Key points are not available for this paper at this time.
In yeast, phosphatidylethanolamine is a target of the Atg8 modifier in ubiquitylation-like reactions essential for autophagy. Three human Atg8 (hAtg8) homologs, LC3, GABARAP, and GATE-16, have been characterized as modifiers in reactions mediated by hAtg7 (an E1-like enzyme) and hAtg3 (an E2-like enzyme) as in yeast Atg8 lipidation, but their final targets have not been identified. The results of a recent study in which COS7 cells were incubated with 14Cethanolamine for 48 h suggested that phosphatidylethanolamine is a target of LC3. However, these results were not conclusive because of the long incubation time. To identify the phospholipid targets of Atg8 homologs, we reconstituted conjugation systems for mammalian Atg8 homologs in vitro using purified recombinant Atg proteins and liposomes. Each purified mutant Atg8 homolog with an exposed C-terminal Gly formed an E1-substrate intermediate with hAtg7 via a thioester bond in an ATP-dependent manner and formed an E2-substrate intermediate with hAtg3 via a thioester bond dependent on ATP and hAtg7. A conjugated form of each Atg8 homolog was observed in the presence of hAtg7, hAtg3, ATP, and liposomes. In addition to phosphatidylethanolamine, in vitro conjugation experiments using synthetic phospholipid liposomes showed that phosphatidylserine is also a target of LC3, GABARAP, and GATE-16. In contrast, thin layer chromatography of phospholipids released on hAtg4B-digestion from endogenous LC3-phospholipid conjugate revealed that phosphatidylethanolamine, but not phosphatidylserine, is the predominant target phospholipid of LC3 in vivo. The discrepancy between in vitro and in vivo reactions suggested that there may be selective factor(s) involved in the endogenous LC3 conjugation system. In yeast, phosphatidylethanolamine is a target of the Atg8 modifier in ubiquitylation-like reactions essential for autophagy. Three human Atg8 (hAtg8) homologs, LC3, GABARAP, and GATE-16, have been characterized as modifiers in reactions mediated by hAtg7 (an E1-like enzyme) and hAtg3 (an E2-like enzyme) as in yeast Atg8 lipidation, but their final targets have not been identified. The results of a recent study in which COS7 cells were incubated with 14Cethanolamine for 48 h suggested that phosphatidylethanolamine is a target of LC3. However, these results were not conclusive because of the long incubation time. To identify the phospholipid targets of Atg8 homologs, we reconstituted conjugation systems for mammalian Atg8 homologs in vitro using purified recombinant Atg proteins and liposomes. Each purified mutant Atg8 homolog with an exposed C-terminal Gly formed an E1-substrate intermediate with hAtg7 via a thioester bond in an ATP-dependent manner and formed an E2-substrate intermediate with hAtg3 via a thioester bond dependent on ATP and hAtg7. A conjugated form of each Atg8 homolog was observed in the presence of hAtg7, hAtg3, ATP, and liposomes. In addition to phosphatidylethanolamine, in vitro conjugation experiments using synthetic phospholipid liposomes showed that phosphatidylserine is also a target of LC3, GABARAP, and GATE-16. In contrast, thin layer chromatography of phospholipids released on hAtg4B-digestion from endogenous LC3-phospholipid conjugate revealed that phosphatidylethanolamine, but not phosphatidylserine, is the predominant target phospholipid of LC3 in vivo. The discrepancy between in vitro and in vivo reactions suggested that there may be selective factor(s) involved in the endogenous LC3 conjugation system. Ubiquitylation and ubiquitylation-like posttranslational reactions play indispensable roles in many cellular functions (1Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2897) Google Scholar, 2Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6825) Google Scholar, 3Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3323) Google Scholar, 4Varshavsky A. Trends Biochem. Sci. 1997; 22: 383-387Abstract Full Text PDF PubMed Scopus (514) Google Scholar, 5Bonifacino J.S. Weissman A.M. Annu. Rev. Cell Dev. Biol. 1998; 14: 19-57Crossref PubMed Scopus (533) Google Scholar, 6Schwartz D.C. Hochstrasser M. Trends Biochem. Sci. 2003; 28: 321-328Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 7Hochstrasser M. Science. 