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Excess Peroxisomes Are Degraded by Autophagic Machinery in Mammals *

Excess Peroxisomes Are Degraded by Autophagic Machinery in Mammals * THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 7, pp. 4035–4041, February 17, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Excess Peroxisomes Are Degraded by Autophagic Machinery in Mammals Received for publication, November 15, 2005 Published, JBC Papers in Press, December 6, 2005, DOI 10.1074/jbc.M512283200 ‡§1 ‡1 ‡§ ¶ ‡ ‡ § Jun-ichi Iwata , Junji Ezaki , Masaaki Komatsu , Sadaki Yokota , Takashi Ueno , Isei Tanida , Tomoki Chiba , § ‡2 Keiji Tanaka , and Eiki Kominami ‡ § From the Department of Biochemistry, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113-8421, the Department of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, and the Biology Laboratory, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Tamaho-machi, Yamanashi 409-38, Japan Peroxisomes are degraded by autophagic machinery termed are degenerated by an autophagy-related process named pexophagy “pexophagy” in yeast; however, whether this is essential for peroxi- during glucose or ethanol adaptation (10–14). Yeast genetics of some degradation in mammals remains unknown. Here we have pexophagy revealed that most autophagy-related (Atg) genes play indis- shown that Atg7, an essential gene for autophagy, plays a pivotal role pensable roles in this selective degradation of peroxisomes as well as in the degradation of excess peroxisomes in mammals. Following autophagy (8, 9, 13, 15, 16). In mammals, however, whether or not the induction of peroxisomes by a 2-week treatment with phthalate autophagic machinery is involved in the degradation of excess peroxi- esters in control and Atg7-deficient livers, peroxisomal degradation somes biosynthesized in response to drug cues remains a mystery. In was monitored within 1 week after discontinuation of phthalate particular, there is no direct evidence for the degradation of disused esters. Although most of the excess peroxisomes in the control liver peroxisomes by the autophagic machinery, and it is not clear whether were selectively degraded within 1 week, this rapid removal was such a degradation process, if any, is selective or non-selective. It has exclusively impaired in the mutant liver. Furthermore, morpholog- also been reported that selective degradation of mitochondria may ical analysis revealed that surplus peroxisomes, but not mutant occur via autophagy-related mechanism in yeast (17, 18). Therefore, hepatocytes, were surrounded by autophagosomes in the control. selectivity in the organelle turnover via autophagy is an important issue. Our results indicated that the autophagic machinery is essential for Among the many Atg genes that regulate autophagy, Atg7, which the selective clearance of excess peroxisomes in mammals. This is encodes a ubiquitin-activating enzyme (E1)-like enzyme common to the first direct evidence for the contribution of autophagic machin- two ubiquitylation-like conjugations, the LC3 (Atg8 in yeast) and Atg12 ery in peroxisomal degradation in mammals. conjugation systems, is a critical gene for autophagosome formation in yeast and mammalian cells (19–26). It has been reported that in yeast, Atg7/Apg7/Gsa7 is essential for pexophagy in addition to autophagy Reorganization of organelles constitutively or suddenly occurs in (19, 22, 24). During mammalian autophagy, LC3-I (a cytosolic form of eukaryotic cells as an adaptation to environmental changes accompa- LC3) is lipidated to LC3-II (its autophagosomal membrane-bound nying the cell cycle, development, and differentiation (1). Such alter- form) by Atg7 (an E1-like enzyme) and Atg3 (a ubiquitin carrier protein ations are stringently regulated by biogenesis and/or degradation. In the (E2)-like enzyme) (21, 27). Recently, we have established conditional last decade, much attention was paid to the study of organelle assembly, knock-out-mice of Atg7 and have shown that Atg7 is indispensable for an interest linked with the translocation of proteins into the organelles mammalian autophagy and that the autophagy deficiency in liver leads (2). One focus of that work was peroxisomes. Peroxisomes are single to marked accumulation of cytoplasmic proteins (20). In the normal membrane-bound organelles that contribute to an array of metabolic liver, LC3 is continuously synthesized to form LC3-I, and LC3-I is sub- pathways and are specifically and markedly induced by a group of non- sequently conjugated with phosphatidylethanolamine to form LC3-II genotoxic carcinogens and endogenous steroids in rodents (3-6). during autophagy. LC3-II is then recruited to autophagosomal mem- Indeed, peroxisome proliferators increase the size, number, and branes (21, 28), and the autophagosomal LC3-II is rapidly degraded after enzymes involved in fatty acid metabolism: e.g. peroxisomal thiolase fusion of autophagosome with lysosome (29). This dynamic flow of LC3 (PT), peroxisomal bifunctional protein (BF), and fatty acid -oxidation is completely inhibited in Atg7-deficient liver and, as a consequence, of peroxisomes (7, 8). However, the mechanistic basis of peroxisome more LC3-I accumulates in the mutant liver (20). Considering that dele- turnover remains poorly understood (8, 9). tion of yeast Atg7/Gsa7 gene results in a defect of pexophagy in P. pas- In yeast species, such as Pichia pastoris, Hansenula polymorpha, Can- toris (24), the liver-specific Atg7-conditional knock-out mice will be an dida boidinii, and Saccharomyces cerevisiae, proliferating peroxisomes advantageous tool in investigating the degradation of peroxisomes in mammals. * This work was supported by Grants-in-aid 15032263, 16790195, 15590254, 09680629, In this study, we analyzed the clearance of surplus peroxisomes using and 1270040 from the Ministry of Education, Culture, Sports, Science and Technology the conditional-knock-out mice of Atg7 (20). The results indicated that of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”in autophagy is essential for the degradation of accumulated peroxisomes accordance with 18 U.S.C. Section 1734 solely to indicate this fact. in the mouse liver. Both authors contributed equally to this work. To whom correspondence should be addressed: Dept. of Biochemistry, Juntendo Uni- versity