Key points are not available for this paper at this time.
Atg5 is covalently modified with a ubiquitin-like modifier, Atg12, and the Atg12-Atg5 conjugate further forms a complex with the multimeric protein Atg16. The Atg12-Atg5·Atg16 multimeric complex plays an essential role in autophagy, the bulk degradation system conserved in all eukaryotes. We have reported here the crystal structure of Atg5 complexed with the N-terminal region of Atg16 at 1.97Ä resolution. Atg5 comprises two ubiquitin-like domains that flank a helix-rich domain. The N-terminal region of Atg16 has a helical structure and is bound to the groove formed by these three domains. In vitro analysis showed that Arg-35 and Phe-46 of Atg16 are crucial for the interaction. Atg16, with a mutation at these residues, failed to localize to the pre-autophagosomal structure and could not restore autophagy in Atg16-deficient yeast strains. Furthermore, these Atg16 mutants could not restore a severe reduction in the formation of the Atg8-phosphatidylethanolamine conjugate, another essential factor for autophagy, in Atg16-deficient strains under starvation conditions. These results taken together suggest that the direct interaction between Atg5 and Atg16 is crucial to the performance of their roles in autophagy. Atg5 is covalently modified with a ubiquitin-like modifier, Atg12, and the Atg12-Atg5 conjugate further forms a complex with the multimeric protein Atg16. The Atg12-Atg5·Atg16 multimeric complex plays an essential role in autophagy, the bulk degradation system conserved in all eukaryotes. We have reported here the crystal structure of Atg5 complexed with the N-terminal region of Atg16 at 1.97Ä resolution. Atg5 comprises two ubiquitin-like domains that flank a helix-rich domain. The N-terminal region of Atg16 has a helical structure and is bound to the groove formed by these three domains. In vitro analysis showed that Arg-35 and Phe-46 of Atg16 are crucial for the interaction. Atg16, with a mutation at these residues, failed to localize to the pre-autophagosomal structure and could not restore autophagy in Atg16-deficient yeast strains. Furthermore, these Atg16 mutants could not restore a severe reduction in the formation of the Atg8-phosphatidylethanolamine conjugate, another essential factor for autophagy, in Atg16-deficient strains under starvation conditions. These results taken together suggest that the direct interaction between Atg5 and Atg16 is crucial to the performance of their roles in autophagy. Autophagy mediates the bulk degradation of cytoplasmic components in lysosomes/vacuoles (1Seglen P.O. Bohley P. Experientia (Basel). 1992; 48: 158-172Crossref PubMed Scopus (370) Google Scholar, 2Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. J. Cell Biol. 1992; 119: 301-311Crossref PubMed Scopus (969) Google Scholar) and plays a critical role in numerous biological processes such as neurodegeneration and pathogen infection, as well as in the survival response during neonatal starvation (3Hara T. Nakamura K. Matsui M. Yamamoto A. Nakahara Y. Suzuki-Migishima R. Yokoyama M. Mishima K. Saito I. Okano H. Mizushima N. Nature. 2006; 441: 885-889Crossref PubMed Scopus (3162) Google Scholar, 4Komatsu M. Waguri S. Chiba T. Murata S. Iwata J. Tanida I. Ueno T. Koike M. Uchiyama Y. Kominami E. Tanaka K. Nature. 2006; 441: 880-884Crossref PubMed Scopus (2874) Google Scholar, 5Ogawa M. Yoshimori T. Suzuki T. Sagara H. Mizushima N. Sasakawa C. Science. 2005; 307: 727-731Crossref PubMed Scopus (712) Google Scholar, 6Nakagawa I. Amano A. Mizushima N. Yamamoto A. Yamaguchi H. Kamimoto T. Nara A. Funao J. Nakata M. Tsuda K. Hamada S. Yoshimori T. Science. 2004; 306: 1037-1040Crossref PubMed Scopus (948) Google Scholar, 7Kuma A. Hatano M. Matsui M. Yamamoto A. Nakaya H. Yoshimori T. Ohsumi Y. Tokuhisa T. Mizushima N. Nature. 