- Email: [email protected]
Role of the Apg12 conjugation system in mammalian autophagy
The International Journal of Biochemistry & Cell Biology 35 (2003) 553–561
Review
Role of the Apg12 conjugation system in mammalian autophagy Noboru Mizushima a,b , Tamotsu Yoshimori c , Yoshinori Ohsumi b,∗ a
b
PRESTO, Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan Department of Cell Biology, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji, Okazaki 444-8585, Japan c Department of Cell Genetics, National Institute of Genetics, Mishima 411-8540, Japan Received 1 October 2002; received in revised form 18 November 2002; accepted 18 November 2002
Abstract The Apg12 system is one of the ubiquitin-like protein conjugation systems conserved in eukaryotes. It was first discovered in yeast during systematic analyses of the apg mutants defective in autophagy, which is the intracellular bulk degradation system. Covalent attachment of Apg12–Apg5 is essential for autophagy. Enzymes catalyzing this conjugation reaction were also identified based on the apg mutant analyses. These are Apg7 and Apg10, corresponding to E1 and E2 enzymes, respectively. Studies using mammalian cells further revealed the function of the Apg12 system. The Apg12–Apg5 conjugate localizes to elongating autophagic isolation membranes. Apg12 conjugation of Apg5 is required for elongation of the isolation membrane to form a complete spherical autophagosome. Discovery of the Apg12 system has facilitated our understanding of the molecular mechanism of autophagosome formation. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Autophagy; Autophagosome; Ubiquitin-like system; Apg12; LC3
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Autophagy as an intracellular bulk degradation system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. The yeast Apg12 conjugation system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. The Apg12 system in mammalian cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Role of Apg12–Apg5 in mammalian autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Since the lysosome was discovered by de Duve in 1950s (de Duve, 1959), it has been thought to ∗ Corresponding author. Tel.: +81-564-55-7515; fax.: +81-564-55-7516. E-mail address: [email protected] (Y. Ohsumi).
553 554 555 556 557 558 558
be a central organelle for intracellular degradation. Soon after that, the phenomenon of self eating, called “autophagy”, was evidenced by Ashford and Porter (1962). However, the molecular mechanism of autophagy has not been understood for a long time. In the past decade, the genetic approach using the yeast Saccharomyces cerevisiae was introduced in this research field and it has provided several unexpected insights
1357-2725/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 2 ) 0 0 3 4 3 - 6
554
N. Mizushima et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 553–561
(Khalfan & Klionsky, 2002; Klionsky & Emr, 2000; Reggiori & Klionsky, 2002). One of the most remarkable findings is the discovery of two ubiquitin-like conjugation systems, the Apg12 (Mizushima, Sugita, Yoshimori, & Ohsumi, 1998) and Aut7/Apg8 systems (Ichimura et al., 2000; Kirisako et al., 2000). What is significant of this discovery is not only that it brought new insights into the mechanism of autophagy but also it extended our understanding of ubiquitin-like molecules. This review focuses on studies that have addressed the function of the Apg12 conjugation systems, particularly in mammalian cells.
2. Autophagy as an intracellular bulk degradation system Most intracellular short-lived proteins are selectively degraded by the ubiquitin-proteasome pathway (Hershko & Ciechanover, 1998; Hochstrasser, 1996), while long-lived proteins are mostly degraded in the lysosomes (Mortimore & Poso, 1987). Three pathways from the cytoplasm to lysosomes have been proposed: macroautophagy, microautophagy and chaperon-mediated autophagy (Blommaart, Luiken,
& Meijer, 1997; Dunn, 1994). Among them, macroautophagy is believed to be responsible for majority of the intracellular protein degradation, particularly starvation-induced proteolysis (Mortimore & Poso, 1987). In macroautophagy (simply referred to autophagy hereafter), cytoplasmic constituents are first enveloped by cap-shaped cisternae known as isolation membrane (Fig. 1). Closure of the isolation membrane results in formation of double membrane structures called autophagosomes, which are also known as initial autophagic vacuoles (AVi). Autophagosomes, then, fuse with endosomes to become amphisomes or intermediate AV (AVi/d) (Berg, Fengsrud, Stromhaug, Berg, & Seglen, 1998; Liou, Geuze, Geelen, & Slot, 1997; Nara et al., 2002; Tooze et al., 1990). Eventually, autolysosomes or degrading AV (AVd) are generated by the fusion of the outer membranes of the autophagosomes and lysosomes. Lysosomal hydrolases degrade the cytoplasm-derived contents of the autophagosome, together with its inner membrane. Autophagy is up-regulated by nutrient starvation, most effectively by amino acid withdrawal, in cultured cells. It is also induced in liver and other tissues in fasting animals. Autophagy is thought to play
Fig. 1. The intracellular protein degradation systems in mammalian cells. (A) Scheme of autophagy. A portion of cytoplasm is enclosed by autophagic isolation membrane to form an autophagosome. The outer membrane of the autophagosome then fuses with lysosome to degrade the inside materials. Organelles such as mitochondria can be also degraded by this pathway. (B) The ubiquitin-proteasome system. Proteins to be degraded are tagged with multi-ubiquitin chains. The C-terminal glycine residue is activated by the E1 ubiquitin-activating enzyme then conjugated to proteins, most of which are short-lived, by the actions E2 ubiquitin-conjugating enzymes and E3 ubiquitin-ligase (complex). The ubiquitinated proteins are specifically recognized by 26S proteasome and then degraded.
N. Mizushima et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 553–561
crucial role in homeostasis (Mortimore & Poso, 1987), and also participate in various physiological processes, such as cellular remodeling (Bolender & Weibel, 1973; Masaki, Yamamoto, & Tashiro, 1987), differentiation (Tsukada & Ohsumi, 1993), production of pulmonary surfactant (Hariri et al., 2000), and non-apoptotic cell death during embryogenesis (Clarke, 1990). Defective autophagy may cause mammary tumors (Liang et al., 1999) and a specific type of myopathy (Nishino et al., 2000; Tanaka et al., 2000).
3. The yeast Apg12 conjugation system Autophagy is found among eukaryotes including yeast (Takeshige, Baba, Tsuboi, Noda, & Ohsumi, 1992). The genetical approach using the yeast Saccharomyces cerevisiae shed light on molecular basis of autophagy. Based on the autophagy-defective mutants (Tsukada & Ohsumi, 1993; Thumm et al., 1994), at least 16 genes (APG and AUT genes) required for autophagosome formation was identified. Among these, two novel ubiquitin-like conjugation systems were found to be essential for autophagy (Ohsumi, 2001).
555
The first was the Apg12 conjugation system (Mizushima et al., 1998) (Fig. 2). Apg12 is a 186-amino acid protein without apparent homology to known proteins. Intriguingly, most endogenous Apg12 is conjugated to another protein, Apg5 (Kuma, Mizushima, Ishihara, & Ohsumi, 2002). The mode of conjugation of Apg12–Apg5 is quite similar to that of ubiquitin. The carboxy-terminal residue of Apg12 was glycine, which is activated by Apg7 in an ATP-dependent manner (Mizushima et al., 1998). Then, Apg12 forms a conjugate with Apg7 through a high energy thioester bond (Kim, Dalton, Eggerton, Scott, & Klionsky, 1999; Yuan, Stromhaug, & Dunn, 1999; Tanida et al., 1999). Apg7 shows homology with E1 ubiquitin-activating enzyme within the region encompassing the putative ATP-binding site (GxGxxG) and the active site cysteine (Mizushima et al., 1998). Most Apg7 exists as homo-dimer (Komatsu et al., 2001). Apg12 is then transferred to Apg10 to form a thioester again (Shintani et al., 1999). The function of Apg10 is likely equivalent to that of E2 ubiquitin-conjugating enzymes, although Apg10 has no homology to E2 enzymes. Finally, the carboxyterminal glycine of Apg12 is covalently attached
Fig. 2. The Apg12 and Aut7/LC3 systems. (A) Apg12 is activated by Apg7 and conjugated to Apg7 via a high energy thioester bond. Subsequently, Apg12 forms a thioester with Apg10. Apg12 is finally conjugated to Apg5 via an isopeptide bond. This process is conserved among eukaryotes. In yeast, the Apg12–Apg5 conjugates are multimerized by Apg16 to form the 350 kDa Apg12–Apg5·Apg16 complex. In mammalian cells, the Apg12–Apg5 conjugate forms an 800 kDa complex with Apg16L. (B) After processed by Aut2/Apg4, Aut7/Apg8 and its mammalian homologue LC3 are activated by Apg7 and then form thioesters with Aut1/Apg3. In yeast, Aut7 is conjugated with phosphatidylethanolamine (PE). In mammalian cells, the target substrate of LC3 has not been identified. The conjugated form of LC3 is thought to be the LC3-II form (see text). Aut7-PE is deconjugated again by Aut2.