School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Tel.: EXPERIMENTAL PROCEDURES 81-3-5802-1031; Fax: 81-3-5802-5889; E-mail: [email protected]. The abbreviations used are: PT, peroxisomal thiolase; BF, bifunctional protein; DEHP, Reagents—Phthalate esters (diethylhexyl phthalate (DEHP)), corn oil, diethylhexyl phthalate; MLP, mitochondrial/lysosomal/peroxisomal; Atg, autophagy- related; BiP, binding protein; pIpC, polyinosinic acid-polycytidylic acid. and leupeptin were purchased from Sigma. FEBRUARY 17, 2006• VOLUME 281 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4035 This is an Open Access article under the CC BY license. Selective Degradation of Excess Peroxisomes FIGURE 1. The recovery process of excess peroxisomes induced by DEHP treatment. A, wild-type mice were treated with DEHP for 2 weeks (2 w DEHP) and then chased for 1 week (2 w DEHP 1w). Untreated and treated mice were dissected, and liver homogenates were fractionated into MLP, microsomal (Ms), and cytosolic (Cyt) fractions. The protein amount in each fraction was measured. Data are mean  S.D. values of five mice in each group; *, p  0.02 and ***, p  0.001. B, wild-type mice were treated as described in A. The vehicle 4036 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 7 •FEBRUARY 17, 2006 Selective Degradation of Excess Peroxisomes Animals and Treatment Regimen—C57B6J mice were used as wild- fixed in 1% reduced osmium tetroxide with 3,3-diaminobendine reac- type mice. Male mice received DEHP (1,150 mg/kg/day) or vehicle (corn tion. All tissue slices were then dehydrated in graded series of ethanol oil, 5 ml/kg/day) via sonde daily for 2 weeks, and the mice were subse- and embedded in Epon. Thin sections were cut with a diamond knife quently fed on a normal diet for 1 week to investigate the changes in using an ultramicrotome (Reichert, Vienna, Austria). Sections were proliferated peroxisomes during the recovery process according to the contrasted with 40 mM lead citrate for 5 min and examined with a protocol reported previously (7). For detection of autophagosomes by Hitachi H7500 electron microscope (Hitachi, Tokyo, Japan). electron microscopy, mice were injected with leupeptin (2 mg/100 g of Quantitative Analysis of Peroxisomes—For each tissue slice, 20 digital body weight) after administration of DEHP. All animals were sacrificed electron micrographs were acquired at 5,000 magnification, enlarged by deep anesthesia. 2.7-fold, and printed by a laser printer. Using the printed figure, we Deletion of Atg7 in Mouse Liver—Atg7 conditional knock-out mice measured the area of peroxisomes and that of the cytoplasmic area of and the heterozygotes were prepared as described previously (20). hepatocytes using a SigmaScan scientific measurement system Briefly, creatine expression in the liver was induced by intraperitoneal equipped with a computer (Jandel Scientific, San Rafael, CA). The rela- injection of polyinosinic acid-polycytidylic acid (pIpC). pIpC was tive total area of peroxisomes was calculated using the following for- injected three times at a 48-h interval. mula: (number of peroxisomes in the average area of peroxisomes/cy- F/ F/F 2 2 Preparation of the Fractions—Livers from Atg7 :Mx1 and Atg7 : toplasmic area) and expressed in m /100 m of cytoplasmic area. Mx1 mice were treated with DEHP or corn oil for 2 weeks, and at 1 week Statistical Analysis—The statistical significance of differences after treatment, they were dissected. Subfractionation of the livers was between experimental and control groups was determined by the two- accomplished by differential centrifugation according to the method of tailed Student’s t test. A p value of 0.05 was considered statistically de Duve et al. (30). Briefly, 20% homogenates were prepared in 0.25 M significant. sucrose, 10 mM HEPES-NaOH, pH 7.4 (homogenizing buffer). The RESULTS homogenate of the liver was centrifuged at 650 g for 5 min to remove nuclei and unbroken cells. The pellets were resuspended in the same Selective Degradation of Excess Peroxisomes—Phthalate ester (DEHP) volume of homogenizing buffer and were then recentrifuged. The and its active metabolite mono-ethylhexyl phthalate can cause marked supernatants from these two centrifugations were combined and used increases in both the size and the number of peroxisomes and induce as postnuclear supernatant fractions. Postnuclear supernatant fractions peroxisomal enzymes in the liver (7). Utilizing these phenomena, we were centrifuged at 10,000  g for 20 min, and pellets were used as the first investigated the specific proliferation of peroxisomes and the rapid mitochondrial/lysosomal/peroxisomal (MLP) fractions. The post-MLP recovery after removal of the drugs in mice. Wild-type mice were supernatants were further centrifuged at 105,000  g for 60 min to treated with DEHP for 2 weeks and then chased for 1 week as described precipitate microsomal fractions in pellet form. All procedures were under “Experimental Procedures.” The mice were dissected at each performed at 4 °C. period, and the liver cell lysates were fractionated into MLP, microso- Immunoblot Analysis—Immunoblotting was performed as described mal, and cytosolic fractions. DEHP administration for 2 weeks was asso- previously (19). The antibody against Mn -superoxide dismutase was ciated with about 2-fold increase in the amount of total protein in MLP, kindly provided by Prof. Naoyuki Taniguchi (Osaka University, Japan). but not in microsomal or cytosolic fractions, as compared with The antibodies for Atg7 (19), LC3 (20), BF (31), PT (32), and the -sub- untreated mice, and the amount almost returned to the basal level at 1 unit of ATP synthase (33) were prepared as described previously. The week after discontinuation of DEHP (Fig. 1A). These changes were not antibodies against tubulin and BiP were purchased from Chemicon observed in mice treated with the vehicle (data not shown). Quantitative International, Inc. (Temecula, CA) and Affinity BioReagents, Inc. densitometric analysis of immunoblotting data revealed that PT and BF, (Golden, CO), respectively. marker proteins of peroxisomes, increased significantly after adminis- Histological Examination—Livers were dissected, fixed in 4% tration of DEHP but not the vehicle, and both diminished significantly paraformaldehyde, frozen, embedded, and sectioned. For immunohis- to basal levels