Structure
Previews A Small Switch Has a Large Effect on Autophagy Winfried Weissenhorn1,2,3,* and Marie-Odile Fauvarque1,4,5,* 1University
Grenoble Alpes, 38000 Grenoble, France UVHCI, 38000 Grenoble, France 3Unit of Virus Host-Cell Interactions, University Grenoble Alpes-EMBL-CNRS, 6 rue Jules Horowitz, 38042 Grenoble, France 4INSERM, U1038, 38054 Grenoble, France 5CEA, DSV, iRTSV, BGE, Gen&Chem, 38054 Grenoble, France *Correspondence: [email protected] (W.W.), [email protected] (M.-O.F.) http://dx.doi.org/10.1016/j.str.2013.12.006 2CNRS,
Atg8 family proteins and Atg13 are important regulators of autophagy. In this issue of Structure, Suzuki and colleagues describe crystal structures of light chain 3 (LC3; Atg8) in complex with the Atg13 LC3 interacting region (LIR) motif and identify a subtle switch of side chain conformation regulating the LC3-LIR interaction and autophagosome formation in vivo. Autophagy is at the basis of cellular homeostasis in eukaryotes. A fine balance regulates biosynthesis and degradation of molecules and whole compartments under high nutrient as well as under starvation conditions when the cell can rescue itself by shutting down part of itself in a process known as macroautophagy. A second type of autophagy, selective autophagy, is active at a basal level and is an essential tool for each cell to deal with toxic molecules, such as protein aggregates, damaged organelles, or pathogens. This cellular self-degradation process is initiated by the generation of a double membrane structure, the phagophore. It grows into a cup-like membrane structure in the cytoplasm that encircles intracellular material, such as cellular components (mitochondria, peroxysomes, ribosomes, part of the cytoplasm, proteins, and aggregates) or pathogens generating the autophagosome, which fuses with the vacuole (yeast) or lysosome, leading to the hydrolysis of its content and the generation of new metabolic precursors (Figure 1). Besides controlling cellular homeostasis, autophagy processes are recognized as playing important roles during the immune defense against pathogens, and dysfunction of autophagy has been linked to cancer and neurodegenerative diseases (Mizushima et al., 2008). Therefore, there is a crucial need to improve our knowledge on the regulatory, molecular, and structural mechanisms underlying this fascinating aspect of cell physiology. A complex sum of events regulates autophagy initiation and completion
(Boya et al., 2013). Without specific induction, autophagy is a repressed phenomenon that is maintained at a low basal level. One of the main signaling pathways responsible for this repression is the mTor (TOR in yeast) pathway, which promotes protein translation and enhanced metabolism. In the case of a poor supply of nutrients, the mTor pathway is repressed and autophagy is activated as an immediate response (Figure 1). A different set of events leads to selective autophagy, the degradation of ubiquinated cargo, which is recruited by yeast Atg19 or mammalian p62 to the autophagosome (Figure 1) (Shaid et al., 2013). Genetic studies in yeast revealed the sequential activation of Atg proteins encoded by 36 ATG genes. First, Atg9 positive vesicles fuse and form a membrane sheet that develops into a cup-shaped elongated structure, the phagophore, which engulfs cargo and closes to form the autophagosome. Atg proteins are recruited to the pre-autophagosomal structure (PAS) in close proximity to the vacuole and are visible by light microscopy. Atg17, Atg29, and Atg31 are constitutively recruited to the PAS under nutrient-rich and limiting conditions where they form the platform for the assembly of the Atg1 complex (Ragusa et al., 2012). The Atg1 complex is inactivated under normal physiological conditions through phosphorylation of Atg13 by the target of rapamycin complex 1 (TORC1) and protein kinase A (Kamada et al., 2000; Stephan et al., 2009). Upon starvation or invasion of certain pathogens, Atg13 is dephos-