556
N. Mizushima et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 553–561
to lysine 149 of Apg5 via an isopeptide bond between the carboxyl group of the glycine residue and the ε-amino group of the lysine residue (Mizushima et al., 1998). Formation of the Apg12–Apg5 conjugate is essential for proceeding of autophagy. The second one is the Aut7/Apg8 system (Fig. 2). The C-terminal arginine residue of Aut7 is cleaved off by Aut2/Apg4, a novel cysteine protease (Kirisako et al., 2000). The exposed glycine residue of Aut7 is conjugated to phosphatidylethanolamine (PE), which is catalyzed by Apg7 and Aut1/Apg3 (Ichimura et al., 2000). Apg7 is shared with the Apg12 system whereas Aut1, which shows partial homology to Apg10, is a specific conjugating enzyme for the Aut7 system. Subsequently, Apg8-PE is deconjugated, again by Aut2. The cycle of conjugation and deconjugation is important for normal progression of autophagy (Kirisako et al., 2000). The Apg12–Apg5 conjugate further interacts with a small coiled-coil protein, Apg16 (Mizushima, Noda, & Ohsumi, 1999). Apg16 forms a homo-oligomer through its coiled-coil region. As each Apg16 molecule interacts with Apg5, Apg16 homo-oligomers crosslink multiple Apg12–Apg5 conjugates. As a result, Apg12–Apg5 and Apg16 form a ∼350 kDa protein complex, thought to contain four sets of Apg12– Apg5 and Apg16 (Kuma, Mizushima, Ishihara, & Ohsumi, 2002). Using an in vivo regulated oligo merization system, it was demonstrated that formation of this ∼350 kDa complex is essential for autophagy. While most Apg12–Apg5·Apg16 complex freely exists in the cytosol (Kuma, Mizushima, Ishihara, & Ohsumi, 2002), a small portion localizes to a punctate structure near the vacuole, termed the pre-autophagosomal structure (PAS) (Kim, Huang, Stromhaug, & Klionsky, 2002; Suzuki et al., 2001). Apg1, Apg2, Aut7/Apg8, Apg9 and Apg14 are also targeted to this structure, from which autophagosomes seem to be generated. A study using the apg5 temperature sensitive mutant showed that Apg5 is directly required for autophagosome formation (George et al., 2000). In the strains defective in the Apg12 conjugation system, PE conjugation of Aut7 is severely reduced and recruitment of Aut7 to PAS is abolished (Kim, Huang, & Klionsky, 2001; Suzuki et al., 2001). Thus, the functions of these two ubiquitin-like systems are closely, probably directly linked.
4. The Apg12 system in mammalian cells Both the Apg12 and Aut7 system are highly conserved in mammals. In mouse and human, there seems only one orthologue for each component of the Apg12 system. Apg12 is conjugated to Apg5 (Mizushima, Sugita, Yoshimori, & Ohsumi, 1998), which is catalyzed by Apg7 (Tanida, Tanida-Miyake, Ueno, & Kominami, 2001) and Apg10 (Mizushima, Yoshimori, & Ohsumi, 2002). In mammalian cells, the Apg12–Apg5 conjugate is a component of ∼800 kDa protein complex. Purification of this complex revealed an additional novel protein that interacts with Apg5 (Mizushima et al., manuscript submitted). It is a 63–74 kD protein with several spliced isoforms, which is quite larger than yeast Apg16 (17 kD). As the N-terminal region of this novel protein contains several features similar to yeast Apg16, it was designated Apg16L (Apg16-like). Apg16L, however, has a large C-terminal domain containing seven WD repeats, absent from yeast Apg16. By a database search, Apg16L homologues are found in all eukaryotes except Saccharomyces cerevisiae and Pichia pastoris (Sakai, personal communication), which have Apg16 without WD repeats. Significance of the WD repeats in Apg16L has not been revealed. In contrast to Apg12, at least three Aut7 homologues were identified in mammals: microtubuleassociated protein 1 (MAP1) light chain 3 (LC3) (Mann & Hammarback, 1994), Golgi-associated ATPase Enhancer of 16 kDa (GATE-16) (Sagiv, LegesseMiller, Porat, & Elazar, 2000) and ␥-aminobutyric acid (GABA)A -receptor-associated protein (GABARAP) (Wang, Bedford, Brandon, Moss, & Olsen, 1999). Among them, LC3 was shown to localize on autophagosome membrane (Kabeya et al., 2000). The precise function and localization of the other two homologues are unclear. GABARAP was suggested to be involved in the GABAA -receptor clustering (Chen, Wang, Vicini, & Olsen, 2000) or transport (Kneussel et al., 2000). GATE-16 has been suggested to be an intra-Golgi transport modulator that interacts with N-ethylmaleimide-sensitive factor (NSF) and Golgi v-SNARE GOS-28 (Sagiv, Legesse-Miller, Porat, & Elazar, 2000). However, transfection experiments showed that both GABARAP and GATE-16 are able to reside on autophagosomal membranes (our unpublished observation). Further analyses are
N. Mizushima et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 553–561
required to reveal the roles of these three molecules. All these Aut7 homologues are processed by mammalian Aut2/Apg4 homologues to expose the conserved C-terminal glycine (our unpublished observation). They are further catalyzed by mammalian Apg7 (Tanida, Tanida-Miyake, Ueno, & Kominami, 2001) and Aut1/Apg3 homologues (Tanida, TanidaMiyake, Komatsu, Ueno, & Kominami, 2002). Function of mammalian Apg12 system was examined using mouse embryonic stem (ES) cells, because genetic approach is feasible and size of autophagosome is larger than most of other cell lines. A gene targeting study demonstrated that Apg5 is required for autophagosome formation also in mammalian cells (Mizushima et al., 2001). LC3 cannot target to the membrane in APG5−/− cells (Mizushima et al., 2001). In wild-type cells, LC3 is detected in two forms on immunoblotting: 18 kDa LC3-I and 16 kDa LC3-II (Kabeya et al., 2000). Since LC3-II is a membrane bound form, it was speculated as a PE-conjugated form. Strikingly, in APG5−/− cells, LC3-II is not generated at all (Mizushima et al., 2001). Thus, functional relationship between the Apg12 and LC3 system is clearly evidenced also in mammalian cells. However, direct interaction between Apg12 and Apg5 and LC3 is not observed (our unpublished observation) while weak interaction between Apg12–Apg5 and Aut7 is suggested in yeast (Kim, Huang, Stromhaug, & Klionsky, 2002). In addition, possible inter-dependence between mammalian Apg12 and LC3 conjugation reaction is suggested (Tanida, Nishitani, Nemoto, Ueno, & Kominami, 2002; Tanida, Tanida-Miyake, Komatsu, Ueno, & Kominami, 2002).
5. Role of Apg12–Apg5 in mammalian autophagy It should be noted that most Apg5 is conjugated with Apg12 and most Apg12–Apg5 forms an ∼800 kDa complex with Apg16L irrespective of whether autophagy is induced or not. Thus, the conjugation and complex formation would not be a trigger of autophagy, rather provide basic machinery involved in autophagosome formation. Localization of Apg12–Apg5 was examined in ES cells in detail using GFP-fused Apg5 (Mizushima et al., 2001). A small
557
Fig. 3. Model of autophagosome formation in mammalian cells. The Apg12–Apg5 conjugate and Apg16L localize to the isolation membrane throughout its elongation process. LC3 is recruited to the membrane in the Apg5-dependent manner. Apg12–Apg5 and Apg16L dissociate from the membrane upon completion of autophagosome formation, while LC3 remains on the autophagosome membrane. Apg5 and its modification by Apg12 are required for elongation of the isolation membrane.
fraction of cytosolic Apg12–Apg5·Apg16L complex localizes to the isolation membrane throughout its elongation process (Fig. 3). Apg12–Apg5 initially associates with a small crescent-shaped vesicle evenly. As the membrane elongates, Apg12–Apg5 shows asymmetric localization; most Apg12–Apg5 associates with the outer side of the isolation membrane. Finally, Apg12–Apg5 dissociates from the membrane upon completion of autophagome formation. Apg16L always behaves with Apg12–Apg5, suggesting that these proteins associates and dissociated from the membrane as the 800 kDa complex (manuscript submitted). LC3 also targets to the isolation membrane in Apg5-dependent manner, and remains on the autophagosomal membrane even after Apg12–Apg5 dissociates (Fig. 3) (Kabeya et al., 2000; Mizushima et al., 2001). The significance of the Apg12 conjugation was also determined using the APG5−/− ES cells. In the mammalian Apg12 system, Apg12 is conjugated Lys130 of Apg5 (Mizushima et al., 1998). When the Apg5K130R mutant, in which Lys130 is replaced with Arg, is expressed in APG5−/− ES cells, Apg5 is no longer conjugated with Apg12. Unexpectedly, the small vesicles (autophagosome precursors), to which Apg5K130R mutant and Apg16L associate, are generated in these cells (Mizushima et al., 2001). However, the membrane does not elongate to form autophagosomes. Thus, the covalent modification of Apg5 with Apg12 is not required for membrane targeting of Apg5 and Apg16L but is essential for involvement of Apg5 in elongation
558
N. Mizushima et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 553–561
of the isolation membranes. Modification of Apg5 with Apg12 is also required for targeting of LC3 to the isolation membranes (Mizushima et al., 2001). Thus, the Apg12–Apg5 conjugate plays an essential role in isolation membrane development, together with LC3. How autophagosomes are generated has yet been poorly understood. The detection of endoplasmic reticulum (ER) marker proteins suggested that the isolation membranes are derived from the ER (Arstila & Trump, 1968; Dunn, 1990; Ericsson, 1969; Ueno, Muno, & Kominami, 1991). Involvement of post-Golgi membranes was also suggested (Locke & Sykes, 1975; Yamamoto, Masaki, & Tashiro, 1990). Finally, there were reports indicating unique feature of isolation membrane that is different from any known organelles (Baba, Osumi, & Ohsumi, 1995; Baba, Takeshige, Baba, & Ohsumi, 1994; Hirsimaki & Reunanen, 1980; Reunanen & Hirsimaki, 1983; Stromhaug, Berg, Fengsrud, & Seglen, 1998). The studies on mammalian Apg12–Apg5 provided valuable insights into autophagosome formation. First, visualization of GFP-Apg5 in living cells directly demonstrated that autophagosomes are generated by elongation of small membrane structures, autophagosome precursors, not derived from pre-existing large membrane such as ER cisternae (Mizushima et al., 2001). Second, genetical manipulation of ES cells, particularly expression of the conjugation-defective Apg5 mutant, dissected the autophagosome formation into at least two steps, formation of the precursor vesicle and its elongation process (Mizushima et al., 2001). The Apg12–Apg5 conjugate and probably LC3 are required for the latter step. What is the precursor vesicle remains to be an unsolved question. Treatment of cells with 3-methyladenine, which is widely used as an autophagy inhibitor (Seglen & Gordon, 1982), abolishes the formation of any Apg5-positive structures (Mizushima et al., 2001). 3-methyladenine is now considered as a phosphatidylinositol 3-kinase (PI3K) inhibitor (Petiot, Ogier-Denis, Blommaart, Meijer, & Codogno, 2000). Indeed, the autophagosome precursors are not formed by treatment with wortmannin, a well-known PI3K inhibitor, suggesting that PI3K activity is required for early stage of autophagosome formation (Mizushima et al., 2001). Although, it is not known whether a structure equivalent to the yeast PAS exists in mammalian cells, these results are quite consistent with those observed in yeast;
a PI3K complex made up of Apg6, Apg14, Vps15 and Vps34 is required for PAS formation in yeast (Kihara, Noda, Ishihara, & Ohsumi, 2001; Suzuki et al., 2001). It is also proposed that yeast autophagosomes are generated through distinct processes, nucleation, assembly and elongation (Abeliovich, Dunn, Kim, & Klionsky, 2000; Noda, Suzuki, & Ohsumi, 2002).