at 1 week after DEHP discontinuation (Fig. 1, B and C). In tochemical analysis, the sections were blocked with 5% normal goat comparison, the levels of mitochondrial proteins, the -subunit of ATP serum in phosphate-buffered saline containing 0.2% Triton X-100 and synthase and manganese superoxide dismutase, and the endoplasmic then incubated with anti-PT antibody and Alexa Fluor 488-labeled sec- reticulum marker, BiP, remained unchanged during the same manipu- ond antibody (Molecular Probes, Eugene, OR). Fluorescence images lations (Fig. 1B). Immunofluorescence analysis using anti-PT antibody were obtained using a fluorescence microscope (Q550FV; Leica, Ger- revealed that a 2-week administration of DEHP, but not the vehicle, many) equipped with cooled charge-coupled device camera (CTR MIC; resulted in the appearance of numerous dots representing peroxisomes, Leica). Pictures were taken using Leica Qfluoro software (Leica). and most of these dots disappeared at 1 week after discontinuation of Electron Microscopy—Livers were perfusion-fixed with the fixative DEHP (Fig. 1D). Considered together, these results indicate that DEHP- through the portal vein for 10 min. The fixative consisted of 2% induced peroxisomes are selectively degraded following removal of the paraformaldehyde, 1% glutaraldehyde, and 0.1 M HEPES-KOH buffer peroxisome proliferator. (pH 7.4). To visualize peroxisomes, some liver slices were incubated in Impairment of Degradation of Proliferated Peroxisomes in Autoph- alkaline 3,3-diaminobendine medium consisting of 2 mg/ml 3,3-diami- agy-deficient Liver—Next, to examine the effects of autophagy defi- nobendine, 0.02% hydrogen peroxide, and 0.2 M glycine-NaOH buffer ciency on peroxisome degradation, we took advantage of the condi- F/F (pH 10.0) for1hat room temperature. Then they were postfixed with tional knock-out mice, Atg7 :Mx1 (mutant mice), and their F/ 1% reduced osmium tetroxide for 1 h. The other tissue slices were post- littermates, Atg7 :Mx1 mice (control mice), the systems of which control mice were treated with corn oil for 2 weeks (2 w vehicle). Untreated and treated mice were sacrificed, and the livers were dissected out and homogenized, and then the postnuclear supernatant fractions were subjected to immunoblotting with anti-PT, BF, -subunit ATP synthase, Mn -superoxide dismutase (SOD), BiP, and tubulin antibodies. Tubulin was used as a control. Data shown are representative of three separate experiments. C, quantitative densitometry of immunoblotting data in B was performed, and the ratios between each of PT, BF, and ATP synthase and tubulin were plotted; **, p 0.01, ***, p 0.001. D, wild-type mice were treated with DEHP as described in A, and the frozen sections of livers were stained with anti-PT antibody to detect peroxisomes. Magnification, 400. FEBRUARY 17, 2006• VOLUME 281 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4037 Selective Degradation of Excess Peroxisomes F/ F/F FIGURE 2. The recovery process of excess peroxisomes is impaired in Atg7-deficient liver. A, Atg7 :Mx1 (F/:Mx1) and Atg7 :Mx1 (F/F:Mx1) mice were treated with DEHP for 2 weeks (2 w DEHP) and then chased for 1 week (2 w DEHP 1w). Both genotype mice were sacrificed at each time point. The liver was dissected out and homogenized, and then the postnuclear supernatant fractions were subjected to immunoblotting using anti-Atg7, LC3, BF, PT, -subunit ATP synthase, Mn -superoxide dismutase (SOD), BiP, and tubulin antibodies. Tubulin was used as control. Data shown are representative of three separate experiments. B, quantitative densitometry of Western blotting shown in A was performed, and PT/tubulin, BF/tubulin, -subunit ATP synthase/tubulin, and Mn -superoxide dismutase/tubulin ratios were plotted; *, p  0.02, **, p  0.01, NS; not significant. were recently established by our group (20). Autophagy is impaired membrane-bound form of LC3) and accumulation of LC3-I (a cytosolic F/F following pIpC injection in Atg7 :Mx1 mouse livers. Indeed, we veri- form of LC3) in the liver. It is generally accepted that LC3-II is a marker F/F F/ fied that Atg7 protein deletion in Atg7 :Mx1 but not Atg7 :Mx1 protein of autophagosomal membranes (21). Although both forms were livers was pIpC injection-dependent (Fig. 2A). Furthermore, we also detected in the control liver, only LC3-I accumulated in the mutant liver F/F tested the loss of Atg7 activity by investigating the lack of LC3-II (a (Fig. 2A), indicating impairment of autophagy in mutant Atg7 :Mx1 4038 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 7 •FEBRUARY 17, 2006 Selective Degradation of Excess Peroxisomes FIGURE 3. Accumulation of excess peroxisomes in Atg7-deficient liver. Immunofluo- F/ rescent detection of peroxisomes with anti-PT antibody in the Atg7 :Mx1 (A–C, F/: F/F F/ F/F Mx1) and Atg7 :Mx1 (D–F, F/F:Mx1) liver is shown. Atg7 :Mx1 and Atg7 :Mx1 mice were treated with DEHP for 2 weeks (B and E, 2 w DEHP) and then chased for 1 week (C and F, 2 w DEHP 1w). Untreated (A and D) and treated mice were sacrificed, and the livers were isolated. The frozen sections of livers were immunostained with anti-PT antibody. Magnification, 400. mouse liver (20). In the control livers, although LC3-II were induced by the proliferated peroxisomes (Fig. 2A, indicated by 2 w DEHP), it was decreased almost to the basal levels at 1 week after withdrawal of DEHP (Fig. 2A), suggesting that autophagy was induced to remove surplus peroxisomes. After a 2-week treatment with DEHP, the livers were dis- sected, and total proteins in the lysates of mutant and control livers were separated by SDS-PAGE and subjected to immunoblot analyses. Similar to the results obtained with wild-type mice (Fig. 1), BF and PT increased profoundly after the treatment as compared with mice prior to DEHP administration and then decreased almost to the basal levels at 1 week F/ after discontinuation in Atg7 :Mx1 livers (Fig. 2, A and B). Although FIGURE 4. Electron microscopic evaluation of livers of Atg7-deficient mice treated F/F this increase was also detected in mutant Atg7 :Mx1 livers, the F/ with DEHP. A–F, electron micrographs of the liver of representative Atg7 :Mx1 mice F/F increased PT and BT proteins did not return to the basal levels following (F/:Mx1) and Atg7 :Mx1 (F/F:Mx1) mice treated with DEHP for 2 weeks (B and E, 2w DEHP) and then fed on normal