2000; 289: 563-564Crossref PubMed Scopus (101) Google Scholar, 8Ohsumi Y. Nat. Rev. Mol. Cell. Biol. 2001; 2: 211-216Crossref PubMed Scopus (1035) Google Scholar). Ubiquitin is synthesized as a precursor, which is cleaved to expose a C-terminal Gly (9Wing S.S. Int. J. Biochem. Cell Biol. 2003; 35: 590-605Crossref PubMed Scopus (161) Google Scholar) and subsequently activated by reaction with a ubiquitin-activating enzyme (E1). 2The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; PE, phosphatidylethanolamine; DOPE, dioleoyl-phosphatidylethanolamine; PS, phosphatidylserine; DOPS, dioleoyl-phosphatidylserine; PC, phosphatidylcholin; POPC, 1-palmitoyl-2-oleoyl-phosphatidylcholine; DTT, dithiothreitol; Atg8-PE Atg8-phosphatidylethanolamine conjugate; hAtg7, human Atg7 homolog (E1-like enzyme); hAtg3, human Atg3p homolog (E2-like enzyme); hAtg8, human Atg8 homolog; GABARAP, human γ-aminobutryric acid type A (GABAA) receptor-associated protein; GABARAP-PL, a GABARAP-phospholipid conjugate; GATE-16, human Golgi-associated ATPase enhancer of 16 kDa; GATE-16-II, a GATE-16-phospholipid conjugate; LC3, human microtubule-associated protein 1 light chain 3; LC3-II, a LC3-phospholipid conjugate; GST, glutathione S-transferase. 2The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; PE, phosphatidylethanolamine; DOPE, dioleoyl-phosphatidylethanolamine; PS, phosphatidylserine; DOPS, dioleoyl-phosphatidylserine; PC, phosphatidylcholin; POPC, 1-palmitoyl-2-oleoyl-phosphatidylcholine; DTT, dithiothreitol; Atg8-PE Atg8-phosphatidylethanolamine conjugate; hAtg7, human Atg7 homolog (E1-like enzyme); hAtg3, human Atg3p homolog (E2-like enzyme); hAtg8, human Atg8 homolog; GABARAP, human γ-aminobutryric acid type A (GABAA) receptor-associated protein; GABARAP-PL, a GABARAP-phospholipid conjugate; GATE-16, human Golgi-associated ATPase enhancer of 16 kDa; GATE-16-II, a GATE-16-phospholipid conjugate; LC3, human microtubule-associated protein 1 light chain 3; LC3-II, a LC3-phospholipid conjugate; GST, glutathione S-transferase. Ubiquitin and E1 form an E1-substrate intermediate via a thioester bond between the E1 active site Cys and the C-terminal Gly of ubiquitin. Thereafter, the activated ubiquitin is transferred to a ubiquitin-conjugating enzyme (E2), which forms an E2-substrate intermediate with ubiquitin via a thioester bond between the E2 active site Cys and the C-terminal Gly of ubiquitin. Finally, the ubiquitin is conjugated to a target protein via an amide bond between the C-terminal Gly of ubiquitin and a Lys within the target protein by a ubiquitin ligase (E3). These enzyme-reaction systems (E1, E2, and E3) are essentially conserved among the other ubiquitylation-like modifications of ubiquitin-like proteins (10Jentsch S. Pyrowolakis G. Trends Cell Biol. 2000; 10: 335-342Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Yeast Atg8 is a unique ubiquitin-like protein essential for autophagy; whereas the targets of ubiquitin and the other ubiquitin-like proteins are proteins, the target of Atg8 is a phospholipid, phosphatidylethanolamine (PE). Genetic analyses have indicated that Atg8 is cleaved by Atg4, a predicted cysteine protease, to expose its C-terminal Gly. Thereafter, Atg8 is activated by Atg7, an E1-like enzyme, and transferred to Atg3p, an E2-like enzyme. Finally, Atg8 is covalently conjugated to PE through an amide bond between the C-terminal Gly of Atg8 and an amino group in the hydrophilic head within PE (11Ichimura 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 (1489) Google Scholar, 12Kirisako 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 (731) Google Scholar), and the target has been identified by mass spectroscopy of the purified Atg8-PE conjugate. Recently, the Atg8 lipidation system has been reconstituted in vitro. These experiments indicated that a mutant Atg8, in which the C-terminal Gly is exposed, as well as Atg7, Atg3, and PE-containing liposomes, are the minimum reaction units in yeast (13Ichimura 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 (173) Google Scholar). In mammals, at least three Atg8 homologs, LC3, GABARAP, and GATE-16, have been identified (Fig. 1A), all of which have structural ubiquitin folds (14Sugawara K. Suzuki N.N. Fujioka Y. Mizushima N. Ohsumi Y. Inagaki F. Genes Cells. 