2004; 432: 1032-1036Crossref PubMed Scopus (2423) Google Scholar). In autophagy, a double membrane structure called an autophagosome sequesters a portion of cytoplasm and fuses with the lysosome/vacuole to deliver its contents into the organelle lumen. Atg5 was identified together with other Atg proteins by genetic screening in the yeast Saccharomyces cerevisiae (8Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1427) Google Scholar). Because Atg5 has little sequence homology with proteins with known functions, it is difficult to predict its structure and function from the sequence. Thus far, biochemical analyses have shown that Lys-149 of Atg5 is conjugated to Atg12, a ubiquitin-like (Ubl) 2The abbreviations used are: Ubl, ubiquitin-like; PE, phosphatidylethanolamine; LC3, microtubule-associated protein light chain 3; GST, glutathione S-transferase; API, aminopeptidase I; PAS, pre-autophagosomal structure; CA, casamino acid; SD, synthetic defined; PI3K, phosphoinositide 3-kinase; PI3P, phosphoinositide 3-phosphate; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; HR, helixrich; -N, nitrogen-depleted; -NC, nitrogen- and carbon-depleted. modifier dependent on ATP and two enzymes Atg7 (E1-like) and Atg10 (E2-like) (9Mizushima N. Noda T. Yoshimori T. Tanaka Y. Ishii T. George M.D. Klionsky D.J. Ohsumi M. Ohsumi Y. Nature. 1998; 395: 395-398Crossref PubMed Scopus (1297) Google Scholar, 10Shintani T. Mizushima N. Ogawa Y. Matsuura A. Noda T. Ohsumi Y. EMBO J. 1999; 18: 5234-5241Crossref PubMed Scopus (235) Google Scholar). Compared with other ubiquitin-like modifications, the Atg12-modification is unique in that it is irreversible and constitutive. The majority of Atg5 and Atg12 exist as Atg12-Atg5 conjugates irrespective of whether autophagy is induced or not and behave as a single protein (11Kuma A. Mizushima N. Ishihara N. Ohsumi Y. J. Biol. Chem. 2002; 277: 18619-18625Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). In yeast, the Atg12-Atg5 conjugate localizes to the pre-autophagosomal structure (PAS), a putative center for autophagosome formation (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar). All Atg proteins involved in Atg12-Atg5 conjugation are also conserved in mammals. Localization studies of the Atg12-Atg5 conjugate in embryonic stem cells using green fluorescent protein-fused Atg5 showed that the Atg12-Atg5 conjugate is translocated from the cytosol to the isolation membranes upon nutrient deprivation and, immediately upon completion of autophagosome formation, the Atg12-Atg5 conjugate dissociates from the membrane, suggesting that it plays a significant role in autophagosome formation (13Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1164) Google Scholar). In addition to the covalent interaction with Atg12, Atg5 interacts non-covalently with a multimeric protein, Atg16 (14Mizushima N. Noda T. Ohsumi Y. EMBO J. 1999; 18: 3888-3896Crossref PubMed Scopus (342) Google Scholar). Atg16 was originally obtained from a two-hybrid screen using Atg12 as a bait and was later confirmed to interact with Atg5 (but not Atg12) via its N-terminal region. Because Atg16 self-assembles via its C-terminal coiled-coil motif (residues 58-123), the Atg12-Atg5 conjugate forms a multimeric complex with Atg16 (11Kuma A. Mizushima N. Ishihara N. Ohsumi Y. J. Biol. Chem. 2002; 277: 18619-18625Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 14Mizushima N. Noda T. Ohsumi Y. EMBO J. 1999; 18: 3888-3896Crossref PubMed Scopus (342) Google Scholar). Further, as the majority of Atg12-Atg5 conjugates form a complex with Atg16 constitutively, the conjugates should function as a complex with Atg16 during autophagosome formation. In fact, the Atg12-Atg5 conjugate cannot localize to the PAS in Δatg16 yeast strains (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar, 15Kim J. Huang W.P. Klionsky D.J. J. Cell Biol. 2001; 152: 51-64Crossref PubMed Scopus (188) Google Scholar). In mammals, Atg16L, a functional counterpart of yeast Atg16, was shown to localize to the isolation membranes together with the Atg12-Atg5 conjugate during autophagosome formation (16Mizushima N. Kuma A. Kobayashi Y. Yamamoto A. Matsubae M. Takao T. Natsume T. Ohsumi Y. Yoshimori