diet for 1 week (C and F, 2 w DEHP  1w). The vehicle the discontinuation of DEHP (Fig. 2, A and B). In contrast to peroxiso- control mice of each genotype were treated with corn oil for 2 weeks (A and D). The mal proteins, the levels of mitochondrial (-subunit of ATP synthase hepatocytes of both genotypes contained a high number of peroxisomes (P) after DEHP treatment (B and E). Note that induced peroxisomes were retained at 1 week after dis- and Mn -superoxide dismutase) and endoplasmic reticulum (BiP) F/F continuation of DEHP in Atg7 :Mx1 hepatocytes, in contrast to the decreased number markers did not change under these conditions (Fig. 2, A and B). These F/ in Atg7 :Mx1 hepatocytes (C and F). Bars,1 m. The total area of peroxisomes relative results indicate selective impairment of degradation of excess peroxiso- to the cytoplasmic area was determined in each genotype (n 10). M, mitochondria; G, F/ F/F F/F morphometric analysis of peroxisomes in Atg7 :Mx1 and Atg7 :Mx1 mice. mal proteins in autophagy-deficient Atg7 :Mx1 liver. We further confirmed the impairment of peroxisome degradation in autophagy-deficient liver by immunofluorescence analysis using structures disappeared after 1 week of discontinuation of DEHP in the anti-PT antibody (Fig. 3). The PT-positive dots representing peroxi- control, but not mutant, hepatocytes (Fig. 4, C and F). The relative total somes were markedly increased following a 2-week DEHP treatment in area of peroxisomes was determined, and the mean values are shown in both genotype livers, as compared with untreated mice (Fig. 3, A and D Fig. 4G. Although the relative total area of peroxisomes increased in versus B and E). Although the dots almost disappeared to the basal levels both groups after a 2-week DEHP administration, the area decreased to at 7 days after discontinuation of DEHP in the control (Fig. 3C), most of the basal level in control hepatocytes, but not in mutant hepatocytes, at the peroxisome dots remained visible in mutant liver after the same 1 week after DEHP withdrawal from the diet (Fig. 2G). intervention (Fig. 3F). The data are in agreement with the biochemical After discontinuation of DEHP, we detected only a few autophago- results shown in Fig. 2. Based on these results, we concluded that auto- some-like structures in control hepatocytes, probably due to the rapid phagy is essential for selective degradation of excess peroxisomes. turnover of autophagosomes by lysosome (Fig. 4C). Considering the Engulfment of Excess Peroxisomes by Autophagosomal Membranes in selective degradation of peroxisomal marker proteins, PT and BF (Fig. Control Hepatocytes—Finally, we used electron microscopy to explore 2), autophagosomes that selectively enwrap peroxisomes could be F/F F/ the level of the peroxisomes in Atg7 :Mx1 and Atg7 :Mx1 livers (Fig. observed by electron microscopic analysis when lysosomal proteolysis is 4). Consistent with the results of immunofluorescent analysis, numer- inhibited. Therefore, we examined whether proliferated peroxisomes ous peroxisomes were detected following a 2-week DEHP treatment in enclosed by autophagosomal membranes can be detected under the both wild and mutant hepatocytes (Fig. 4, B and E), and most of these condition of inhibited autophagic proteolysis. Injection of leupeptin, a FEBRUARY 17, 2006• VOLUME 281 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4039 Selective Degradation of Excess Peroxisomes micropexophagy. Our data using electron microscopy revealed that autophagosomes preferentially surrounded excess peroxisomes in con- trol hepatocytes (Fig. 5), suggesting that DEHP-induced peroxisomes are degraded mainly through the process of macropexophagy. Thus, we could show the selective role of autophagic machinery in the clearance of surplus peroxisomes after induction of peroxisomes by phthalate esters. Recent studies provided evidence for the involvement of the autoph- agic machinery in selective sequestration of proteins in the cell. For example, the precursor form of aminopeptidase I (prApe1) is a selective cargo molecule of autophagy in yeast (36), and cytosolic acetaldehyde F/ dehydrogenase (Ald6p) is preferentially transported to vacuoles via FIGURE 5. Excess peroxisomes are surrounded by autophagosome. Atg7 :Mx1 mice were treated with DEHP for 2 weeks and then injected with leupeptin as described autophagosomes in yeast (37). Consistently, the autophagic machinery under “Experimental Procedures.” The mice were sacrificed, and the livers were dis- could also selectively eliminate pathogenic group A Streptococci invad- sected out and processed for electron microscopic examination. These images show ing the cells (38). These reports strongly suggest that autophagosomes representative autophagosomes surrounding peroxisomes. Four typical electron micro- graphs are represented. Arrowheads indicate the engulfment of peroxisome(s) by iso- sequester the cytosolic protein(s) and invading pathogens in a highly lated membranes. Bars,1 m. selective manner. We recently reported that Atg7-deficient hepatocytes exhibit impaired constitutive autophagy responsible for selective degra- lysosomal cysteine proteinase inhibitor, into a 2-week DEHP-treated dation of ubiquitinated proteins (20). Our previous findings together F/ control Atg7 :Mx1 mouse resulted in marked accumulation of auto- with the present results suggest that the autophagic process eliminates phagosomes, and some peroxisomes were surrounded by a double- abnormal and/or excess proteins and organelles including peroxisomes membrane structure, autophagosome, in control hepatocytes (Fig. 5). in a selective manner even under normal conditions. How the autoph- F/F No autophagosome was identified in hepatocytes of Atg7 :Mx1 mice agy machinery recognizes these organelles to degrade them awaits fur- (data not shown). These lines of evidence indicated that the autophagic ther investigation. machinery mediated is essential for selective clearance of excess peroxi- somes, as it is so for starvation-induced autophagy in the mouse liver. Acknowledgment—We thank Tsuguka Kouno for technical assistance. DISCUSSION Most cellular components, if not all, are regulated quantitatively to REFERENCES maintain cell homeostasis. For this regulation, there are growing lines of 1. Lazarow, P. B., and Fujiki, Y. (1985) Annu. Rev. Cell Biol. 1, 489–530 evidence for the importance of the balance between biosynthesis and 2. Heiland, I., and Erdmann, R. (2005) FEBS J. 272, 2362–2372 degradation. Peroxisomes, a typical cellular component, are dynamic 3. Reddy, J. K., Azarnoff, D. L., Hignite, C. E. (1980) Nature 283, 397–398 4. Reddy, J. 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Science 306, 1037–1040 FEBRUARY 17, 2006• VOLUME 281 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4041 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Excess Peroxisomes Are Degraded by Autophagic Machinery in Mammals *