phorylated and triggers the assembly of the Atg1 complex, a dimer of Atg1Atg13-Atg17-Atg31-Atg29 pentamers, which may act as a scaffold to facilitate the fusion of Atg9 vesicles forming the initial membrane sheet that develops into the phagophore (Stanley et al., 2014). Besides controlling assembly of the Atg1 complex and its human counterpart Ulk1 (Hosokawa et al., 2009), Atg13 interacts with Atg8, which has seven homologs in human cells, including the microtubule associated protein 1 light chain 3 (LC3) family members LC3A, LC3B, and LC3C. Atg8/LC3 activation and lipid anchoring on the phagophore is an indispensable step for phagophore maturation (Nakatogawa et al., 2007). In selective autophagy, binding of autophagy adaptors to Atg8/LC3 is mediated by the LC3 interacting region (LIR), which triggers the incorporation of ubiquitinated cargo into the autophagosome (Johansen and Lamark, 2011) (Figure 1). LIR motifs utilize two sets of hydrophobic residues (Trp/Phe/Tyr and Leu/Ile/Val) to contact two hydrophobic surfaces, the W- and L-sites on Atg8 proteins (Noda et al., 2010), which are also present in other Atg molecules, including Atg13, allowing for Atg8 activation in response to starvation. The LIR binding interface thus constitutes a major entry point for controlling autophagy. In this issue of Structure, Suzuki et al. (2014) characterize the molecular details of the interaction of LC3 family members and the LIR of Atg13. They mapped the minimal Atg13 binding region to residues 441 to 447 and showed that LC3A and LC3C interact with the LIR with a KD of
Structure 22, January 7, 2014 ª2014 Elsevier Ltd All rights reserved 1
Structure
Previews
10–18 mM, while the affinity Noticeably, yeast does not of the LIR to LC3B is approxseem to require this level imately three times lower. of regulation, because the Comparison of the crystal corresponding Atg8 lysine structures of unliganded and residue (Lys46) does not liganded LC3 isoforms cover the LIR binding site demonstrate that the Atg13 (Kumeta et al., 2010). The LIR interacts with the L- and next step in the puzzle will W-sites similar to what was be to find out why, how, reported for the LC3B/p62 and when mammalian cells interaction (Ichimura et al., employ their seven Atg8 2008). Although the overall homologs during diverse structural changes between autophagic processes. unliganded and liganded REFERENCES LC3 isoforms are small, Lys49 of LC3A undergoes a Boya, P., Reggiori, F., and Colarge rotamer rearrangement, dogno, P. (2013). Nat. Cell Biol. 15, which is important for ac713–720. cessing the hydrophobic Hosokawa, N., Hara, T., Kaizuka, T., interaction surface. Notably, Kishi, C., Takamura, A., Miura, Y., Iemura, S., Natsume, T., Takehana, the Lys49 movement controlK., Yamada, N., et al. (2009). Mol. ling LIR binding is conserved Biol. Cell 20, 1981–1991. among mammalian LC3 hoIchimura, Y., Kumanomidou, T., Sou, mologs. The authors suggest Y.S., Mizushima, T., Ezaki, J., Ueno, that the L-site makes the first T., Kominami, E., Yamane, T., Tanaka, K., and Komatsu, M. (2008). contact to the LIR, which J. Biol. Chem. 283, 22847–22857. leads to a structural rearrangement of the Lys49 Johansen, T., and Lamark, T. (2011). Autophagy 7, 279–296. side chain and subsequent opening up of the W-site. Kamada, Y., Funakoshi, T., Shintani, T., Nagano, K., Ohsumi, M., and Consistent with the idea that Ohsumi, Y. (2000). J. Cell Biol. 150, Figure 1. The LIR Binding Interface Plays a Central Role during Early Lys49 acts as a gate keeper, 1507–1513. Steps in Autophagy replacement of Lys49 by The TOR pathway promotes protein translation and enhanced metabolism Kumeta, H., Watanabe, M., NakatoAla increased the binding while maintaining the autophagy initiator complex (Atg1/ULK1) in a phosgawa, H., Yamaguchi, M., Ogura, K., affinity of the LIR peptide, phorylated repressed state. Lack of nutrients induces the repression of Adachi, W., Fujioka, Y., Noda, N.N., Ohsumi, Y., and Inagaki, F. (2010). TORC1 and dephosphorylation of Atg13. Atg13 then regulates the assembly whereas mutation of Lys51, J. Biomol. NMR 47, 237–241. of the Atg1 complex (Atg1-Atg13-Atg17-Atg31-Atg29), which catalyzes the which contacts LIR, deformation of the phagophore from Atg9-containing vesicles. Atg8/LC3 family Mizushima, N., Levine, B., Cuervo, creased binding. However, proteins are concomitantly anchored to the phagophore membrane via A.M., and Klionsky, D.J. (2008). sequential activation of another set of Atg proteins, resulting in the conjugation when the mutants were Nature 451, 1069–1075. of phosphatidylethanolamine (PE). Membrane-anchored Atg8/LC3 recruits tested in vivo, neither