T. J. Cell Sci. 2003; 116: 1679-1688Crossref PubMed Scopus (575) Google Scholar). Although the significance of the Atg12-Atg5·Atg16 complex in autophagosome formation has been shown, its molecular function is still not clearly understood. One identified function of the Atg12-Atg5 conjugate is to promote the formation of the Atg8-phosphatidylethanolamine (PE) conjugate, the other conjugate essential for autophagosome formation, and the targeting of the Atg8-PE conjugate to the PAS (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar). Atg8 is a ubiquitin-like modifier that is first processed by a cysteine protease, Atg4 (17Kirisako T. Ichimura Y. Okada H. Kabeya Y. Mizushima N. Yoshimori T. Ohsumi M. Takao T. Noda T. Ohsumi Y. J. Cell Biol. 2000; 151: 263-276Crossref PubMed Scopus (742) Google Scholar). The processed Atg8 is conjugated to PE dependent on ATP and two enzymes, Atg7 (E1-like) and Atg3 (E2-like) (18Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. Nature. 2000; 408: 488-492Crossref PubMed Scopus (1542) Google Scholar). Although Atg3, Atg7, Atg8 (processed form), and ATP are sufficient for Atg8-PE conjugation in vitro (19Ichimura Y. Imamura Y. Emoto K. Umeda M. Noda T. Ohsumi Y. J. Biol. Chem. 2004; 279: 40584-40592Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), the Atg12-Atg5 conjugate is also required for Atg8-PE conjugation in vivo (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar). The expression of Atg8 is dramatically enhanced upon nutrient depletion. Under such conditions, Atg16 is also required for the efficient formation of Atg8-PE (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar). However, the molecular mechanism by which the Atg12-Atg5 conjugate and Atg16 promote Atg8-PE formation is not clear. Recently, we reported the crystal structure of plant Atg12 and revealed that Atg12 is a ubiquitin-fold protein (20Suzuki N.N. Yoshimoto K. Fujioka Y. Ohsumi Y. Inagaki F. Autophagy. 2005; 1: 119-126Crossref PubMed Scopus (93) Google Scholar). However, structural information on Atg5 and Atg16 has been thoroughly lacking, preventing us from elucidating the molecular functions of the Atg12-Atg5·Atg16 complex. In this report, we describe the first structure of Atg5 in complex with the N-terminal region of Atg16. Furthermore, based on structural information, Atg16 mutants that lost the binding affinity to Atg5 were constructed and used to clarify the significance of the direct interaction between Atg5 and Atg16 in autophagy. Protein Expression and Purification—Expression and purification of Atg5·Atg16-(1-46) and Atg5·Atg16-(1-57) as well as the construction of the expression vector of hexahistidine-tagged Atg5 were described previously (21Matsushita M. Suzuki N.N. Fujioka Y. Ohsumi Y. Inagaki F. Acta Crystallogr. F. Struct. Biol. Crystalliz. Comm. 2006; 62: 1021-1023Crossref PubMed Scopus (12) Google Scholar). The expression vectors of GST-fused Atg16 and its mutants were constructed as follows. The full-length ATG16 gene was amplified by PCR and inserted into pGEX-6P-1 (GE Healthcare). Mutations leading to the indicated amino acid substitutions were introduced by PCR-mediated site-directed mutagenesis. All of the constructs were sequenced to confirm their identities and were expressed in Escherichia coli strain BL21 (DE3) cells. After cell lysis, GST-fused Atg16 and its mutants were purified by sequential chromatography using a glutathione-Sepharose 4B column (GE Healthcare) and a Superdex200 gel filtration column (GE Healthcare). Hexahistidine-tagged Atg5 was purified by sequential chromatography using a nickel-nitrilotriacetic acid column (Qiagen) and a Superdex75 gel filtration column (GE Healthcare). Diffraction Data of and Atg5·Atg16-(1-57) was and crystal forms were obtained as described previously (21Matsushita M. Suzuki N.N. Fujioka Y. Ohsumi Y. Inagaki F. Acta Crystallogr. F. Struct. Biol. Crystalliz. Comm. 2006; 62: 1021-1023Crossref PubMed Scopus (12) Google Scholar). form was used for and crystal form was used for