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American Society for Biochemistry and Molecular Biology
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0021-9258
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10.1074/jbc.m512283200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 7, pp. 4035–4041, February 17, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Excess Peroxisomes Are Degraded by Autophagic Machinery in Mammals Received for publication, November 15, 2005 Published, JBC Papers in Press, December 6, 2005, DOI 10.1074/jbc.M512283200 ‡§1 ‡1 ‡§ ¶ ‡ ‡ § Jun-ichi Iwata , Junji Ezaki , Masaaki Komatsu , Sadaki Yokota , Takashi Ueno , Isei Tanida , Tomoki Chiba , § ‡2 Keiji Tanaka , and Eiki Kominami ‡ § From the Department of Biochemistry, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113-8421, the Department of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, and the Biology Laboratory, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Tamaho-machi, Yamanashi 409-38, Japan Peroxisomes are degraded by autophagic machinery termed are degenerated by an autophagy-related process named pexophagy “pexophagy” in yeast; however, whether this is essential for peroxi- during glucose or ethanol adaptation (10–14). Yeast genetics of some degradation in mammals remains unknown. Here we have pexophagy revealed that most autophagy-related (Atg) genes play indis- shown that Atg7, an essential gene for autophagy, plays a pivotal role pensable roles in this selective degradation of peroxisomes as well as in the degradation of excess peroxisomes in mammals. Following autophagy (8, 9, 13, 15, 16). In mammals, however, whether or not the induction of peroxisomes by a 2-week treatment with phthalate autophagic machinery is involved in the degradation of excess peroxi- esters in control and Atg7-deficient livers, peroxisomal degradation somes biosynthesized in response to drug cues remains a mystery. In was monitored within 1 week after discontinuation of phthalate particular, there is no direct evidence for the degradation of disused esters. Although most of the excess peroxisomes in the control liver peroxisomes by the autophagic machinery, and it is not clear whether were selectively degraded within 1 week, this rapid removal was such a degradation process, if any, is selective or non-selective. It has exclusively impaired in the mutant liver. Furthermore, morpholog- also been reported that selective degradation of mitochondria may ical analysis revealed that surplus peroxisomes, but not mutant occur via autophagy-related mechanism in yeast (17, 18). Therefore, hepatocytes, were surrounded by autophagosomes in the control. selectivity in the organelle turnover via autophagy is an important issue. Our results indicated that the autophagic machinery is essential for Among the many Atg genes that regulate autophagy, Atg7, which the selective clearance of excess peroxisomes in mammals. This is encodes a ubiquitin-activating enzyme (E1)-like enzyme common to the first direct evidence for the contribution of autophagic machin- two ubiquitylation-like conjugations, the LC3 (Atg8 in yeast) and Atg12 ery in peroxisomal degradation in mammals. conjugation systems, is a critical gene for autophagosome formation in yeast and mammalian cells (19–26). It has been reported that in yeast, Atg7/Apg7/Gsa7 is essential for pexophagy in addition to autophagy Reorganization of organelles constitutively or suddenly occurs in (19, 22, 24). During mammalian autophagy, LC3-I (a cytosolic form of eukaryotic cells as an adaptation to environmental changes accompa- LC3) is lipidated to LC3-II (its autophagosomal membrane-bound nying the cell cycle, development, and differentiation (1). Such alter- form) by Atg7 (an E1-like enzyme) and Atg3 (a ubiquitin carrier protein ations are stringently regulated by biogenesis and/or degradation. In the (E2)-like enzyme) (21, 27). Recently, we have established conditional last decade, much attention was paid to the study of organelle assembly, knock-out-mice of Atg7 and have shown that Atg7 is indispensable for an interest linked with the translocation of proteins into the organelles mammalian autophagy and that the autophagy deficiency in liver leads (2). One focus of that work was peroxisomes. Peroxisomes are single to marked accumulation of cytoplasmic proteins (20). In the normal membrane-bound organelles that contribute to an array of metabolic liver, LC3 is continuously synthesized to form LC3-I, and LC3-I is sub- pathways and are specifically and markedly induced by a group of non- sequently conjugated with phosphatidylethanolamine to form LC3-II genotoxic carcinogens and endogenous steroids in rodents (3-6). during autophagy. LC3-II is then recruited to autophagosomal mem- Indeed, peroxisome proliferators increase the size, number, and branes (21, 28), and the autophagosomal LC3-II is rapidly degraded after enzymes involved in fatty acid metabolism: e.g. peroxisomal thiolase fusion of autophagosome with lysosome (29). This dynamic flow of LC3 (PT), peroxisomal bifunctional protein (BF), and fatty acid -oxidation is completely inhibited in Atg7-deficient liver and, as a consequence, of peroxisomes (7, 8). However, the mechanistic basis of peroxisome more LC3-I accumulates in the mutant liver (20). Considering that dele- turnover remains poorly understood (8, 9). tion of yeast Atg7/Gsa7 gene results in a defect of pexophagy in P. pas- In yeast species, such as Pichia pastoris, Hansenula polymorpha, Can- toris (24), the liver-specific Atg7-conditional knock-out mice will be an dida boidinii, and Saccharomyces cerevisiae, proliferating peroxisomes advantageous tool in investigating the degradation of peroxisomes in mammals. * This work was supported by Grants-in-aid 15032263, 