vacuoles after 24 h of starvation. This further suggested that the action of N-acetyl-l-cysteine on mitophagy involved its effect on glutathione metabolism but not its scavenging properties.TABLE 1Effect of NAC on cellular metabolic parameters
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