structure of of for Atg5·Atg16-(1-46) and Atg5·Atg16-(1-57) for was described previously (21Matsushita M. Suzuki N.N. Fujioka Y. Ohsumi Y. Inagaki F. Acta Crystallogr. F. Struct. Biol. Crystalliz. Comm. 2006; 62: 1021-1023Crossref PubMed Scopus (12) Google Scholar) and for as well as the and for Atg5·Atg16-(1-46) were on and using from an for the crystal of Atg5·Atg16-(1-46) at three were on the at the All were and with PubMed Scopus Google and in to the is the of the and is the in to the of to the and the of a to the for and to the and the of a to the for and to the and the of a to the for and of is the of the and is the in to the in to the to the and the of a to the for and in a of protein of from in a was by the of and Atg5·Atg16-(1-46) using the P. J. M. T. Acta Crystallogr. Biol. Crystallogr. 1998; PubMed Scopus Google Scholar). After were using the between and for and for were identified from the using the and the were using the between and for a crystal at three and were with the After the obtained were used to the for was using M. Acta Crystallogr. A. PubMed Scopus Google Scholar) and P. K. Acta Crystallogr. Biol. Crystallogr. 2004; PubMed Scopus Google Scholar), and was using The structure of Atg5·Atg16-(1-57) was by the molecular using the structure of Atg5·Atg16-(1-46) as a In Atg16 and its mutants were with hexahistidine-tagged Atg5 in and at for a of glutathione-Sepharose 4B was by further at for After three with proteins were with glutathione in at The were to and with In significance of the complex formation between Atg16 and Atg5 in vivo was as follows. were introduced by site-directed using or the gene (14Mizushima N. Noda T. Ohsumi Y. EMBO J. 1999; 18: 3888-3896Crossref PubMed Scopus (342) Google Scholar) as of the was confirmed by These were introduced into cells on a Klionsky D.J. Biol. PubMed Scopus Google Scholar) or cells Atg16 mutants were in synthetic casamino to and to After for in cells were The were by the acid in and by with using and were using an system (GE with a and Atg8 by gel was used (17Kirisako T. Ichimura Y. Okada H. Kabeya Y. Mizushima N. Yoshimori T. Ohsumi M. Takao T. Noda T. Ohsumi Y. J. Cell Biol. 2000; 151: 263-276Crossref PubMed Scopus (742) Google Scholar). of was using cells the Atg16 in of were to After for in cells were by fluorescent Atg16 mutants were by site-directed using a the gene (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar) as a of the was confirmed by The were introduced into cells. with the were in to and to After for in the cells were under an with a were using and processed using of the Atg5·Atg16-(1-57) the N-terminal (residues of Atg16 was reported to sufficient for complex formation with Atg5 (14Mizushima N. Noda T. Ohsumi Y. EMBO J. 1999; 18: 3888-3896Crossref PubMed Scopus (342) Google Scholar), we constructed two of Atg16, and were to form a complex with Atg5 not We and Atg5·Atg16-(1-46) and Atg5·Atg16-(1-57) (21Matsushita M. Suzuki N.N. Fujioka Y. Ohsumi Y. Inagaki F. Acta Crystallogr. F. Struct. Biol. Crystalliz. Comm. 2006; 62: 1021-1023Crossref PubMed Scopus (12) Google Scholar) and their The of the two were for of we to the structure of the Atg5·Atg16-(1-57) complex The structure of Atg5·Atg16-(1-57) was of to an of and a of The to amino of Atg5 and of Atg16 were with and of Atg5 and of Atg16 and were from the The Atg5·Atg16-(1-57) complex has an and of Atg5 and a of with of Atg5 the of Atg5 comprises two ubiquitin-like domains that flank a helix-rich domain. We the and C-terminal ubiquitin-like domains and and the helix-rich between and The with and were and In addition to the three domains and two Atg5 has an at its of Atg5 domains is shown in and a and two which is a conserved in all ubiquitin of and of Atg5 with the Protein Data using the revealed that domains structural to ubiquitin and ubiquitin-like a of yeast and a plant of yeast Atg12) with a of and a of for the sequence of Atg5 in which and are all to and and other conserved are The of ubiquitin and are also based on their Although