16790195, 15590254, 09680629, In this study, we analyzed the clearance of surplus peroxisomes using and 1270040 from the Ministry of Education, Culture, Sports, Science and Technology the conditional-knock-out mice of Atg7 (20). The results indicated that of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”in autophagy is essential for the degradation of accumulated peroxisomes accordance with 18 U.S.C. Section 1734 solely to indicate this fact. in the mouse liver. Both authors contributed equally to this work. To whom correspondence should be addressed: Dept. of Biochemistry, Juntendo Uni- versity School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Tel.: EXPERIMENTAL PROCEDURES 81-3-5802-1031; Fax: 81-3-5802-5889; E-mail: [email protected]. The abbreviations used are: PT, peroxisomal thiolase; BF, bifunctional protein; DEHP, Reagents—Phthalate esters (diethylhexyl phthalate (DEHP)), corn oil, diethylhexyl phthalate; MLP, mitochondrial/lysosomal/peroxisomal; Atg, autophagy- related; BiP, binding protein; pIpC, polyinosinic acid-polycytidylic acid. and leupeptin were purchased from Sigma. FEBRUARY 17, 2006• VOLUME 281 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4035 This is an Open Access article under the CC BY license. Selective Degradation of Excess Peroxisomes FIGURE 1. The recovery process of excess peroxisomes induced by DEHP treatment. A, wild-type mice were treated with DEHP for 2 weeks (2 w DEHP) and then chased for 1 week (2 w DEHP 1w). Untreated and treated mice were dissected, and liver homogenates were fractionated into MLP, microsomal (Ms), and cytosolic (Cyt) fractions. The protein amount in each fraction was measured. Data are mean  S.D. values of five mice in each group; *, p  0.02 and ***, p  0.001. B, wild-type mice were treated as described in A. The vehicle 4036 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 7 •FEBRUARY 17, 2006 Selective Degradation of Excess Peroxisomes Animals and Treatment Regimen—C57B6J mice were used as wild- fixed in 1% reduced osmium tetroxide with 3,3-diaminobendine reac- type mice. Male mice received DEHP (1,150 mg/kg/day) or vehicle (corn tion. All tissue slices were then dehydrated in graded series of ethanol oil, 5 ml/kg/day) via sonde daily for 2 weeks, and the mice were subse- and embedded in Epon. Thin sections were cut with a diamond knife quently fed on a normal diet for 1 week to investigate the changes in using an ultramicrotome (Reichert, Vienna, Austria). Sections were proliferated peroxisomes during the recovery process according to the contrasted with 40 mM lead citrate for 5 min and examined with a protocol reported previously (7). For detection of autophagosomes by Hitachi H7500 electron microscope (Hitachi, Tokyo, Japan). electron microscopy, mice were injected with leupeptin (2 mg/100 g of Quantitative Analysis of Peroxisomes—For each tissue slice, 20 digital body weight) after administration of DEHP. All animals were sacrificed electron micrographs were acquired at 5,000 magnification, enlarged by deep anesthesia. 2.7-fold, and printed by a laser printer. Using the printed figure, we Deletion of Atg7 in Mouse Liver—Atg7 conditional knock-out mice measured the area of peroxisomes and that of the cytoplasmic area of and the heterozygotes were prepared as described previously (20). hepatocytes using a SigmaScan scientific measurement system Briefly, creatine expression in the liver was induced by intraperitoneal equipped with a computer (Jandel Scientific, San Rafael, CA). The rela- injection of polyinosinic acid-polycytidylic acid (pIpC). pIpC was tive total area of peroxisomes was calculated using the following for- injected three times at a 48-h interval. mula: (number of peroxisomes in the average area of peroxisomes/cy- F/ F/F 2 2 Preparation of the Fractions—Livers from Atg7 :Mx1 and Atg7 : toplasmic area) and expressed in m /100 m of cytoplasmic area. Mx1 mice were treated with DEHP or corn oil for 2 weeks, and at 1 week Statistical Analysis—The statistical significance of differences after treatment, they were dissected. Subfractionation of the livers was between experimental and control groups was determined by the two- accomplished by differential centrifugation according to the method of tailed Student’s t test. A p value of 0.05 was considered statistically de Duve et al. (30). Briefly, 20% homogenates were prepared in 0.25 M significant. sucrose, 10 mM HEPES-NaOH, pH 7.4 (homogenizing buffer). The RESULTS homogenate of the liver was centrifuged at 650 g for 5 min to remove nuclei and unbroken cells. The pellets were resuspended in the same Selective Degradation of Excess Peroxisomes—Phthalate ester (DEHP) volume of homogenizing buffer and were then recentrifuged. The and its active metabolite mono-ethylhexyl phthalate can cause marked supernatants from these two centrifugations were combined and used increases in both the size and the number of peroxisomes and induce as postnuclear supernatant fractions. Postnuclear supernatant fractions peroxisomal enzymes in the liver (7). Utilizing these phenomena, we were centrifuged at 10,000  g for 20 min, and pellets were used as the first investigated the specific proliferation of peroxisomes and the rapid mitochondrial/lysosomal/peroxisomal (MLP) fractions. The post-MLP recovery after removal of the drugs in mice. Wild-type mice were supernatants were further centrifuged at 105,000  g for 60 min to treated with DEHP for 2 weeks and then chased for 1 week as described precipitate microsomal fractions in pellet form. All procedures were under “Experimental Procedures.” The mice were dissected at each performed at 4 °C. period, and the liver cell lysates were fractionated into MLP, microso- Immunoblot Analysis—Immunoblotting was performed as described mal, and cytosolic fractions. DEHP administration for 2 weeks was asso- previously (19). The antibody against Mn -superoxide dismutase was ciated with about 2-fold increase in the amount of total protein in MLP, kindly provided by Prof. Naoyuki Taniguchi (Osaka University, Japan). but not in microsomal or cytosolic fractions, as compared with The antibodies for Atg7 (19), LC3 (20), BF (31), PT (32), and the -sub- untreated mice, and the amount almost returned to the basal level at 1 unit of ATP synthase (33) were prepared as described previously. The week after discontinuation of