6. Future prospects Although it is suggested that the Apg12–Apg5 conjugate functions in elongation step of isolation membrane, the exact molecular mechanism of Apg12–Apg5 is still unknown. The characteristic distribution of Apg12–Apg5 on elongating isolation membrane suggests it may function as a coat protein, although the mode of membrane dynamics is quite different from conventional membrane budding. The association–dissociation cycle of Apg12–Apg5 is also similar to behavior of coat proteins during vesicle budding. Alternatively, they may act as a receptor for LC3 itself or membrane containing LC3. All these possibilities should be addressed. Another obvious question is why Apg12 and Apg5 must be conjugated post-translationally, instead of being synthesized as single molecule. These two proteins are conjugated immediately after the synthesis and the conjugation seems to be irreversible (our unpublished observation). Thus, the Apg12–Apg5 conjugate always behaves as if it was a single protein. This is a quite unique feature of the Apg12 conjugation system compared with other Ubl system. All of ubiquitin, SUMO/Smt3, Nedd8/Rub1 and Aut7/Apg8 conjugations are reversible, and deconjugation processes are suggested to be important (Kirisako et al., 2000; Li & Hochstrasser, 1999). Understanding the purpose of this irreversible conjugation would provide new insights into Ubl systems that are well conserved through evolution.
References Abeliovich, H., Dunn Jr., W. A., Kim, J., & Klionsky, D. J. (2000). Dissection of autophagosome biogenesis into distinct nucleation and expansion steps. Journal of Cell Biology, 151, 1025–1033.
N. Mizushima et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 553–561 Arstila, A. U., & Trump, B. F. (1968). Studies on cellular autophagocytosis. The formation of autophagic vacuoles in the liver after glucagon administration. American Journal of Pathology, 53, 687–733. Ashford, T. P., & Porter, K. R. (1962). Cytoplasmic components in hepatic cell lysosomes. Journal of Cell Biology, 12, 198–202. Baba, M., Osumi, M., & Ohsumi, Y. (1995). Analysis of the membrane structure involved in autophagy in yeast by freeze-replica method. Cell Structure and Function, 20, 465– 471. Baba, M., Takeshige, K., Baba, N., & Ohsumi, Y. (1994). Ultrastructural analysis of the autophagic process in yeast: Detection of autophagosomes and their characterization. Journal of Cell Biology, 124, 903–913. Berg, T. O., Fengsrud, M., Stromhaug, P. E., Berg, T., & Seglen, P. O. (1998). Isolation and characterization of rat liver amphisomes. Evidence for fusion of autophagosomes with both early and late endosomes. The Journal of Biological Chemistry, 273, 21883–21892. Blommaart, E. F. C., Luiken, J. J. F. P., & Meijer, A. J. (1997). Autophagic proteolysis: Control and specificity. Histochemical Journal, 29, 365–385. Bolender, R. P., & Weibel, E. R. (1973). A morphometric study of the removal of phenobarbital-induced membranes from hepatocytes after cessation of treatment. Journal of Cell Biology, 56, 746–761. Chen, L., Wang, H., Vicini, S., & Olsen, W. (2000). The gamma-aminobutyric acid type A (GABAA) receptor-associated protein (GABARAP) promotes GABAA receptor clustering and modulates the channel kinetics. Proceedings of the National Academy of Sciences of the United States of America, 97, 11557–11562. Clarke, P. G. (1990). Developmental cell death: Morphological diversity and multiple mechanisms. Anatomy and Embryology (Berlin), 181, 195–213. de Duve, C. (1959). Lysosomes, a new group of cytoplasmic particles. In T. Hayashi (Ed.), Subcellular Particles (pp. 128–159). New York: Ronald. Dunn Jr., W. A. (1994). Autophagy and related mechanisms of lysosome-mediated protein degradation. Trends in Cell Biology, 4, 139–143. Dunn Jr., W. A. (1990). Studies on the mechanisms of autophagy: Formation of the autophagic vacuole. Journal of Cell Biology, 110, 1923–1933. Ericsson, J. L. (1969). Studies on induced cellular autophagy. II. Characterization of the membranes bordering autophagosomes in parenchymal liver cells. Experimental Cell Research, 56, 393–405. George, M. D., Baba, M., Scott, S. V., Mizushima, N., Garrison, B. S., Ohsumi, Y., & Klionsky, D. J. (2000). Apg5p functions in the sequestration step in the cytoplasm-to-vacuole targeting and macroautophagy pathways. Molecular Biology of the Cell, 11, 969–982. Hariri, M., Millane, G., Guimond, M. P., Guay, G., Dennis, J. W., & Nabi, I. R. (2000). Biogenesis of multilamellar bodies via autophagy. Molecular Biology of the Cell, 11, 255–268. Hershko, A., & Ciechanover, A. (1998). The ubiquitin system. Annual Review of Biochemistry, 67, 425–479.