2004; 9: 611-618Crossref PubMed Scopus (138) Google Scholar, 15Paz Y. Elazar Z. Fass D. J. Biol. Chem. 2000; 275: 25445-25450Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 16Bavro V.N. Sola M. Bracher A. Kneussel M. Betz H. Weissenhorn W. EMBO Rep. 2002; 3: 183-189Crossref PubMed Scopus (60) Google Scholar). In vivo and in vitro biochemical analyses have shown that human Atg4B is an authentic cysteine protease essential for cleavage of the C terminus of each Atg8 homolog to expose the C-terminal Gly (17Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1103) Google Scholar, 18Tanida I. Sou Y.S. Ezaki J. Minematsu-Ikeguchi N. Ueno T. Kominami E. J. Biol. Chem. 2004; 279: 36268-36276Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). After cleavage, each Atg8 homolog is activated by human Atg7 (hAtg7), transferred to human Atg3 (hAtg3), and finally forms a conjugate as a modifier (19Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5395) Google Scholar, 20Tanida I. Komatsu M. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2003; 300: 637-644Crossref PubMed Scopus (84) Google Scholar). We have recently demonstrated that LC3 and GABARAP form protein-phospholipid conjugates, LC3-II and GABARAP-PL, respectively (18Tanida I. Sou Y.S. Ezaki J. Minematsu-Ikeguchi N. Ueno T. Kominami E. J. Biol. Chem. 2004; 279: 36268-36276Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Incubation of COS7 cells with 14Cethanolamine for 48 h has been reported to result in incorporation of radioactivity into the LC3-phospholipid conjugate (LC3-II), suggesting that the target of LC3, similar to yeast Atg8, is PE (17Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1103) Google Scholar). The metabolic pathways of the synthesis and degradation of phospholipids, however, are such that 48-h labeling with 14Cethanolamine cannot lead to conclusive results. Although the target of the third homolog, GATE-16, is not yet known, a conjugated form of GATE-16 (GATE-16-II) shows biochemical characteristics similar to those of LC3-II and GABARAP-PL, suggesting that the target of GATE-16 may also be a phospholipid. However, there are differences among the conjugated forms of the three Atg8 homologs. For example, the amount of LC3-II in rat tissues cannot be correlated with the amounts of the other conjugates. In HEK293 cells, conjugation of LC3, but not of GATE-16 or GABARAP, is facilitated by overexpression of hAtg7 and hAtg3. In HeLa cells, LC3-II and GABARAP-PL, but not GATE-16-II, accumulate in the presence of the protease inhibitors, E64d and pepstatin A, under nutrient-rich conditions. In the livers of mice lacking Atg7, the amounts of the unlipidated forms of all three Atg8 homologs increase, suggesting that conjugation of all three molecules is active in the mouse liver (21Komatsu M. Waguri S. Ueno T. Iwata J. Murata S. Tanida I. Ezaki J. Mizushima N. Ohsumi Y. Uchiyama Y. Kominami E. Tanaka K. Chiba T. J. Cell Biol. 2005; 169: 425-434Crossref PubMed Scopus (1899) Google Scholar). In the present study, we focused on whether hAtg7, hAtg3, and phospholipid(s) are the minimum essential requirements needed for conjugation of the three mammalian Atg8 homologs and on the nature of the targets of these three human Atg8 homologs. We hypothesized that PE and phosphatidylserine (PS) may be targets, because the hydrophilic heads of both PE and PS have amino groups, which are required in ubiquitylation-like reactions. We reconstituted the conjugation systems for the mammalian Atg8 homologs in vitro using purified recombinant hAtg7, hAtg3, LC3, GABARAP, and GATE-16, as well as synthetic phospholipid liposomes. Furthermore, we directly analyzed the in vivo target phospholipid of endogenous LC3-II purified from HeLa cells by TLC. Strain, Cells, Culture, Biochemical Materials, and Molecular Biological Techniques—Molecular biological and biochemical techniques were performed as described previously (18Tanida I. Sou Y.S. Ezaki J. Minematsu-Ikeguchi N. Ueno T. Kominami E. J. Biol. Chem. 2004; 279: 36268-36276Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). The Escherichia coli strain JM109 was used for plasmid construction and protein expression. Total lipid was extracted from HeLa cells as described (22Bligh E.G. Dyer W.J. J. Biochem. Physiol. PubMed Scopus Google Scholar). The plasmid was from The synthetic phospholipids, and were from of human Atg7, Atg3, LC3, GATE-16, and GABARAP, and the mutant human Atg8 homologs (Fig. the were into by and biological techniques (Fig. human LC3, GABARAP, Atg3, and Atg7 have been described (18Tanida I. Sou Y.S. Ezaki J. Minematsu-Ikeguchi N. Ueno T. Kominami E. J. Biol. Chem. 2004; 279: 36268-36276Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, I. E. Komatsu M. Ueno T. Kominami E. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, I. E. Ueno T. Kominami E. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). To GATE-16, a was with purified and in the was by a To LC3 and GABARAP, the was through and GABARAP unlipidated GABARAP, and GATE-16 unlipidated GATE-16. of Atg in E. coli and proteins were in E. coli and purified using and protease to the In for and via recombinant proteins were in and ATP, and 1 were to the The was incubated at for 1 the reaction was by the addition of an of in the or presence of a and the were incubated for at I. G. EMBO J. 1997; PubMed Scopus Google Scholar, M. Chiba T. K. S. Tanida I. N. Ueno T. Kominami E. T. Tanaka K. EMBO J. 2004; PubMed Scopus Google Scholar). of Total from HeLa and of in HeLa cells were extracted as described (13Ichimura 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 (173) Google Scholar, E.G. Dyer W.J. J. Biochem. Physiol. PubMed Scopus Google Scholar, N. 2003; PubMed Scopus Google Scholar) and as a To lipid the was with and the were in under at for The lipid were to a final of 1 phospholipids in a of at and for at After at for the was used as liposomes (13Ichimura 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 (173) Google Scholar, N. 2003; PubMed Scopus Google Scholar, J. Chem. Scopus Google Scholar). In for LC3, GABARAP, and GATE-16 mutant homolog with exposed Gly and hAtg7 hAtg3 and liposomes were in in the presence of ATP, and 1 and incubated at for 1 To the an of was and the was for of to LC3-II in HeLa cells was in the presence of the protease inhibitors, E64d and pepstatin A, as described (18Tanida I. Sou Y.S. Ezaki J. Minematsu-Ikeguchi N. Ueno T. Kominami E. J. Biol. Chem. 2004; 279: 36268-36276Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). After of an from the HeLa cells 1 LC3-II was from in (18Tanida I. Sou Y.S. Ezaki J. Minematsu-Ikeguchi N. Ueno T. Kominami E. J. Biol. Chem. 2004; 279: 36268-36276Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar) and purified by chromatography on an LC3-II was with recombinant a protease (17Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1103) Google Scholar, 18Tanida I. Sou Y.S. Ezaki J. Minematsu-Ikeguchi N. Ueno T. Kominami E. J. Biol. Chem. 2004; 279: 36268-36276Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar) from E. and the was incubated at for phospholipids were extracted from reaction using (22Bligh E.G. Dyer W.J. J. Biochem. Physiol. PubMed Scopus Google Scholar) and subsequently by on using as a The were in a and with J. PubMed Scopus Google Scholar) to phospholipids under light In between hAtg7 and Each Atg8 and and HeLa cells, all of the C of LC3, GABARAP, and GATE-16 are cleaved to expose a C-terminal Gly which is essential for ubiquitylation-like reactions. for in vitro E1-substrate hAtg7 and a of mutant Atg8 homologs were as proteins in E. coli and purified using and protease (Fig. of the proteins on showed that all three been purified to (Fig. We focused on the of the E1-substrate intermediate between hAtg7 and by hAtg7 and and at for 1 h in the presence or of ATP After the proteins were by under and was by using In the presence of ATP, a of to the intermediate was (Fig. intermediate was in the presence of a that the intermediate was formed via a thioester intermediate was observed we used a mutant in which