supLIR-containing interaction partners such as Atg13 or cargo-binding adaptors Nakatogawa, H., Ichimura, Y., and ported autophagosome forsuch as Atg19 (or p62 in mammals), which in turn bind to ubiquitinated cargo. Ohsumi, Y. (2007). Cell 130, 165–178. mation in nutrient-rich conThe elongating phagophore then adopts a cup-like shape that closes to form Noda, N.N., Ohsumi, Y., and Inathe autophagosome engulfing its content. Upon fusion of autophagosomes ditions and both showed gaki, F. (2010). FEBS Lett. 584, and vacuoles (lysosomes), their content is degraded, which produces meta1379–1385. reduced activity under starbolic precursors and contributes, in vertebrates to pathogens destruction, vation conditions. This was antigen presentation and tissue remodeling. Ragusa, M.J., Stanley, R.E., and Hurley, J.H. (2012). Cell 151, 1501– surprising, because one 1512. would expect that the increased Atg13 binding to Atg8, as mutants on selective autophagosome Shaid, S., Brandts, C.H., Serve, H., and Dikic, I. demonstrated for the Lys49Ala mutation formation is even more dramatic than (2013). Cell Death Differ. 20, 21–30. in vitro, would favor autophagosome for- its effect under starvation conditions, Stanley, R.E., Ragusa, M.J., and Hurley, J.H. mation. The authors conclude that both consistent with the proposal that Atg8- (2014). Trends Cell Biol. 24, 73–81. mutants induce a defect in autophago- LIR interactions also serve to recruit Stephan, J.S., Yeh, Y.Y., Ramachandran, V., some formation and that sole binding cargo such as protein aggregates, Deminoff, S.J., and Herman, P.K. (2009). of Atg8 to Atg13 is not sufficient. If the mitochondria and pathogens to the auto- Proc. Natl. Acad. Sci. USA 106, 17049–17054. affinity is either too high or too low the phagosome. Thus, a small conserved Suzuki, H., Tabata, K., Morita, E., Kawasaki, M., Kato, dynamic process of autophagosome gate keeper, a lysine side chain, deter- R., Dobson, R.C., Yoshimori, T., and Wakatsuki, S. formation is disturbed. The effect of both mines the fate of a mammalian cell. (2014). Structure 22, this issue, 47–58.
2 Structure 22, January 7, 2014 ª2014 Elsevier Ltd All rights reserved
Previews A Small Switch Has a Large Effect on Autophagy Winfried Weissenhorn1,2,3,* and Marie-Odile Fauvarque1,4,5,* 1University
Grenoble Alpes, 38000 Grenoble, France UVHCI, 38000 Grenoble, France 3Unit of Virus Host-Cell Interactions, University Grenoble Alpes-EMBL-CNRS, 6 rue Jules Horowitz, 38042 Grenoble, France 4INSERM, U1038, 38054 Grenoble, France 5CEA, DSV, iRTSV, BGE, Gen&Chem, 38054 Grenoble, France *Correspondence: [email protected] (W.W.), [email protected] (M.-O.F.) http://dx.doi.org/10.1016/j.str.2013.12.006 2CNRS,
Atg8 family proteins and Atg13 are important regulators of autophagy. In this issue of Structure, Suzuki and colleagues describe crystal structures of light chain 3 (LC3; Atg8) in complex with the Atg13 LC3 interacting region (LIR) motif and identify a subtle switch of side chain conformation regulating the LC3-LIR interaction and autophagosome formation in vivo. Autophagy is at the basis of cellular homeostasis in eukaryotes. A fine balance regulates biosynthesis and degradation of molecules and whole compartments under high nutrient as well as under starvation conditions when the cell can rescue itself by shutting down part of itself in a process known as macroautophagy. A second type of autophagy, selective autophagy, is active at a basal level and is an essential tool for each cell to deal with toxic molecules, such as protein aggregates, damaged organelles, or pathogens. This cellular self-degradation process is initiated by the generation of a double membrane structure, the phagophore. It grows into a cup-like membrane structure in the cytoplasm that encircles intracellular material, such as cellular components (mitochondria, peroxysomes, ribosomes, part of the cytoplasm, proteins, and aggregates) or pathogens generating the autophagosome, which fuses with the vacuole (yeast) or lysosome, leading to the hydrolysis of its content and the generation of new metabolic precursors (Figure 1). Besides controlling cellular homeostasis, autophagy processes are recognized as playing important roles during the immune defense against pathogens, and dysfunction of autophagy has been linked to cancer and neurodegenerative diseases (Mizushima et al., 2008). Therefore, there is a crucial need to improve our knowledge on the regulatory, molecular, and structural mechanisms underlying this fascinating aspect of cell physiology. A complex sum of events regulates autophagy initiation and completion