559
Hirsimaki, P., & Reunanen, H. (1980). Studies on vinblastineinduced autophagocytosis in mouse liver. II. Origin of membranes and acquisition of acid phosphatase. Histochemistry, 67, 139–153. Hochstrasser, M. (1996). Ubiquitin-dependent protein degradation. Annual Review of Genetics, 30, 405–439. Ichimura, Y., Kirisako, T., Takao, T., Satomi, Y., Shimonishi, Y., Ishihara, N., Mizushima, N., Tanida, I., Kominami, E., Ohsumi, M., Noda, T., & Ohsumi, Y. (2000). A ubiquitin-like system mediates protein lipidation. Nature, 408, 488–492. Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y., & Yoshimori, T. (2000). LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO Journal, 19, 5720–5728. Khalfan, W. A., & Klionsky, D. J. (2002). Molecular machinery required for autophagy and the cytoplasm to vacuole targeting (Cvt) pathway in S. cerevisiae. Current Opinion in Cell Biology, 14, 468–475. Kihara, A., Noda, T., Ishihara, N., & Ohsumi, Y. (2001). Two distinct Vps34 phosphatidylinositol 3-kinase complexs function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae. Journal of Cell Biology, 152, 519–530. Kim, J., Dalton, V. M., Eggerton, K. P., Scott, S. V., & Klionsky, D. J. (1999). Apg7p/Cvt2p is required for the Cvt, macroautophagy, and peroxisome degradation pathway. Molecular Biology of the Cell, 10, 1337–1351. Kim, J., Huang, W. -P., & Klionsky, D. J. (2001). Membrane recruitment of Aut7p in the autophagy and cytoplasm to vacuole targeting pathways requires Aut1p, Aut2p, and autophagy conjugation complex. Journal of Cell Biology, 152, 51–64. Kim, J., Huang, W. -P., Stromhaug, P. E., & Klionsky, D. J. (2002). Convergence of multiple autophagy and Cvt components to a perivacuolar membrane compartment prior to de novo vesicle formation. The Journal of Biological Chemistry, 277, 763–773. Kirisako, T., Ichimura, Y., Okada, H., Kabeya, Y., Mizushima, N., Yoshimori, T., Ohsumi, M., Takao, T., Noda, T., & Ohsumi, Y. (2000). The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway. Journal of Cell Biology, 151, 263–275. Klionsky, D. J., & Emr, S. D. (2000). Autophagy as a regulated pathway of cellular degradation. Science, 290, 1717–1721. Kneussel, M., Haverkamp, S., Fuhrmann, J. C., Wang, H., Wassle, H., Olsen, R. W., & Betz, H. (2000). The gamma-aminobutyric acid type A receptor (GABAAR)-associated protein GABAPAR interacts with gephyrin but is not involved in ireceptor anchoring at the symapse. Proceedings of the National Academy of Sciences of the United States of America, 97, 8594–8599. Komatsu, M., Tanida, I., Ueno, T., Ohsumi, M., Ohsumi, Y., & Kominami, E. (2001). The C-terminal region of an Apg7p/Cvt2p is required for homodimerization and is essential for its E1 activity and E1-E2 complex formation. The Journal of Biological Chemistry, 276, 9846–9854. Kuma, A., Mizushima, N., Ishihara, N., & Ohsumi, Y. (2002). Formation of the ∼350 kD Apg12–Apg5-Apg16 multimeric complex, mediated by Apg16 oligomerization, is essential for
560
N. Mizushima et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 553–561
autophagy in yeast. The Journal of Biological Chemistry, 277, 18619–18625. Li, S. J., & Hochstrasser, M. (1999). A new protease required for cell-cycle progression in yeast. Nature, 398, 246–251. Liang, X. H., Jackson, S., Seaman, M., Brown, K., Kempkes, B., Hibshoosh, H., & Levine, B. (1999). Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature, 402, 672– 676. Liou, W., Geuze, H. J., Geelen, M. J., & Slot, J. W. (1997). The autophagic and endocytic pathways converge at the nascent autophagic vacuoles. Journal of Cell Biology, 136, 61–70. Locke, M., & Sykes, A. K. (1975). The role of the Golgi complex in the isolation and digestion of organelles. Tissue Cell, 7, 143–158. Mann, S. S., & Hammarback, J. A. (1994). Molecular characterization of light chain 3. The Journal of Biological Chemistry, 269, 11492–11497. Masaki, R., Yamamoto, A., & Tashiro, Y. (1987). Cytochrome P-450 and NADPH-cytochrome P-450 reductase are degraded in the autolysosomes in rat liver. Journal of Cell Biology, 104, 1207–1215. Mizushima, N., Noda, T., Yoshimori, T., Tanaka, Y., Ishii, T., George, M. D., Klionsky, D. J., Ohsumi, M., & Ohsumi, Y. (1998). A protein conjugation system essential for autophagy. Nature, 395, 395–398. Mizushima, N., Yamamoto, A., Hatano, M., Kobayashi, Y., Kabeya, Y., Suzuki, K., Tokuhisa, T., Ohsumi, Y., & Yoshimori, T. (2001). Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. Journal of Cell Biology, 152, 657–667. Mizushima, N., Noda, T., & Ohsumi, Y. (1999). Apg16p is required for the function of the Apg12p-Apg5p conjugate in the yeast autophagy pathway. EMBO Journal, 18, 3888–3896. Mizushima, N., Sugita, H., Yoshimori, T., & Ohsumi, Y. (1998). A new protein conjugation system in human. The counterpart of the yeast Apg12p conjugation system essential for autophagy. The Journal of Biological Chemistry, 273, 33889–33892. Mizushima, N., Yoshimori, T., Ohsumi, Y. (2002) Mouse Apg10 as an Apg12 conjugating enzyme: Analysis by the conjugation-mediated yeast two-hybrid method. FEBS Letters, 532, 450–454. Mortimore, G., & Poso, E. A. R. (1987). Intracellular protein catabolism and its control during nutrient deprivation and supply. Annual Review of Nutrition, 7, 539–564. Nara, A., Mizushima, N., Yamamoto, A., Kabeya, Y., Ohsumi, Y., Yoshimori, T. (2002) SKD1 AAA ATPase-dependent endosomal transport is involved in autolysosome formation. Cell Structure and Function, 27, 29–37. Nishino, I., Fu, J., Tanji, K., Yamada, T., Shimojo, S., Koori, T., Mora, M., Riggs, J. E., Oh, S. J., Koga, Y., Sue, C. M., Yamamoto, A., Murakami, N., Shanske, S., Byrne, E., Bonilla, E., Nonaka, I., DiMauro, S., & Hirano, M. (2000). Primary LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and myopathy (Danon disease). Nature, 406, 906–910. Noda, T., Suzuki, K., & Ohsumi, Y. (2002). Yeast autophagosomes: De novo formation of a membrane structure. Trends in Cell Biology, 12, 231–235.
Ohsumi, Y. (2001). Molecular dissection of autophagy: Two ubiquitin-like systems. Natural Review in Molecular and Cellular Biology, 2, 211–216. Petiot, A., Ogier-Denis, E., Blommaart, E. F., Meijer, A. J., & Codogno, P. (2000). Distinct classes of phosphatidylinositol 3 -kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. The Journal of Biological Chemistry, 275, 992–998. Reggiori, F., & Klionsky, D. J. (2002). Autophagy in the eukaryotic cell. Eukaryotic Cell, 1, 11–21. Reunanen, H., & Hirsimaki, P. (1983). Studies on vinblastineinduced autophagocytosis in mouse liver. IV. Origin of membranes. Histochemistry, 79, 59–67. Sagiv, Y., Legesse-Miller, A., Porat, A., & Elazar, Z. (2000). GATE-16, a membrane transport modulator, interacts with NSF and the Golgi v-SNARE GOS-28. EMBO Journal, 19, 1494– 1504. Seglen, P. O., & Gordon, P. B. (1982). 3-Methyladenine: Specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proceedings of the National Academy of Sciences of the United States of America, 79, 1889– 1892. Shintani, T., Mizushima, N., Ogawa, Y., Matsuura, A., Noda, T., & Ohsumi, Y. (1999). Apg10p, a novel protein-conjugating enzyme essential for autophagy in yeast. EMBO Journal, 18, 5234–5241. Stromhaug, P. E., Berg, T. O., Fengsrud, M., & Seglen, P. O. (1998). Purification and characterization of autophagosomes from rat hepatocytes. Biochemical Journal, 335, 217–224. Suzuki, K., Kirisako, T., Kamada, Y., Mizushima, N., Noda, T., & Ohsumi, Y. (2001). The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation. EMBO Journal, 20, 5971–5981. Takeshige, K., Baba, M., Tsuboi, S., Noda, T., & Ohsumi, Y. (1992). Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction. Journal of Cell Biology, 119, 301–311. Tanaka, Y., Guhde, G., Suter, A., Eskelinen, E. -L., Hartmann, D., Lullmann-Rauch, R., Janssen, P. M. L., Blanz, J., von Figura, K., & Saftig, P. (2000). Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice. Nature, 406, 902–906. Tanida, I., Mizushima, N., Kiyooka, M., Ohsumi, M., Ueno, T., Ohsumi, Y., & Kominami, E. (1999). Apg7p/Cvt2p: A novel protein-activating enzyme essential for autophagy. Molecular Biology of the Cell, 10, 1367–1379. Tanida, I., Nishitani, T., Nemoto, T., Ueno, T., & Kominami, E. (2002). Mammalian Apg12p, but not the Apg12p·Apg5p conjugate, facilitates LC3 processing. Biochemical and Biophysical Research Communications, 296, 1164–1170. Tanida, I., Tanida-Miyake, E., Komatsu, M., Ueno, T., & Kominami, E. (2002). Human Apg3p/Aut1p homologue is an authentic E2 enzyme for multiple substrates, GATE-16, GABARAP, and MAP-LC3, and facilitates the conjugation of hApg12p to hApg5p. The Journal of Biological Chemistry, 277, 13739–13744. Tanida, I., Tanida-Miyake, E., Ueno, T., & Kominami, E. (2001). The human homolog of Saccharomyces cerevisiae Apg7p is
N. Mizushima et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 553–561 a protein-activating enzyme for multiple substrates including human Apg12p, GATE-16, GABARAP, and MAP-LC3. The Journal of Biological Chemistry, 276, 1701–1706. Thumm, M., Egner, R., Koch, B., Schlumpberger, M., Straub, M., Veenhuis, M., & Wolf, D. H. (1994). Isolation of autophagocytosis mutants of Saccharomyces cerevisiae. FEBS Letters, 349, 275–280. Tooze, J., Hollinshead, M., Ludwig, T., Howell, K., Hoflack, B., & Kern, H. (1990). In exocrine pancreas, the basolateral endocytic pathway converges with the autophagic pathway immediately after the early endosome. Journal of Cell Biology, 111, 329– 345. Tsukada, M., & Ohsumi, Y. (1993). Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Letters, 333, 169–174.
561
Ueno, T., Muno, D., & Kominami, E. (1991). Membrane markers of endoplasmic reticulum preserved in autophagic vacuolar membranes isolated from leupeptin-administered rat liver. The Journal of Biological Chemistry, 266, 18995–18999. Wang, H., Bedford, F. K., Brandon, N. J., Moss, S. J., & Olsen, R. W. (1999). GABAA -receptor-associated protein links GABAA receptors and the cytoskeleton. Nature, 397, 69–72. Yamamoto, A., Masaki, R., & Tashiro, Y. (1990). Characterization of the isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes by lectin cytochemistry. Journal of Histochemistry and Cytochemistry, 38, 573–580. Yuan, W., Stromhaug, P. E., & Dunn Jr., W. A. (1999). Glucose-induced microautophagy of peroxysomes in Pichia pastoris requires a unique E1-like protein. Molecular Biology of the Cell, 10, 1353–1366.