the sequence identities of and with ubiquitin and are that the ubiquitin are that the two ubiquitin-like domains are of the conserved in Atg5 comprises three and that form a of with the using the showed that is to the of a Struct. Biol. 2003; PubMed Scopus Google Scholar) with a of and a of for also structural to of ubiquitin conjugation to a ubiquitin binding with a of and a of for the of are conserved Atg5 that is also of the conserved in Atg5 the conjugation for Atg12, is on of and its chain of the Atg5 Atg5 has a the of Atg5 is and have been two and form with other and are into a all the forms the interaction of is on the of and is by three and of a of with binding is to that between ubiquitin and the motif I. EMBO J. 2003; PubMed Scopus Google Scholar), the of the bound is Compared with the interaction between and between and and between and are However, and form with other and of interact with and of HR, of interacts with and of also interacts with and of a crucial role in the three domains. the and of which plays a role in these is and to the of and In LC3, the is involved in the interaction with two N-terminal and K. Suzuki N.N. Fujioka Y. Mizushima N. Ohsumi Y. Inagaki F. 2004; PubMed Scopus Google Scholar). and of and and of are also conserved in and are involved in the interaction between the ubiquitin region and of the ubiquitin in Atg5 and the for the construction of the of in addition to the three and are also conserved Atg5 is not conserved at all has residues, and which are conserved Atg5 and interact with and interact with the conserved and of these on a and to the In addition to the described the by to critical roles in the formation of the Atg5 has two conserved and interacts with conserved residues, and of HR, interacts with a conserved of of which is also interacts with Thus which is to the of plays an essential role in the of the three domains. In addition to the and of also interact with the in Atg16 of the between Atg5 and comprises an (residues and its (residues The of Atg16 is bound to the groove formed by and the is bound to The N-terminal (residues of Atg16 were in the crystal and not interact with The between Atg5 and Atg16 are shown in The of and of Atg16 form with In the of and are at the groove at the of and of Atg5 and form with these three and together with and of which the are well conserved Atg5 In addition to the Atg16 also forms with and of and and of form an on which and of Atg16 are Furthermore, and of form a in which Phe-46 of Atg16 is In of Atg16 have with which is with the that is sufficient for complex formation with the of Atg16 crucial for the interaction with an in vitro was using hexahistidine-tagged Atg5 and GST-fused Atg16 Atg16 in were with shown in Arg-35 and were crucial for the interaction between Atg5 and Atg16. of the for Atg8 and the significance of the direct interaction between Atg5 and Atg16, we the in vivo of two Atg16 and that binding affinity to Atg5 in a the Atg16 which binding affinity to Atg5 in was also The of Atg16 mutants was first by the of the of which is into via the targeting or by autophagy under and starvation conditions, The protein is processed into a form which was not in Δatg16 was in cells the Atg16 to a as Atg16 In was little of in cells the or mutants of Atg16 the of in was using an of was in cells and Δatg16 cells or Atg16 and such was in Δatg16 cells or the or mutants of Atg16 and These results the that direct interaction between Atg5 and Atg16 is crucial for autophagy. The Atg12-Atg5·Atg16 complex localizes to the PAS and plays a crucial role in autophagosome formation (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar), Atg5 and Atg16 (but not Atg12) is for the we whether Atg16 mutants localize to the PAS using fluorescent Atg16 in Δatg16 yeast strains. Atg16 to a structure to the which to the PAS The Atg16 also to a structure In Atg16 and mutants not localize to and in the cytosol two These results suggest that the direct interaction between Atg5 and Atg16 is essential for the