Human Light Chain 3/MAP1LC3B Is Cleaved at Its Carboxyl-terminal Met121 to Expose Gly120 for Lipidation and Targeting to Autophagosomal Membranes

Human light chain 3/MAP1LC3B, an autophagosomal ortholog of yeast Atg8, is conjugated to phospholipid (PL) via ubiquitylation-like reactions mediated by human Atg7 and Atg3. Since human Atg4B was found to cleave the carboxyl terminus of MAP1LC3B in vitro, we hypothesized that this exposes its carboxyl-terminal Gly120. It was recently reported, however, that when Myc-MAP1LC3B-His is expressed in HEK293 cells, its carboxyl terminus is not cleaved. (Tanida, I., Sou, Y.-s., Ezaki, J., Minematsu-Ikeguchi, N., Ueno, T., and Kominami, E. (2004) J. Biol. Chem. 279, 36268–36276). To clarify this contradiction, we sought to determine whether the carboxyl terminus of MAP1LC3B is cleaved to expose Gly120 for further ubiquitylation-like reactions. When MAP1LC3B-3xFLAG and Myc-MAP1LC3B-His were expressed in HEK293 cells, their carboxyl termini were cleaved, whereas there was little cleavage of mutant proteins MAP1LC3BG120A-3xFLAG and Myc-MAP1LC3BG120A-His, containing Ala in place of Gly120. An in vitro assay showed that Gly120 is essential for carboxyl-terminal cleavage by human Atg4B as well as for formation of the intermediates Atg7-MAP1LC3B (ubiquitin-activating enzyme-substrate) and Atg3-MAP1LC3B (ubiquitin carrier protein-substrate). Recombinant MAP1LC3B-PL was fractionated into the 100,000 × g pellet in a manner similar to that shown for endogenous MAP1LC3B-PL. RNA interference of MAP1LC3B mRNA resulted in a decrease in both endogenous MAP1LC3B-PL and MAP1LC3B. These results indicate that the carboxyl terminus of MAP1LC3B is cleaved to expose Gly120 for further ubiquitylation-like reactions. Human light chain 3/MAP1LC3B, an autophagosomal ortholog of yeast Atg8, is conjugated to phospholipid (PL) via ubiquitylation-like reactions mediated by human Atg7 and Atg3. Since human Atg4B was found to cleave the carboxyl terminus of MAP1LC3B in vitro, we hypothesized that this exposes its carboxyl-terminal Gly120. It was recently reported, however, that when Myc-MAP1LC3B-His is expressed in HEK293 cells, its carboxyl terminus is not cleaved. (Tanida, I., Sou, Y.-s., Ezaki, J., Minematsu-Ikeguchi, N., Ueno, T., and Kominami, E. (2004) J. Biol. Chem. 279, 36268–36276). To clarify this contradiction, we sought to determine whether the carboxyl terminus of MAP1LC3B is cleaved to expose Gly120 for further ubiquitylation-like reactions. When MAP1LC3B-3xFLAG and Myc-MAP1LC3B-His were expressed in HEK293 cells, their carboxyl termini were cleaved, whereas there was little cleavage of mutant proteins MAP1LC3BG120A-3xFLAG and Myc-MAP1LC3BG120A-His, containing Ala in place of Gly120. An in vitro assay showed that Gly120 is essential for carboxyl-terminal cleavage by human Atg4B as well as for formation of the intermediates Atg7-MAP1LC3B (ubiquitin-activating enzyme-substrate) and Atg3-MAP1LC3B (ubiquitin carrier protein-substrate). Recombinant MAP1LC3B-PL was fractionated into the 100,000 × g pellet in a manner similar to that shown for endogenous MAP1LC3B-PL. RNA interference of MAP1LC3B mRNA resulted in a decrease in both endogenous MAP1LC3B-PL and MAP1LC3B. These results indicate that the carboxyl terminus of MAP1LC3B is cleaved to expose Gly120 for further ubiquitylation-like reactions. Autophagy is the bulk degradation of proteins and organelles essential for cellular maintenance and viability. Autophagy has significant associations with neurodegenerative diseases, cardiomyopathies, cancer, programmed cell death, and bacterial and viral infections (1Klionsky D.J. Autophagy. Landes Bioscience, Georgetown, TX2004Google Scholar). Among the autophagosomal marker proteins are Atg8/Apg8/Aut7 in yeast and light chain 3 (LC3) 1The abbreviations used are: LC3, light chain 3; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; h, human; PL, phospholipid; GABARAP, γ-aminobutyric acid type A receptor-associated protein; GFP, green fluorescent protein; TRX, thioredoxin. in mammals (2Kirisako T. Baba M. Ishihara N. Miyazawa K. Ohsumi M. Yoshimori T. Noda T. Ohsumi Y. J. Cell Biol. 1999; 147: 435-446Crossref PubMed Scopus (719) Google Scholar, 3Kabeya 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 (5510) Google Scholar), both of which are modified via ubiquitylation-like reactions, allowing them to localize to