Review
Role of the Apg12 conjugation system in mammalian autophagy Noboru Mizushima a,b , Tamotsu Yoshimori c , Yoshinori Ohsumi b,∗ a
b
PRESTO, Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan Department of Cell Biology, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji, Okazaki 444-8585, Japan c Department of Cell Genetics, National Institute of Genetics, Mishima 411-8540, Japan Received 1 October 2002; received in revised form 18 November 2002; accepted 18 November 2002
Abstract The Apg12 system is one of the ubiquitin-like protein conjugation systems conserved in eukaryotes. It was first discovered in yeast during systematic analyses of the apg mutants defective in autophagy, which is the intracellular bulk degradation system. Covalent attachment of Apg12–Apg5 is essential for autophagy. Enzymes catalyzing this conjugation reaction were also identified based on the apg mutant analyses. These are Apg7 and Apg10, corresponding to E1 and E2 enzymes, respectively. Studies using mammalian cells further revealed the function of the Apg12 system. The Apg12–Apg5 conjugate localizes to elongating autophagic isolation membranes. Apg12 conjugation of Apg5 is required for elongation of the isolation membrane to form a complete spherical autophagosome. Discovery of the Apg12 system has facilitated our understanding of the molecular mechanism of autophagosome formation. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Autophagy; Autophagosome; Ubiquitin-like system; Apg12; LC3
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Autophagy as an intracellular bulk degradation system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. The yeast Apg12 conjugation system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. The Apg12 system in mammalian cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Role of Apg12–Apg5 in mammalian autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Since the lysosome was discovered by de Duve in 1950s (de Duve, 1959), it has been thought to ∗ Corresponding author. Tel.: +81-564-55-7515; fax.: +81-564-55-7516. E-mail address: [email protected] (Y. Ohsumi).
553 554 555 556 557 558 558
be a central organelle for intracellular degradation. Soon after that, the phenomenon of self eating, called “autophagy”, was evidenced by Ashford and Porter (1962). However, the molecular mechanism of autophagy has not been understood for a long time. In the past decade, the genetic approach using the yeast Saccharomyces cerevisiae was introduced in this research field and it has provided several unexpected insights
1357-2725/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 2 ) 0 0 3 4 3 - 6
554
N. Mizushima et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 553–561
(Khalfan & Klionsky, 2002; Klionsky & Emr, 2000; Reggiori & Klionsky, 2002). One of the most remarkable findings is the discovery of two ubiquitin-like conjugation systems, the Apg12 (Mizushima, Sugita, Yoshimori, & Ohsumi, 1998) and Aut7/Apg8 systems (Ichimura et al., 2000; Kirisako et al., 2000). What is significant of this discovery is not only that it brought new insights into the mechanism of autophagy but also it extended our understanding of ubiquitin-like molecules. This review focuses on studies that have addressed the function of the Apg12 conjugation systems, particularly in mammalian cells.
2. Autophagy as an intracellular bulk degradation system Most intracellular short-lived proteins are selectively degraded by the ubiquitin-proteasome pathway (Hershko & Ciechanover, 1998; Hochstrasser, 1996), while long-lived proteins are mostly degraded in the lysosomes (Mortimore & Poso, 1987). Three pathways from the cytoplasm to lysosomes have been proposed: macroautophagy, microautophagy and chaperon-mediated autophagy (Blommaart, Luiken,
& Meijer, 1997; Dunn, 1994). Among them, macroautophagy is believed to be responsible for majority of the intracellular protein degradation, particularly starvation-induced proteolysis (Mortimore & Poso, 1987). In macroautophagy (simply referred to autophagy hereafter), cytoplasmic constituents are first enveloped by cap-shaped cisternae known as isolation membrane (Fig. 1). Closure of the isolation membrane results in formation of double membrane structures called autophagosomes, which are also known as initial autophagic vacuoles (AVi). Autophagosomes, then, fuse with endosomes to become amphisomes or intermediate AV (AVi/d) (Berg, Fengsrud, Stromhaug, Berg, & Seglen, 1998; Liou, Geuze, Geelen, & Slot, 1997; Nara et al., 2002; Tooze et al., 1990). Eventually, autolysosomes or degrading AV (AVd) are generated by the fusion of the outer membranes of the autophagosomes and lysosomes. Lysosomal hydrolases degrade the cytoplasm-derived contents of the autophagosome, together with its inner membrane. Autophagy is up-regulated by nutrient starvation, most effectively by amino acid withdrawal, in cultured cells. It is also induced in liver and other tissues in fasting animals. Autophagy is thought to play
Fig. 1. The intracellular protein degradation systems in mammalian cells. (A) Scheme of autophagy. A portion of cytoplasm is enclosed by autophagic isolation membrane to form an autophagosome. The outer membrane of the autophagosome then fuses with lysosome to degrade the inside materials. Organelles such as mitochondria can be also degraded by this pathway. (B) The ubiquitin-proteasome system. Proteins to be degraded are tagged with multi-ubiquitin chains. The C-terminal glycine residue is activated by the E1 ubiquitin-activating enzyme then conjugated to proteins, most of which are short-lived, by the actions E2 ubiquitin-conjugating enzymes and E3 ubiquitin-ligase (complex). The ubiquitinated proteins are specifically recognized by 26S proteasome and then degraded.
N. Mizushima et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 553–561
crucial role in homeostasis (Mortimore & Poso, 1987), and also participate in various physiological processes, such as cellular remodeling (Bolender & Weibel, 1973; Masaki, Yamamoto, & Tashiro, 1987), differentiation (Tsukada & Ohsumi, 1993), production of pulmonary surfactant (Hariri et al., 2000), and non-apoptotic cell death during embryogenesis (Clarke, 1990). Defective autophagy may cause mammary tumors (Liang et al., 1999) and a specific type of myopathy (Nishino et al., 2000; Tanaka et al., 2000).
3. The yeast Apg12 conjugation system Autophagy is found among eukaryotes including yeast (Takeshige, Baba, Tsuboi, Noda, & Ohsumi, 1992). The genetical approach using the yeast Saccharomyces cerevisiae shed light on molecular basis of autophagy. Based on the autophagy-defective mutants (Tsukada & Ohsumi, 1993; Thumm et al., 1994), at least 16 genes (APG and AUT genes) required for autophagosome formation was identified. Among these, two novel ubiquitin-like conjugation systems were found to be essential for autophagy (Ohsumi, 2001).
555
The first was the Apg12 conjugation system (Mizushima et al., 1998) (Fig. 2). Apg12 is a 186-amino acid protein without apparent homology to known proteins. Intriguingly, most endogenous Apg12 is conjugated to another protein, Apg5 (Kuma, Mizushima, Ishihara, & Ohsumi, 2002). The mode of conjugation of Apg12–Apg5 is quite similar to that of ubiquitin. The carboxy-terminal residue of Apg12 was glycine, which is activated by Apg7 in an ATP-dependent manner (Mizushima et al., 1998). Then, Apg12 forms a conjugate with Apg7 through a high energy thioester bond (Kim, Dalton, Eggerton, Scott, & Klionsky, 1999; Yuan, Stromhaug, & Dunn, 1999; Tanida et al., 1999). Apg7 shows homology with E1 ubiquitin-activating enzyme within the region encompassing the putative ATP-binding site (GxGxxG) and the active site cysteine (Mizushima et al., 1998). Most Apg7 exists as homo-dimer (Komatsu et al., 2001). Apg12 is then transferred to Apg10 to form a thioester again (Shintani et al., 1999). The function of Apg10 is likely equivalent to that of E2 ubiquitin-conjugating enzymes, although Apg10 has no homology to E2 enzymes. Finally, the carboxyterminal glycine of Apg12 is covalently attached
Fig. 2. The Apg12 and Aut7/LC3 systems. (A) Apg12 is activated by Apg7 and conjugated to Apg7 via a high energy thioester bond. Subsequently, Apg12 forms a thioester with Apg10. Apg12 is finally conjugated to Apg5 via an isopeptide bond. This process is conserved among eukaryotes. In yeast, the Apg12–Apg5 conjugates are multimerized by Apg16 to form the 350 kDa Apg12–Apg5·Apg16 complex. In mammalian cells, the Apg12–Apg5 conjugate forms an 800 kDa complex with Apg16L. (B) After processed by Aut2/Apg4, Aut7/Apg8 and its mammalian homologue LC3 are activated by Apg7 and then form thioesters with Aut1/Apg3. In yeast, Aut7 is conjugated with phosphatidylethanolamine (PE). In mammalian cells, the target substrate of LC3 has not been identified. The conjugated form of LC3 is thought to be the LC3-II form (see text). Aut7-PE is deconjugated again by Aut2.