DEHP (Fig. 1A). These changes were not antibodies against tubulin and BiP were purchased from Chemicon observed in mice treated with the vehicle (data not shown). Quantitative International, Inc. (Temecula, CA) and Affinity BioReagents, Inc. densitometric analysis of immunoblotting data revealed that PT and BF, (Golden, CO), respectively. marker proteins of peroxisomes, increased significantly after adminis- Histological Examination—Livers were dissected, fixed in 4% tration of DEHP but not the vehicle, and both diminished significantly paraformaldehyde, frozen, embedded, and sectioned. For immunohis- to basal levels at 1 week after DEHP discontinuation (Fig. 1, B and C). In tochemical analysis, the sections were blocked with 5% normal goat comparison, the levels of mitochondrial proteins, the -subunit of ATP serum in phosphate-buffered saline containing 0.2% Triton X-100 and synthase and manganese superoxide dismutase, and the endoplasmic then incubated with anti-PT antibody and Alexa Fluor 488-labeled sec- reticulum marker, BiP, remained unchanged during the same manipu- ond antibody (Molecular Probes, Eugene, OR). Fluorescence images lations (Fig. 1B). Immunofluorescence analysis using anti-PT antibody were obtained using a fluorescence microscope (Q550FV; Leica, Ger- revealed that a 2-week administration of DEHP, but not the vehicle, many) equipped with cooled charge-coupled device camera (CTR MIC; resulted in the appearance of numerous dots representing peroxisomes, Leica). Pictures were taken using Leica Qfluoro software (Leica). and most of these dots disappeared at 1 week after discontinuation of Electron Microscopy—Livers were perfusion-fixed with the fixative DEHP (Fig. 1D). Considered together, these results indicate that DEHP- through the portal vein for 10 min. The fixative consisted of 2% induced peroxisomes are selectively degraded following removal of the paraformaldehyde, 1% glutaraldehyde, and 0.1 M HEPES-KOH buffer peroxisome proliferator. (pH 7.4). To visualize peroxisomes, some liver slices were incubated in Impairment of Degradation of Proliferated Peroxisomes in Autoph- alkaline 3,3-diaminobendine medium consisting of 2 mg/ml 3,3-diami- agy-deficient Liver—Next, to examine the effects of autophagy defi- nobendine, 0.02% hydrogen peroxide, and 0.2 M glycine-NaOH buffer ciency on peroxisome degradation, we took advantage of the condi- F/F (pH 10.0) for1hat room temperature. Then they were postfixed with tional knock-out mice, Atg7 :Mx1 (mutant mice), and their F/ 1% reduced osmium tetroxide for 1 h. The other tissue slices were post- littermates, Atg7 :Mx1 mice (control mice), the systems of which control mice were treated with corn oil for 2 weeks (2 w vehicle). Untreated and treated mice were sacrificed, and the livers were dissected out and homogenized, and then the postnuclear supernatant fractions were subjected to immunoblotting with anti-PT, BF, -subunit ATP synthase, Mn -superoxide dismutase (SOD), BiP, and tubulin antibodies. Tubulin was used as a control. Data shown are representative of three separate experiments. C, quantitative densitometry of immunoblotting data in B was performed, and the ratios between each of PT, BF, and ATP synthase and tubulin were plotted; **, p 0.01, ***, p 0.001. D, wild-type mice were treated with DEHP as described in A, and the frozen sections of livers were stained with anti-PT antibody to detect peroxisomes. Magnification, 400. FEBRUARY 17, 2006• VOLUME 281 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4037 Selective Degradation of Excess Peroxisomes F/ F/F FIGURE 2. The recovery process of excess peroxisomes is impaired in Atg7-deficient liver. A, Atg7 :Mx1 (F/:Mx1) and Atg7 :Mx1 (F/F:Mx1) mice were treated with DEHP for 2 weeks (2 w DEHP) and then chased for 1 week (2 w DEHP 1w). Both genotype mice were sacrificed at each time point. The liver was dissected out and homogenized, and then the postnuclear supernatant fractions were subjected to immunoblotting using anti-Atg7, LC3, BF, PT, -subunit ATP synthase, Mn -superoxide dismutase (SOD), BiP, and tubulin antibodies. Tubulin was used as control. Data shown are representative of three separate experiments. B, quantitative densitometry of Western blotting shown in A was performed, and PT/tubulin, BF/tubulin, -subunit ATP synthase/tubulin, and Mn -superoxide dismutase/tubulin ratios were plotted; *, p  0.02, **, p  0.01, NS; not significant. were recently established by our group (20). Autophagy is impaired membrane-bound form of LC3) and accumulation of LC3-I (a cytosolic F/F following pIpC injection in Atg7 :Mx1 mouse livers. Indeed, we veri- form of LC3) in the liver. It is generally accepted that LC3-II is a marker F/F F/ fied that Atg7 protein deletion in Atg7 :Mx1 but not Atg7 :Mx1 protein of autophagosomal membranes (21). Although both forms were livers was pIpC injection-dependent (Fig. 2A). Furthermore, we also detected in the control liver, only LC3-I accumulated in the mutant liver F/F tested the loss of Atg7 activity by investigating the lack of LC3-II (a (Fig. 2A), indicating impairment of autophagy in mutant Atg7 :Mx1 4038 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 7 •FEBRUARY 17, 2006 Selective Degradation of Excess Peroxisomes FIGURE 3. Accumulation of excess peroxisomes in Atg7-deficient liver. Immunofluo- F/ rescent detection of peroxisomes with anti-PT antibody in the Atg7 :Mx1 (A–C, F/: F/F F/ F/F Mx1) and Atg7 :Mx1 (D–F, F/F:Mx1) liver is shown. Atg7 :Mx1 and Atg7 :Mx1 mice were treated with DEHP for 2 weeks (B and E, 2 w DEHP) and then chased for 1 week (C and F, 2 w DEHP 1w). Untreated (A and D) and treated mice were sacrificed, and the livers were isolated. The frozen sections of livers were immunostained with anti-PT antibody. Magnification, 400. mouse liver (20). In the control livers, although LC3-II were induced by the proliferated peroxisomes (Fig. 2A, indicated by 2 w DEHP), it was decreased almost to the basal levels at 1 week after withdrawal of DEHP (Fig. 2A), suggesting that autophagy was induced to remove surplus peroxisomes. After a 2-week treatment with DEHP, the livers were dis- sected, and total proteins in the lysates of mutant and control livers were separated by SDS-PAGE and subjected to immunoblot analyses. Similar to the results obtained with wild-type mice (Fig. 1), BF and PT increased profoundly