(Boya et al., 2013). Without specific induction, autophagy is a repressed phenomenon that is maintained at a low basal level. One of the main signaling pathways responsible for this repression is the mTor (TOR in yeast) pathway, which promotes protein translation and enhanced metabolism. In the case of a poor supply of nutrients, the mTor pathway is repressed and autophagy is activated as an immediate response (Figure 1). A different set of events leads to selective autophagy, the degradation of ubiquinated cargo, which is recruited by yeast Atg19 or mammalian p62 to the autophagosome (Figure 1) (Shaid et al., 2013). Genetic studies in yeast revealed the sequential activation of Atg proteins encoded by 36 ATG genes. First, Atg9 positive vesicles fuse and form a membrane sheet that develops into a cup-shaped elongated structure, the phagophore, which engulfs cargo and closes to form the autophagosome. Atg proteins are recruited to the pre-autophagosomal structure (PAS) in close proximity to the vacuole and are visible by light microscopy. Atg17, Atg29, and Atg31 are constitutively recruited to the PAS under nutrient-rich and limiting conditions where they form the platform for the assembly of the Atg1 complex (Ragusa et al., 2012). The Atg1 complex is inactivated under normal physiological conditions through phosphorylation of Atg13 by the target of rapamycin complex 1 (TORC1) and protein kinase A (Kamada et al., 2000; Stephan et al., 2009). Upon starvation or invasion of certain pathogens, Atg13 is dephos-
phorylated and triggers the assembly of the Atg1 complex, a dimer of Atg1Atg13-Atg17-Atg31-Atg29 pentamers, which may act as a scaffold to facilitate the fusion of Atg9 vesicles forming the initial membrane sheet that develops into the phagophore (Stanley et al., 2014). Besides controlling assembly of the Atg1 complex and its human counterpart Ulk1 (Hosokawa et al., 2009), Atg13 interacts with Atg8, which has seven homologs in human cells, including the microtubule associated protein 1 light chain 3 (LC3) family members LC3A, LC3B, and LC3C. Atg8/LC3 activation and lipid anchoring on the phagophore is an indispensable step for phagophore maturation (Nakatogawa et al., 2007). In selective autophagy, binding of autophagy adaptors to Atg8/LC3 is mediated by the LC3 interacting region (LIR), which triggers the incorporation of ubiquitinated cargo into the autophagosome (Johansen and Lamark, 2011) (Figure 1). LIR motifs utilize two sets of hydrophobic residues (Trp/Phe/Tyr and Leu/Ile/Val) to contact two hydrophobic surfaces, the W- and L-sites on Atg8 proteins (Noda et al., 2010), which are also present in other Atg molecules, including Atg13, allowing for Atg8 activation in response to starvation. The LIR binding interface thus constitutes a major entry point for controlling autophagy. In this issue of Structure, Suzuki et al. (2014) characterize the molecular details of the interaction of LC3 family members and the LIR of Atg13. They mapped the minimal Atg13 binding region to residues 441 to 447 and showed that LC3A and LC3C interact with the LIR with a KD of
Structure 22, January 7, 2014 ª2014 Elsevier Ltd All rights reserved 1
Structure
Previews
10–18 mM, while the affinity Noticeably, yeast does not of the LIR to LC3B is approxseem to require this level imately three times lower. of regulation, because the Comparison of the crystal corresponding Atg8 lysine structures of unliganded and residue (Lys46) does not liganded LC3 isoforms cover the LIR binding site demonstrate that the Atg13 (Kumeta et al., 2010). The LIR interacts with the L- and next step in the puzzle will W-sites similar to what was be to find out why, how, reported for the LC3B/p62 and when mammalian cells interaction (Ichimura et al., employ their seven Atg8 2008). Although the overall homologs during diverse structural changes between autophagic processes. unliganded and liganded REFERENCES LC3 isoforms are small, Lys49 of LC3A undergoes a Boya, P., Reggiori, F., and Colarge rotamer rearrangement, dogno, P. (2013). Nat. Cell