556
N. Mizushima et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 553–561
to lysine 149 of Apg5 via an isopeptide bond between the carboxyl group of the glycine residue and the ε-amino group of the lysine residue (Mizushima et al., 1998). Formation of the Apg12–Apg5 conjugate is essential for proceeding of autophagy. The second one is the Aut7/Apg8 system (Fig. 2). The C-terminal arginine residue of Aut7 is cleaved off by Aut2/Apg4, a novel cysteine protease (Kirisako et al., 2000). The exposed glycine residue of Aut7 is conjugated to phosphatidylethanolamine (PE), which is catalyzed by Apg7 and Aut1/Apg3 (Ichimura et al., 2000). Apg7 is shared with the Apg12 system whereas Aut1, which shows partial homology to Apg10, is a specific conjugating enzyme for the Aut7 system. Subsequently, Apg8-PE is deconjugated, again by Aut2. The cycle of conjugation and deconjugation is important for normal progression of autophagy (Kirisako et al., 2000). The Apg12–Apg5 conjugate further interacts with a small coiled-coil protein, Apg16 (Mizushima, Noda, & Ohsumi, 1999). Apg16 forms a homo-oligomer through its coiled-coil region. As each Apg16 molecule interacts with Apg5, Apg16 homo-oligomers crosslink multiple Apg12–Apg5 conjugates. As a result, Apg12–Apg5 and Apg16 form a ∼350 kDa protein complex, thought to contain four sets of Apg12– Apg5 and Apg16 (Kuma, Mizushima, Ishihara, & Ohsumi, 2002). Using an in vivo regulated oligo merization system, it was demonstrated that formation of this ∼350 kDa complex is essential for autophagy. While most Apg12–Apg5·Apg16 complex freely exists in the cytosol (Kuma, Mizushima, Ishihara, & Ohsumi, 2002), a small portion localizes to a punctate structure near the vacuole, termed the pre-autophagosomal structure (PAS) (Kim, Huang, Stromhaug, & Klionsky, 2002; Suzuki et al., 2001). Apg1, Apg2, Aut7/Apg8, Apg9 and Apg14 are also targeted to this structure, from which autophagosomes seem to be generated. A study using the apg5 temperature sensitive mutant showed that Apg5 is directly required for autophagosome formation (George et al., 2000). In the strains defective in the Apg12 conjugation system, PE conjugation of Aut7 is severely reduced and recruitment of Aut7 to PAS is abolished (Kim, Huang, & Klionsky, 2001; Suzuki et al., 2001). Thus, the functions of these two ubiquitin-like systems are closely, probably directly linked.
4. The Apg12 system in mammalian cells Both the Apg12 and Aut7 system are highly conserved in mammals. In mouse and human, there seems only one orthologue for each component of the Apg12 system. Apg12 is conjugated to Apg5 (Mizushima, Sugita, Yoshimori, & Ohsumi, 1998), which is catalyzed by Apg7 (Tanida, Tanida-Miyake, Ueno, & Kominami, 2001) and Apg10 (Mizushima, Yoshimori, & Ohsumi, 2002). In mammalian cells, the Apg12–Apg5 conjugate is a component of ∼800 kDa protein complex. Purification of this complex revealed an additional novel protein that interacts with Apg5 (Mizushima et al., manuscript submitted). It is a 63–74 kD protein with several spliced isoforms, which is quite larger than yeast Apg16 (17 kD). As the N-terminal region of this novel protein contains several features similar to yeast Apg16, it was designated Apg16L (Apg16-like). Apg16L, however, has a large C-terminal domain containing seven WD repeats, absent from yeast Apg16. By a database search, Apg16L homologues are found in all eukaryotes except Saccharomyces cerevisiae and Pichia pastoris (Sakai, personal communication), which have Apg16 without WD repeats. Significance of the WD repeats in Apg16L has not been revealed. In contrast to Apg12, at least three Aut7 homologues were identified in mammals: microtubuleassociated protein 1 (MAP1) light chain 3 (LC3) (Mann & Hammarback, 1994), Golgi-associated ATPase Enhancer of 16 kDa (GATE-16) (Sagiv, LegesseMiller, Porat, & Elazar, 2000) and ␥-aminobutyric acid (GABA)A -receptor-associated protein (GABARAP) (Wang, Bedford, Brandon, Moss, & Olsen, 1999). Among them, LC3 was shown to localize on autophagosome membrane (Kabeya et al., 2000). The precise function and localization of the other two homologues are unclear. GABARAP was suggested to be involved in the GABAA -receptor clustering (Chen, Wang, Vicini, & Olsen, 2000) or transport (Kneussel et al., 2000). GATE-16 has been suggested to be an intra-Golgi transport modulator that interacts with N-ethylmaleimide-sensitive factor (NSF) and Golgi v-SNARE GOS-28 (Sagiv, Legesse-Miller, Porat, & Elazar, 2000). However, transfection experiments showed that both GABARAP and GATE-16 are able to reside on autophagosomal membranes (our unpublished observation). Further analyses are
N. Mizushima et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 553–561
required to reveal the roles of these three molecules. All these Aut7 homologues are processed by mammalian Aut2/Apg4 homologues to expose the conserved C-terminal glycine (our unpublished observation). They are further catalyzed by mammalian Apg7 (Tanida, Tanida-Miyake, Ueno, & Kominami, 2001) and Aut1/Apg3 homologues (Tanida, TanidaMiyake, Komatsu, Ueno, & Kominami, 2002). Function of mammalian Apg12 system was examined using mouse embryonic stem (ES) cells, because genetic approach is feasible and size of autophagosome is larger than most of other cell lines. A gene targeting study demonstrated that Apg5 is required for autophagosome formation also in mammalian cells (Mizushima et al., 2001). LC3 cannot target to the membrane in APG5−/− cells (Mizushima et al., 2001). In wild-type cells, LC3 is detected in two forms on immunoblotting: 18 kDa LC3-I and 16 kDa LC3-II (Kabeya et al., 2000). Since LC3-II is a membrane bound form, it was speculated as a PE-conjugated form. Strikingly, in APG5−/− cells, LC3-II is not generated at all (Mizushima et al., 2001). Thus, functional relationship between the Apg12 and LC3 system is clearly evidenced also in mammalian cells. However, direct interaction between Apg12 and Apg5 and LC3 is not observed (our unpublished observation) while weak interaction between Apg12–Apg5 and Aut7 is suggested in yeast (Kim, Huang, Stromhaug, & Klionsky, 2002). In addition, possible inter-dependence between mammalian Apg12 and LC3 conjugation reaction is suggested (Tanida, Nishitani, Nemoto, Ueno, & Kominami, 2002; Tanida, Tanida-Miyake, Komatsu, Ueno, & Kominami, 2002).
5. Role of Apg12–Apg5 in mammalian autophagy It should be noted that most Apg5 is conjugated with Apg12 and most Apg12–Apg5 forms an ∼800 kDa complex with Apg16L irrespective of whether autophagy is induced or not. Thus, the conjugation and complex formation would not be a trigger of autophagy, rather provide basic machinery involved in autophagosome formation. Localization of Apg12–Apg5 was examined in ES cells in detail using GFP-fused Apg5 (Mizushima et al., 2001). A small
557
Fig. 3. Model of autophagosome formation in mammalian cells. The Apg12–Apg5 conjugate and Apg16L localize to the isolation membrane throughout its elongation process. LC3 is recruited to the membrane in the Apg5-dependent manner. Apg12–Apg5 and Apg16L dissociate from the membrane upon completion of autophagosome formation, while LC3 remains on the autophagosome membrane. Apg5 and its modification by Apg12 are required for elongation of the isolation membrane.
fraction of cytosolic Apg12–Apg5·Apg16L complex localizes to the isolation membrane throughout its elongation process (Fig. 3). Apg12–Apg5 initially associates with a small crescent-shaped vesicle evenly. As the membrane elongates, Apg12–Apg5 shows asymmetric localization; most Apg12–Apg5 associates with the outer side of the isolation membrane. Finally, Apg12–Apg5 dissociates from the membrane upon completion of autophagome formation. Apg16L always behaves with Apg12–Apg5, suggesting that these proteins associates and dissociated from the membrane as the 800 kDa complex (manuscript submitted). LC3 also targets to the isolation membrane in Apg5-dependent manner, and remains on the autophagosomal membrane even after Apg12–Apg5 dissociates (Fig. 3) (Kabeya et al., 2000; Mizushima et al., 2001). The significance of the Apg12 conjugation was also determined using the APG5−/− ES cells. In the mammalian Apg12 system, Apg12 is conjugated Lys130 of Apg5 (Mizushima et al., 1998). When the Apg5K130R mutant, in which Lys130 is replaced with Arg, is expressed in APG5−/− ES cells, Apg5 is no longer conjugated with Apg12. Unexpectedly, the small vesicles (autophagosome precursors), to which Apg5K130R mutant and Apg16L associate, are generated in these cells (Mizushima et al., 2001). However, the membrane does not elongate to form autophagosomes. Thus, the covalent modification of Apg5 with Apg12 is not required for membrane targeting of Apg5 and Apg16L but is essential for involvement of Apg5 in elongation
558
N. Mizushima et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 553–561
of the isolation membranes. Modification of Apg5 with Apg12 is also required for targeting of LC3 to the isolation membranes (Mizushima et al., 2001). Thus, the Apg12–Apg5 conjugate plays an essential role in isolation membrane development, together with LC3. How autophagosomes are generated has yet been poorly understood. The detection of endoplasmic reticulum (ER) marker proteins suggested that the isolation membranes are derived from the ER (Arstila & Trump, 1968; Dunn, 1990; Ericsson, 1969; Ueno, Muno, & Kominami, 1991). Involvement of post-Golgi membranes was also suggested (Locke & Sykes, 1975; Yamamoto, Masaki, & Tashiro, 1990). Finally, there were reports indicating unique feature of isolation membrane that is different from any known organelles (Baba, Osumi, & Ohsumi, 1995; Baba, Takeshige, Baba, & Ohsumi, 1994; Hirsimaki & Reunanen, 1980; Reunanen & Hirsimaki, 1983; Stromhaug, Berg, Fengsrud, & Seglen, 1998). The studies on mammalian Apg12–Apg5 provided valuable insights into autophagosome formation. First, visualization of GFP-Apg5 in living cells directly demonstrated that autophagosomes are generated by elongation of small membrane structures, autophagosome precursors, not derived from pre-existing large membrane such as ER cisternae (Mizushima et al., 2001). Second, genetical manipulation of ES cells, particularly expression of the conjugation-defective Apg5 mutant, dissected the autophagosome formation into at least two steps, formation of the precursor vesicle and its elongation process (Mizushima et al., 2001). The Apg12–Apg5 conjugate and probably LC3 are required for the latter step. What is the precursor vesicle remains to be an unsolved question. Treatment of cells with 3-methyladenine, which is widely used as an autophagy inhibitor (Seglen & Gordon, 1982), abolishes the formation of any Apg5-positive structures (Mizushima et al., 2001). 3-methyladenine is now considered as a phosphatidylinositol 3-kinase (PI3K) inhibitor (Petiot, Ogier-Denis, Blommaart, Meijer, & Codogno, 2000). Indeed, the autophagosome precursors are not formed by treatment with wortmannin, a well-known PI3K inhibitor, suggesting that PI3K activity is required for early stage of autophagosome formation (Mizushima et al., 2001). Although, it is not known whether a structure equivalent to the yeast PAS exists in mammalian cells, these results are quite consistent with those observed in yeast;
a PI3K complex made up of Apg6, Apg14, Vps15 and Vps34 is required for PAS formation in yeast (Kihara, Noda, Ishihara, & Ohsumi, 2001; Suzuki et al., 2001). It is also proposed that yeast autophagosomes are generated through distinct processes, nucleation, assembly and elongation (Abeliovich, Dunn, Kim, & Klionsky, 2000; Noda, Suzuki, & Ohsumi, 2002).