after the treatment as compared with mice prior to DEHP administration and then decreased almost to the basal levels at 1 week F/ after discontinuation in Atg7 :Mx1 livers (Fig. 2, A and B). Although FIGURE 4. Electron microscopic evaluation of livers of Atg7-deficient mice treated F/F this increase was also detected in mutant Atg7 :Mx1 livers, the F/ with DEHP. A–F, electron micrographs of the liver of representative Atg7 :Mx1 mice F/F increased PT and BT proteins did not return to the basal levels following (F/:Mx1) and Atg7 :Mx1 (F/F:Mx1) mice treated with DEHP for 2 weeks (B and E, 2w DEHP) and then fed on normal diet for 1 week (C and F, 2 w DEHP  1w). The vehicle the discontinuation of DEHP (Fig. 2, A and B). In contrast to peroxiso- control mice of each genotype were treated with corn oil for 2 weeks (A and D). The mal proteins, the levels of mitochondrial (-subunit of ATP synthase hepatocytes of both genotypes contained a high number of peroxisomes (P) after DEHP treatment (B and E). Note that induced peroxisomes were retained at 1 week after dis- and Mn -superoxide dismutase) and endoplasmic reticulum (BiP) F/F continuation of DEHP in Atg7 :Mx1 hepatocytes, in contrast to the decreased number markers did not change under these conditions (Fig. 2, A and B). These F/ in Atg7 :Mx1 hepatocytes (C and F). Bars,1 m. The total area of peroxisomes relative results indicate selective impairment of degradation of excess peroxiso- to the cytoplasmic area was determined in each genotype (n 10). M, mitochondria; G, F/ F/F F/F morphometric analysis of peroxisomes in Atg7 :Mx1 and Atg7 :Mx1 mice. mal proteins in autophagy-deficient Atg7 :Mx1 liver. We further confirmed the impairment of peroxisome degradation in autophagy-deficient liver by immunofluorescence analysis using structures disappeared after 1 week of discontinuation of DEHP in the anti-PT antibody (Fig. 3). The PT-positive dots representing peroxi- control, but not mutant, hepatocytes (Fig. 4, C and F). The relative total somes were markedly increased following a 2-week DEHP treatment in area of peroxisomes was determined, and the mean values are shown in both genotype livers, as compared with untreated mice (Fig. 3, A and D Fig. 4G. Although the relative total area of peroxisomes increased in versus B and E). Although the dots almost disappeared to the basal levels both groups after a 2-week DEHP administration, the area decreased to at 7 days after discontinuation of DEHP in the control (Fig. 3C), most of the basal level in control hepatocytes, but not in mutant hepatocytes, at the peroxisome dots remained visible in mutant liver after the same 1 week after DEHP withdrawal from the diet (Fig. 2G). intervention (Fig. 3F). The data are in agreement with the biochemical After discontinuation of DEHP, we detected only a few autophago- results shown in Fig. 2. Based on these results, we concluded that auto- some-like structures in control hepatocytes, probably due to the rapid phagy is essential for selective degradation of excess peroxisomes. turnover of autophagosomes by lysosome (Fig. 4C). Considering the Engulfment of Excess Peroxisomes by Autophagosomal Membranes in selective degradation of peroxisomal marker proteins, PT and BF (Fig. Control Hepatocytes—Finally, we used electron microscopy to explore 2), autophagosomes that selectively enwrap peroxisomes could be F/F F/ the level of the peroxisomes in Atg7 :Mx1 and Atg7 :Mx1 livers (Fig. observed by electron microscopic analysis when lysosomal proteolysis is 4). Consistent with the results of immunofluorescent analysis, numer- inhibited. Therefore, we examined whether proliferated peroxisomes ous peroxisomes were detected following a 2-week DEHP treatment in enclosed by autophagosomal membranes can be detected under the both wild and mutant hepatocytes (Fig. 4, B and E), and most of these condition of inhibited autophagic proteolysis. Injection of leupeptin, a FEBRUARY 17, 2006• VOLUME 281 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4039 Selective Degradation of Excess Peroxisomes micropexophagy. Our data using electron microscopy revealed that autophagosomes preferentially surrounded excess peroxisomes in con- trol hepatocytes (Fig. 5), suggesting that DEHP-induced peroxisomes are degraded mainly through the process of macropexophagy. Thus, we could show the selective role of autophagic machinery in the clearance of surplus peroxisomes after induction of peroxisomes by phthalate esters. Recent studies provided evidence for the involvement of the autoph- agic machinery in selective sequestration of proteins in the cell. For example, the precursor form of aminopeptidase I (prApe1) is a selective cargo molecule of autophagy in yeast (36), and cytosolic acetaldehyde F/ dehydrogenase (Ald6p) is preferentially transported to vacuoles via FIGURE 5. Excess peroxisomes are surrounded by autophagosome. Atg7 :Mx1 mice were treated with DEHP for 2 weeks and then injected with leupeptin as described autophagosomes in yeast (37). Consistently, the autophagic machinery under “Experimental Procedures.” The mice were sacrificed, and the livers were dis- could also selectively eliminate pathogenic group A Streptococci invad- sected out and processed for electron microscopic examination. These images show ing the cells (38). These reports strongly suggest that autophagosomes representative autophagosomes surrounding peroxisomes. Four typical electron micro- graphs are represented. Arrowheads indicate the engulfment of peroxisome(s) by iso- sequester the cytosolic protein(s) and invading pathogens in a highly lated membranes. Bars,1 m. selective manner. We recently reported that Atg7-deficient hepatocytes exhibit impaired constitutive autophagy responsible for selective degra- lysosomal cysteine proteinase inhibitor, into a 2-week DEHP-treated dation of ubiquitinated proteins (20). Our previous findings together F/ control Atg7 :Mx1 mouse resulted in marked accumulation of auto- with the present results suggest that the autophagic process eliminates phagosomes, and some peroxisomes were surrounded by a double- abnormal and/or excess proteins and organelles including peroxisomes membrane structure, autophagosome, in control hepatocytes (Fig. 5). in a selective manner even under normal conditions. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Feb 17, 2006

References