Biol. 15, which is important for ac713–720. cessing the hydrophobic Hosokawa, N., Hara, T., Kaizuka, T., interaction surface. Notably, Kishi, C., Takamura, A., Miura, Y., Iemura, S., Natsume, T., Takehana, the Lys49 movement controlK., Yamada, N., et al. (2009). Mol. ling LIR binding is conserved Biol. Cell 20, 1981–1991. among mammalian LC3 hoIchimura, Y., Kumanomidou, T., Sou, mologs. The authors suggest Y.S., Mizushima, T., Ezaki, J., Ueno, that the L-site makes the first T., Kominami, E., Yamane, T., Tanaka, K., and Komatsu, M. (2008). contact to the LIR, which J. Biol. Chem. 283, 22847–22857. leads to a structural rearrangement of the Lys49 Johansen, T., and Lamark, T. (2011). Autophagy 7, 279–296. side chain and subsequent opening up of the W-site. Kamada, Y., Funakoshi, T., Shintani, T., Nagano, K., Ohsumi, M., and Consistent with the idea that Ohsumi, Y. (2000). J. Cell Biol. 150, Figure 1. The LIR Binding Interface Plays a Central Role during Early Lys49 acts as a gate keeper, 1507–1513. Steps in Autophagy replacement of Lys49 by The TOR pathway promotes protein translation and enhanced metabolism Kumeta, H., Watanabe, M., NakatoAla increased the binding while maintaining the autophagy initiator complex (Atg1/ULK1) in a phosgawa, H., Yamaguchi, M., Ogura, K., affinity of the LIR peptide, phorylated repressed state. Lack of nutrients induces the repression of Adachi, W., Fujioka, Y., Noda, N.N., Ohsumi, Y., and Inagaki, F. (2010). TORC1 and dephosphorylation of Atg13. Atg13 then regulates the assembly whereas mutation of Lys51, J. Biomol. NMR 47, 237–241. of the Atg1 complex (Atg1-Atg13-Atg17-Atg31-Atg29), which catalyzes the which contacts LIR, deformation of the phagophore from Atg9-containing vesicles. Atg8/LC3 family Mizushima, N., Levine, B., Cuervo, creased binding. However, proteins are concomitantly anchored to the phagophore membrane via A.M., and Klionsky, D.J. (2008). sequential activation of another set of Atg proteins, resulting in the conjugation when the mutants were Nature 451, 1069–1075. of phosphatidylethanolamine (PE). Membrane-anchored Atg8/LC3 recruits tested in vivo, neither supLIR-containing interaction partners such as Atg13 or cargo-binding adaptors Nakatogawa, H., Ichimura, Y., and ported autophagosome forsuch as Atg19 (or p62 in mammals), which in turn bind to ubiquitinated cargo. Ohsumi, Y. (2007). Cell 130, 165–178. mation in nutrient-rich conThe elongating phagophore then adopts a cup-like shape that closes to form Noda, N.N., Ohsumi, Y., and Inathe autophagosome engulfing its content. Upon fusion of autophagosomes ditions and both showed gaki, F. (2010). FEBS Lett. 584, and vacuoles (lysosomes), their content is degraded, which produces meta1379–1385. reduced activity under starbolic precursors and contributes, in vertebrates to pathogens destruction, vation conditions. This was antigen presentation and tissue remodeling. Ragusa, M.J., Stanley, R.E., and Hurley, J.H. (2012). Cell 151, 1501– surprising, because one 1512. would expect that the increased Atg13 binding to Atg8, as mutants on selective autophagosome Shaid, S., Brandts, C.H., Serve, H., and Dikic, I. demonstrated for the Lys49Ala mutation formation is even more dramatic than (2013). Cell Death Differ. 20, 21–30. in vitro, would favor autophagosome for- its effect under starvation conditions, Stanley, R.E., Ragusa, M.J., and Hurley, J.H. mation. The authors conclude that both consistent with the proposal that Atg8- (2014). Trends Cell Biol. 24, 73–81. mutants induce a defect in autophago- LIR interactions also serve to recruit Stephan, J.S., Yeh, Y.Y., Ramachandran, V., some formation and that sole binding cargo such as protein aggregates, Deminoff, S.J., and Herman, P.K. (2009). of Atg8 to Atg13 is not sufficient. If the mitochondria and pathogens to the auto- Proc. Natl. Acad. Sci. USA 106, 17049–17054. affinity is either too high or too low the phagosome. Thus, a small conserved Suzuki, H., Tabata, K., Morita, E., Kawasaki, M., Kato, dynamic process of autophagosome gate keeper, a lysine side chain, deter- R., Dobson, R.C., Yoshimori, T., and Wakatsuki, S. formation is disturbed. The effect of both mines the fate of a mammalian cell. (2014). Structure 22, this issue, 47–58.
2 Structure 22, January 7, 2014 ª2014 Elsevier Ltd All rights reserved