the C-terminal Gly been of results were in the for E1-substrate of and (Fig. and of to and were by their in the presence of ATP, in a manner to the was intermediate and were for and These results indicated that hAtg7, each of the Atg8 homologs, and ATP are the minimum essential for the of E1-substrate via thioester In E2-substrate between hAtg3 and Each Atg8 whether each of the Atg8 homologs form an E2-substrate intermediate with hAtg3 in vitro. to the other proteins hAtg3 was as a protein in E. coli and purified with and protease (Fig. To form an E2-substrate intermediate between hAtg3 and we incubated hAtg7 hAtg3 and at for 1 h in the presence or of ATP After the the proteins were on or under and was by with (Fig. In the presence of ATP, a of to an was was not observed in the of hAtg7 or ATP and was to the was of E2-substrate intermediate was that the C-terminal Gly of LC3 is essential for reaction in vitro. results were in the for E2-substrate of and (Fig. and hAtg7 hAtg3 ATP and or were an E2-substrate intermediate between hAtg3 and or was by with the These were to and were dependent on the presence of hAtg7 and and were for and intermediate was that the C-terminal Gly of GABARAP and GATE-16 is required for reaction in vitro. In of the Three Atg8 by hAtg7 and Atg8 is conjugated to PE in vitro and in vivo. is whether hAtg7 and hAtg3 are the minimum required for the conjugation reactions and whether GATE-16 is conjugated to a To these we whether the conjugated forms of the three Atg8 homologs are by hAtg7 and hAtg3 E1 and E2 reactions in vitro. a phospholipid we used liposomes from from HeLa cells, because LC3-II (an LC3-phospholipid and GABARAP-phospholipid are present in HeLa cells under nutrient-rich conditions. We that LC3-II was hAtg7 hAtg3 liposomes and were incubated in the presence of ATP at for 1 as by with (Fig. In the of hAtg7, hAtg3, ATP, or liposomes, or was for conjugate was results were for GABARAP and GATE-16 conjugation reactions (Fig. and was by with all hAtg3, ATP, and were present in the reaction whereas was formed was for (Fig. GATE-16-II, a conjugated form of GATE-16, was by with all hAtg3, ATP, and were present in the reaction but was formed was for (Fig. These results indicated that the minimum essential requirements for in vitro conjugation of the homologs hAtg7, hAtg3, ATP, liposomes, and each LC3, GABARAP, and GATE-16 to both PE and PS in of LC3 and GABARAP have been reported to be to suggesting that these homologs target a Ubiquitin and other modifiers are conjugated to targets via amide between the group in their C-terminal Gly and the chain amino group in a Lys or in the hydrophilic head of PE and PS have amino in their hydrophilic these phospholipids are targets of the human Atg8 homologs. To whether phospholipids are conjugated to the Atg8 homologs, we liposomes of synthetic phospholipids a of DOPE, a of and DOPS, a of and reconstituted the conjugation reactions in vitro (Fig. We focused on the of synthetic liposomes on the conjugation of LC3. liposomes of were used in the in vitro of LC3 LC3-II was observed by with (Fig. In contrast, liposomes and were LC3-II was observed (Fig. 1 and We the of PS in the liposomes for in vitro conjugation by the of in the liposomes from to LC3-II was observed liposomes were used (Fig. with the for in vitro LC3 conjugation These results indicated that PS is a target of LC3. is not for of liposomes, we a of liposomes and their on LC3 conjugation (Fig. a of liposomes DOPE, DOPS, POPC, and DOPE, we observed LC3-II in liposomes (Fig. and liposomes DOPE, we observed LC3-II in the presence of liposomes (Fig. with the for in vitro LC3 conjugation These results indicated that PE is also a target of LC3 conjugation in vitro. We focused on the of synthetic liposomes on in vitro GABARAP there are differences in the amounts of the forms of LC3, GABARAP, and GATE-16 in rat there may be among these homologs their target phospholipids or their However, we observed between LC3 and GABARAP conjugation reactions in vitro (Fig. was observed using liposomes (Fig. but not using liposomes of or of a of and liposomes in the reaction (Fig. indicated that their for in