6. Future prospects Although it is suggested that the Apg12–Apg5 conjugate functions in elongation step of isolation membrane, the exact molecular mechanism of Apg12–Apg5 is still unknown. The characteristic distribution of Apg12–Apg5 on elongating isolation membrane suggests it may function as a coat protein, although the mode of membrane dynamics is quite different from conventional membrane budding. The association–dissociation cycle of Apg12–Apg5 is also similar to behavior of coat proteins during vesicle budding. Alternatively, they may act as a receptor for LC3 itself or membrane containing LC3. All these possibilities should be addressed. Another obvious question is why Apg12 and Apg5 must be conjugated post-translationally, instead of being synthesized as single molecule. These two proteins are conjugated immediately after the synthesis and the conjugation seems to be irreversible (our unpublished observation). Thus, the Apg12–Apg5 conjugate always behaves as if it was a single protein. This is a quite unique feature of the Apg12 conjugation system compared with other Ubl system. All of ubiquitin, SUMO/Smt3, Nedd8/Rub1 and Aut7/Apg8 conjugations are reversible, and deconjugation processes are suggested to be important (Kirisako et al., 2000; Li & Hochstrasser, 1999). Understanding the purpose of this irreversible conjugation would provide new insights into Ubl systems that are well conserved through evolution.
References Abeliovich, H., Dunn Jr., W. A., Kim, J., & Klionsky, D. J. (2000). Dissection of autophagosome biogenesis into distinct nucleation and expansion steps. Journal of Cell Biology, 151, 1025–1033.
N. Mizushima et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 553–561 Arstila, A. U., & Trump, B. F. (1968). Studies on cellular autophagocytosis. The formation of autophagic vacuoles in the liver after glucagon administration. American Journal of Pathology, 53, 687–733. Ashford, T. P., & Porter, K. R. (1962). Cytoplasmic components in hepatic cell lysosomes. Journal of Cell Biology, 12, 198–202. Baba, M., Osumi, M., & Ohsumi, Y. (1995). Analysis of the membrane structure involved in autophagy in yeast by freeze-replica method. Cell Structure and Function, 20, 465– 471. Baba, M., Takeshige, K., Baba, N., & Ohsumi, Y. (1994). Ultrastructural analysis of the autophagic process in yeast: Detection of autophagosomes and their characterization. Journal of Cell Biology, 124, 903–913. Berg, T. O., Fengsrud, M., Stromhaug, P. E., Berg, T., & Seglen, P. O. (1998). Isolation and characterization of rat liver amphisomes. Evidence for fusion of autophagosomes with both early and late endosomes. The Journal of Biological Chemistry, 273, 21883–21892. Blommaart, E. F. C., Luiken, J. J. F. P., & Meijer, A. J. (1997). Autophagic proteolysis: Control and specificity. Histochemical Journal, 29, 365–385. Bolender, R. P., & Weibel, E. R. (1973). A morphometric study of the removal of phenobarbital-induced membranes from hepatocytes after cessation of treatment. Journal of Cell Biology, 56, 746–761. Chen, L., Wang, H., Vicini, S., & Olsen, W. (2000). The gamma-aminobutyric acid type A (GABAA) receptor-associated protein (GABARAP) promotes GABAA receptor clustering and modulates the channel kinetics. Proceedings of the National Academy of Sciences of the United States of America, 97, 11557–11562. Clarke, P. G. (1990). Developmental cell death: Morphological diversity and multiple mechanisms. Anatomy and Embryology (Berlin), 181, 195–213. de Duve, C. (1959). Lysosomes, a new group of cytoplasmic particles. In T. Hayashi (Ed.), Subcellular Particles (pp. 128–159). New York: Ronald. Dunn Jr., W. A. (1994). Autophagy and related mechanisms of lysosome-mediated protein degradation. Trends in Cell Biology, 4, 139–143. Dunn Jr., W. A. (1990). Studies on the mechanisms of autophagy: Formation of the autophagic vacuole. Journal of Cell Biology, 110, 1923–1933. Ericsson, J. L. (1969). Studies on induced cellular autophagy. II. Characterization of the membranes bordering autophagosomes in parenchymal liver cells. Experimental Cell Research, 56, 393–405. George, M. D., Baba, M., Scott, S. V., Mizushima, N., Garrison, B. S., Ohsumi, Y., & Klionsky, D. J. (2000). Apg5p functions in the sequestration step in the cytoplasm-to-vacuole targeting and macroautophagy pathways. Molecular Biology of the Cell, 11, 969–982. Hariri, M., Millane, G., Guimond, M. P., Guay, G., Dennis, J. W., & Nabi, I. R. (2000). Biogenesis of multilamellar bodies via autophagy. Molecular Biology of the Cell, 11, 255–268. Hershko, A., & Ciechanover, A. (1998). The ubiquitin system. Annual Review of Biochemistry, 67, 425–479.