of Atg16 to the The expression of Atg8 is enhanced under starvation conditions, and the Atg8-PE dramatically with that of Atg8-PE formation was in the of the Atg12-Atg5 conjugate (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar). in the of Atg16, Atg8-PE formation was and Atg8 was under starvation not under (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar). we Atg8-PE formation in Δatg16 cells Atg16 In cells the Atg16 Atg8-PE formation was to a to Atg16 under starvation In Atg8 not Atg8-PE was in cells the or mutants of Atg16 under starvation to a as cells with a vector These results suggest that the direct interaction between Atg5 and Atg16 is critical for efficient Atg8-PE formation under starvation conditions. In the yeast S. Atg proteins have been to into a structure to the called the the PAS, isolation the of are to The Atg12-Atg5·Atg16 complex was to to the PAS, suggesting that the complex plays a critical role in autophagosome formation. Thus far, it has been reported that the of the Atg12-Atg5·Atg16 complex to the PAS on Atg5 and Atg16, not Atg12 (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar). However, the significance of the direct complex formation between Atg5 and Atg16 We have shown that the direct interaction between Atg5 and Atg16 is crucial not for the also for autophagy Furthermore, we have also shown that the binding for Atg16 and the conjugation for Atg12 are on the of Atg5 These results suggest that the Atg12-Atg5·Atg16 complex localizes to the PAS using the formed by Atg5 and Atg16. The phosphoinositide which localizes to the PAS K. T. Ohsumi Y. Biol. 2006; PubMed Scopus Google Scholar), is known to essential for targeting the Atg12-Atg5 conjugate to the PAS (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar). Because phosphoinositide it that is on the PAS by and interacts with the or via binding by which the complex is to the Because the of Atg5·Atg16-(1-57) is and the structure known binding for PI3P, the interaction between the complex and PI3P, it is also to Atg3, Atg7, Atg8 (processed form), and ATP are and sufficient for Atg8-PE formation in vitro (19Ichimura Y. Imamura Y. Emoto K. Umeda M. Noda T. Ohsumi Y. J. Biol. Chem. 2004; 279: 40584-40592Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). However, in addition to these the Atg12-Atg5 conjugate is also required for Atg8-PE formation in vivo (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar), and the significance of Atg12 in Atg8-PE formation was shown by analyses T. Ohsumi Y. Autophagy. 2005; 1: PubMed Scopus Google Scholar). The expression of Atg8 is dramatically enhanced upon nutrient depletion. Under such conditions, Atg16 is also required for the efficient formation of Atg8-PE (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar). We here that Atg16 mutants that affinity to Atg5 not promote Atg8-PE formation Because Atg5 as a conjugate with Atg12 in this that Atg16 Atg8-PE formation as a complex with the Atg12-Atg5 of the Atg12-Atg5·Atg16 complex is to that of enzymes in the ubiquitin In to and a protein, the of ubiquitin between The Atg5·Atg16-(1-57) complex structural to enzymes, and the molecular mechanism by which the Atg12-Atg5·Atg16 complex Atg8-PE formation is still Because complex formation between Atg16 and the Atg12-Atg5 conjugate is crucial for the of Atg8-PE formation and targeting of the Atg12-Atg5·Atg16 complex to the PAS, these two to have a Atg12 was shown to interact with Atg3, the for Atg8 by yeast two-hybrid screening P. M. P. A. Y. T. M. M. S. Nature. 2000; PubMed Scopus Google Scholar). these results suggest that the Atg12-Atg5·Atg16 complex to Atg3 and the PAS, the of Atg8 between Atg3 and PE at the is with the that the Atg12-Atg5·Atg16 complex is also crucial for the of Atg8-PE to the However, further studies are required to this the of the PAS We J. Klionsky for the and R. for The were at the in the with the of the
Matsushita et al. (Thu,) studied this question.
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