vitro GABARAP conjugation were similar to those observed for LC3 conjugation (Fig. These results indicated that both PS and PE are targets of GABARAP conjugation in vitro. biochemical of and the results shown in suggested that PS and PE are targets of GATE-16 we used synthetic phospholipid liposomes to GATE-16 and a of liposomes were in the in vitro was observed by with (Fig. The of and liposomes for in vitro GATE-16 conjugation were similar to those observed for LC3 and GABARAP (Fig. These indicated that is a protein-phospholipid conjugate and that the targets of GATE-16 are also PS and In both PS and PE are targets of all three Atg8 homologs GABARAP, and in these protein-phospholipid conjugation reactions. The in of LC3 but the in vitro conjugation LC3 conjugate with both PS and However, is not whether is also the in vivo. The results of a recent study the incubation of 14Cethanolamine in COS7 cells for 48 h suggested that phosphatidylethanolamine is a target of LC3 (17Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1103) Google Scholar). However, the not the that phosphatidylserine may be endogenous target of LC3 in the conjugation as in the of in vitro To identify the endogenous target phospholipid(s) of LC3 in we purified endogenous LC3-II from HeLa cells in the presence of protease inhibitors, E64d and pepstatin A as described (18Tanida I. Sou Y.S. Ezaki J. Minematsu-Ikeguchi N. Ueno T. Kominami E. J. Biol. Chem. 2004; 279: 36268-36276Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar), LC3-II with the enzyme, and analyzed the phospholipids released on reaction with purified endogenous LC3-II was from the with and on an LC3-II was incubated with or mutant and showed that LC3-II to form (Fig. and In contrast, incubation of the mutant with LC3-II not (Fig. and The phospholipids were extracted from the reaction using the (22Bligh E.G. Dyer W.J. J. Biochem. Physiol. PubMed Scopus Google Scholar) and analyzed by TLC. shown in of LC3-II with released PE but not phospholipid were with the with the These results indicated that PE, but not PS, is the target phospholipid conjugated with endogenous LC3 in vivo. The results of the present study indicated that in vitro conjugation systems for the three Atg8 homologs, LC3, GABARAP, and GATE-16, be reconstituted using purified hAtg7 (E1-like hAtg3 (E2-like and synthetic phospholipid liposomes. three homolog formed via ATP-dependent thioester and all three homolog formed via thioester dependent on ATP and hAtg7. Finally, all three Atg8 homologs were conjugated to phospholipids, PS and The lead to three the minimum for human were shown to be hAtg7, hAtg3, the Atg8 homolog GABARAP, or phospholipid or liposomes, and the form of GATE-16, GATE-16-II, is a protein-phospholipid conjugate and be of Finally, PS and PE are targets of all human Atg8 modifiers in vitro. We have demonstrated that the in vivo target phospholipid of endogenous LC3-II is PE, as revealed by is the for the of the target phospholipid of the LC3 conjugation system in vivo. These the discrepancy between in vitro and in vivo in vitro conjugation system E1-like enzyme and E2-like enzyme for conjugation of the three Atg8 homologs with PE or PS as in yeast in vitro conjugation of Atg8 with PE (13Ichimura 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 (173) Google Scholar). However, not the of other as yet (Fig. in selective conjugation of with PE in vivo. and be for is an enzyme, which PE as the of conjugation reaction of activated on Atg3 (E2-like and of to PE We a which LC3 conjugation to PS (Fig. In in vitro all three Atg8 homologs GABARAP, and be conjugated with both PE and However, cellular of the three homologs are in tissues and cells I. Ueno T. Kominami E. Int. J. Biochem. Cell Biol. 2004; PubMed Scopus Google Scholar). the of the forms of the homologs also be the homologs to be may be on tissues and cells, and target phospholipid(s) may be also on which of the three homologs is involved in the of is also a of in vivo lipidation of Atg8 homologs. are under in to for of and is to identify and such factor(s) to of the of mammalian autophagy. We T. for and
Sou et al. (Wed,) studied this question.