559
Hirsimaki, P., & Reunanen, H. (1980). Studies on vinblastineinduced autophagocytosis in mouse liver. II. Origin of membranes and acquisition of acid phosphatase. Histochemistry, 67, 139–153. Hochstrasser, M. (1996). Ubiquitin-dependent protein degradation. Annual Review of Genetics, 30, 405–439. Ichimura, Y., Kirisako, T., Takao, T., Satomi, Y., Shimonishi, Y., Ishihara, N., Mizushima, N., Tanida, I., Kominami, E., Ohsumi, M., Noda, T., & Ohsumi, Y. (2000). A ubiquitin-like system mediates protein lipidation. Nature, 408, 488–492. Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y., & Yoshimori, T. (2000). LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO Journal, 19, 5720–5728. Khalfan, W. A., & Klionsky, D. J. (2002). Molecular machinery required for autophagy and the cytoplasm to vacuole targeting (Cvt) pathway in S. cerevisiae. Current Opinion in Cell Biology, 14, 468–475. Kihara, A., Noda, T., Ishihara, N., & Ohsumi, Y. (2001). Two distinct Vps34 phosphatidylinositol 3-kinase complexs function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae. Journal of Cell Biology, 152, 519–530. Kim, J., Dalton, V. M., Eggerton, K. P., Scott, S. V., & Klionsky, D. J. (1999). Apg7p/Cvt2p is required for the Cvt, macroautophagy, and peroxisome degradation pathway. Molecular Biology of the Cell, 10, 1337–1351. Kim, J., Huang, W. -P., & Klionsky, D. J. (2001). Membrane recruitment of Aut7p in the autophagy and cytoplasm to vacuole targeting pathways requires Aut1p, Aut2p, and autophagy conjugation complex. Journal of Cell Biology, 152, 51–64. Kim, J., Huang, W. -P., Stromhaug, P. E., & Klionsky, D. J. (2002). Convergence of multiple autophagy and Cvt components to a perivacuolar membrane compartment prior to de novo vesicle formation. The Journal of Biological Chemistry, 277, 763–773. Kirisako, T., Ichimura, Y., Okada, H., Kabeya, Y., Mizushima, N., Yoshimori, T., Ohsumi, M., Takao, T., Noda, T., & Ohsumi, Y. (2000). The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway. Journal of Cell Biology, 151, 263–275. Klionsky, D. J., & Emr, S. D. (2000). Autophagy as a regulated pathway of cellular degradation. Science, 290, 1717–1721. Kneussel, M., Haverkamp, S., Fuhrmann, J. C., Wang, H., Wassle, H., Olsen, R. W., & Betz, H. (2000). The gamma-aminobutyric acid type A receptor (GABAAR)-associated protein GABAPAR interacts with gephyrin but is not involved in ireceptor anchoring at the symapse. Proceedings of the National Academy of Sciences of the United States of America, 97, 8594–8599. Komatsu, M., Tanida, I., Ueno, T., Ohsumi, M., Ohsumi, Y., & Kominami, E. (2001). The C-terminal region of an Apg7p/Cvt2p is required for homodimerization and is essential for its E1 activity and E1-E2 complex formation. The Journal of Biological Chemistry, 276, 9846–9854. Kuma, A., Mizushima, N., Ishihara, N., & Ohsumi, Y. (2002). Formation of the ∼350 kD Apg12–Apg5-Apg16 multimeric complex, mediated by Apg16 oligomerization, is essential for
560
N. Mizushima et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 553–561
autophagy in yeast. The Journal of Biological Chemistry, 277, 18619–18625. Li, S. J., & Hochstrasser, M. (1999). A new protease required for cell-cycle progression in yeast. Nature, 398, 246–251. Liang, X. H., Jackson, S., Seaman, M., Brown, K., Kempkes, B., Hibshoosh, H., & Levine, B. (1999). Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature, 402, 672– 676. Liou, W., Geuze, H. J., Geelen, M. J., & Slot, J. W. (1997). The autophagic and endocytic pathways converge at the nascent autophagic vacuoles. Journal of Cell Biology, 136, 61–70. Locke, M., & Sykes, A. K. (1975). The role of the Golgi complex in the isolation and digestion of organelles. Tissue Cell, 7, 143–158. Mann, S. S., & Hammarback, J. A. (1994). Molecular characterization of light chain 3. The Journal of Biological Chemistry, 269, 11492–11497. Masaki, R., Yamamoto, A., & Tashiro, Y. (1987). Cytochrome P-450 and NADPH-cytochrome P-450 reductase are degraded in the autolysosomes in rat liver. Journal of Cell Biology, 104, 1207–1215. Mizushima, N., Noda, T., Yoshimori, T., Tanaka, Y., Ishii, T., George, M. D., Klionsky, D. J., Ohsumi, M., & Ohsumi, Y. (1998). A protein conjugation system essential for autophagy. Nature, 395, 395–398. Mizushima, N., Yamamoto, A., Hatano, M., Kobayashi, Y., Kabeya, Y., Suzuki, K., Tokuhisa, T., Ohsumi, Y., & Yoshimori, T. (2001). Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. Journal of Cell Biology, 152, 657–667. Mizushima, N., Noda, T., & Ohsumi, Y. (1999). Apg16p is required for the function of the Apg12p-Apg5p conjugate in the yeast autophagy pathway. EMBO Journal, 18, 3888–3896. Mizushima, N., Sugita, H., Yoshimori, T., & Ohsumi, Y. (1998). A new protein conjugation system in human. The counterpart of the yeast Apg12p conjugation system essential for autophagy. The Journal of Biological Chemistry, 273, 33889–33892. Mizushima, N., Yoshimori, T., Ohsumi, Y. (2002) Mouse Apg10 as an Apg12 conjugating enzyme: Analysis by the conjugation-mediated yeast two-hybrid method. FEBS Letters, 532, 450–454. Mortimore, G., & Poso, E. A. R. (1987). Intracellular protein catabolism and its control during nutrient deprivation and supply. Annual Review of Nutrition, 7, 539–564. Nara, A., Mizushima, N., Yamamoto, A., Kabeya, Y., Ohsumi, Y., Yoshimori, T. (2002) SKD1 AAA ATPase-dependent endosomal transport is involved in autolysosome formation. Cell Structure and Function, 27, 29–37. Nishino, I., Fu, J., Tanji, K., Yamada, T., Shimojo, S., Koori, T., Mora, M., Riggs, J. E., Oh, S. J., Koga, Y., Sue, C. M., Yamamoto, A., Murakami, N., Shanske, S., Byrne, E., Bonilla, E., Nonaka, I., DiMauro, S., & Hirano, M. (2000). Primary LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and myopathy (Danon disease). Nature, 406, 906–910. Noda, T., Suzuki, K., & Ohsumi, Y. (2002). Yeast autophagosomes: De novo formation of a membrane structure. Trends in Cell Biology, 12, 231–235.
Ohsumi, Y. (2001). Molecular dissection of autophagy: Two ubiquitin-like systems. Natural Review in Molecular and Cellular Biology, 2, 211–216. Petiot, A., Ogier-Denis, E., Blommaart, E. F., Meijer, A. J., & Codogno, P. (2000). Distinct classes of phosphatidylinositol 3 -kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. The Journal of Biological Chemistry, 275, 992–998. Reggiori, F., & Klionsky, D. J. (2002). Autophagy in the eukaryotic cell. Eukaryotic Cell, 1, 11–21. Reunanen, H., & Hirsimaki, P. (1983). Studies on vinblastineinduced autophagocytosis in mouse liver. IV. Origin of membranes. Histochemistry, 79, 59–67. Sagiv, Y., Legesse-Miller, A., Porat, A., & Elazar, Z. (2000). GATE-16, a membrane transport modulator, interacts with NSF and the Golgi v-SNARE GOS-28. EMBO Journal, 19, 1494– 1504. Seglen, P. O., & Gordon, P. B. (1982). 3-Methyladenine: Specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proceedings of the National Academy of Sciences of the United States of America, 79, 1889– 1892. Shintani, T., Mizushima, N., Ogawa, Y., Matsuura, A., Noda, T., & Ohsumi, Y. (1999). Apg10p, a novel protein-conjugating enzyme essential for autophagy in yeast. EMBO Journal, 18, 5234–5241. Stromhaug, P. E., Berg, T. O., Fengsrud, M., & Seglen, P. O. (1998). Purification and characterization of autophagosomes from rat hepatocytes. Biochemical Journal, 335, 217–224. Suzuki, K., Kirisako, T., Kamada, Y., Mizushima, N., Noda, T., & Ohsumi, Y. (2001). The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation. EMBO Journal, 20, 5971–5981. Takeshige, K., Baba, M., Tsuboi, S., Noda, T., & Ohsumi, Y. (1992). Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction. Journal of Cell Biology, 119, 301–311. Tanaka, Y., Guhde, G., Suter, A., Eskelinen, E. -L., Hartmann, D., Lullmann-Rauch, R., Janssen, P. M. L., Blanz, J., von Figura, K., & Saftig, P. (2000). Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice. Nature, 406, 902–906. Tanida, I., Mizushima, N., Kiyooka, M., Ohsumi, M., Ueno, T., Ohsumi, Y., & Kominami, E. (1999). Apg7p/Cvt2p: A novel protein-activating enzyme essential for autophagy. Molecular Biology of the Cell, 10, 1367–1379. Tanida, I., Nishitani, T., Nemoto, T., Ueno, T., & Kominami, E. (2002). Mammalian Apg12p, but not the Apg12p·Apg5p conjugate, facilitates LC3 processing. Biochemical and Biophysical Research Communications, 296, 1164–1170. Tanida, I., Tanida-Miyake, E., Komatsu, M., Ueno, T., & Kominami, E. (2002). Human Apg3p/Aut1p homologue is an authentic E2 enzyme for multiple substrates, GATE-16, GABARAP, and MAP-LC3, and facilitates the conjugation of hApg12p to hApg5p. The Journal of Biological Chemistry, 277, 13739–13744. Tanida, I., Tanida-Miyake, E., Ueno, T., & Kominami, E. (2001). The human homolog of Saccharomyces cerevisiae Apg7p is
N. Mizushima et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 553–561 a protein-activating enzyme for multiple substrates including human Apg12p, GATE-16, GABARAP, and MAP-LC3. The Journal of Biological Chemistry, 276, 1701–1706. Thumm, M., Egner, R., Koch, B., Schlumpberger, M., Straub, M., Veenhuis, M., & Wolf, D. H. (1994). Isolation of autophagocytosis mutants of Saccharomyces cerevisiae. FEBS Letters, 349, 275–280. Tooze, J., Hollinshead, M., Ludwig, T., Howell, K., Hoflack, B., & Kern, H. (1990). In exocrine pancreas, the basolateral endocytic pathway converges with the autophagic pathway immediately after the early endosome. Journal of Cell Biology, 111, 329– 345. Tsukada, M., & Ohsumi, Y. (1993). Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Letters, 333, 169–174.
561
Ueno, T., Muno, D., & Kominami, E. (1991). Membrane markers of endoplasmic reticulum preserved in autophagic vacuolar membranes isolated from leupeptin-administered rat liver. The Journal of Biological Chemistry, 266, 18995–18999. Wang, H., Bedford, F. K., Brandon, N. J., Moss, S. J., & Olsen, R. W. (1999). GABAA -receptor-associated protein links GABAA receptors and the cytoskeleton. Nature, 397, 69–72. Yamamoto, A., Masaki, R., & Tashiro, Y. (1990). Characterization of the isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes by lectin cytochemistry. Journal of Histochemistry and Cytochemistry, 38, 573–580. Yuan, W., Stromhaug, P. E., & Dunn Jr., W. A. (1999). Glucose-induced microautophagy of peroxysomes in Pichia pastoris requires a unique E1-like protein. Molecular Biology of the Cell, 10, 1353–1366.