autophagosomes. The molecular mechanism of Atg8 lipidation has been well characterized in yeast. The carboxyl terminus of Atg8 is cotranslationally cleaved by Atg4/Apg4/Aut2, a cysteine protease, to expose a carboxyl-terminal Gly (4Ichimura 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, 5Kirisako 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). This proteolytic reaction is indispensable for the ensuing conjugation of Atg8 to phosphatidylethanolamine (4Ichimura 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), a reaction mediated by Atg7/Apg7/Cvt2, a ubiquitin-activating enzyme (E1)-like enzyme (4Ichimura 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, 6Kim J. Dalton V.M. Eggerton K.P. Scott S.V. Klionsky D.J. Mol. Biol. Cell. 1999; 10: 1337-1351Crossref PubMed Scopus (178) Google Scholar, 7Tanida I. Mizushima N. Kiyooka M. Ohsumi M. Ueno T. Ohsumi Y. Kominami E. Mol. Biol. Cell. 1999; 10: 1367-1379Crossref PubMed Scopus (327) Google Scholar, 8Yuan W. Stromhaug P.E. Dunn Jr., W.A. Mol. Biol. Cell. 1999; 10: 1353-1366Crossref PubMed Scopus (117) Google Scholar), and by Atg3/Apg3/Aut1, a ubiquitin carrier protein (E2)-like enzyme (4Ichimura 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). Phosphatidylethanolamine-conjugated Atg8 is finally localized to autophagosomes under starvation conditions and to Cvt vesicles under nutrient-rich conditions. In mammalian cells, the Atg8-like modification system for formation of autophagosomes seems to be conserved. The carboxyl terminus of rat LC3 is cleaved, exposing Gly120, a residue that is essential for the cleavage and modification of LC3-I to LC3-II (3Kabeya 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 (5510) Google Scholar). Modification of rat LC3-I to LC3-II is facilitated under starvation conditions. During ultracentrifugation, rat LC3-II is fractionated into the 100,000 × g pellet, and LC3-II localizes to autophagosomes. Human (h) LC3/MAP1LC3B, one of three orthologs of rat MAP1LC3 (microtubule-associated protein 1light chain 3), shows significant homology to rat LC3 (3Kabeya 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 (5510) Google Scholar, 9Tanida I. Tanida-Miyake E. Ueno T. Kominami E. J. Biol. Chem. 2001; 276: 1701-1706Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar), especially at residues 1–120, which are 98.3% identical (see Fig. 1A). hAtg7 and hAtg3 can form E1- and E2-substrate intermediates, respectively, with hLC3/MAP1LC3B (9Tanida I. Tanida-Miyake E. Ueno T. Kominami E. J. Biol. Chem. 2001; 276: 1701-1706Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 10Tanida I. Tanida-Miyake E. Komatsu M. Ueno T. Kominami E. J. Biol. Chem. 2002; 277: 13739-13744Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 11Tanida I. Komatsu M. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2003; 300: 637-644Crossref PubMed Scopus (85) Google Scholar), and transient expression of hAtg7 and hAtg3 along with MAP1LC3B results in an increase in MAP1LC3B-II (11Tanida I. Komatsu M. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2003; 300: 637-644Crossref PubMed Scopus (85) Google Scholar). We recently showed that endogenous MAP1LC3B-II isolated from HeLa cells is an LC3-phospholipid (PL) conjugate (MAP1LC3B-PL); that purified hAtg4B cleaves the carboxyl terminus of MAP1LC3B in vitro; and that, during ultracentrifugation, MAP1LC3B-PL is fractionated into the pellet (11Tanida I. Komatsu M. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2003; 300: 637-644Crossref PubMed Scopus (85) Google Scholar, 12Tanida 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 (286) Google Scholar). Since the exposure of a carboxyl-terminal Gly of a ubiquitin-like modifier (e.g. ubiquitin, SUMO-1, Nedd8, Atg12, Atg8, γ-aminobutyric acid type A receptor-associated protein (GABARAP), and Ufm1) is essential for further ubiquitylation-like reactions, we hypothesized that the carboxyl-terminal Gly120 of MAP1LC3B is exposed in vivo and that MAP1LC3B is an authentic modifier, as is rat LC3. In contrast to our finding that the carboxyl terminus of MAP1LC3B is cleaved to expose Gly120, it was recently reported that recombinant Myc-MAP1LC3B-His protein does not undergo carboxyl-terminal cleavage and that human MAP1LC3B is post-translationally modified at Lys122 without any carboxyl-terminal cleavage (13He H. Dang Y. Dai F. Guo Z. Wu J. She X. Pei Y. Chen Y. Ling W. Wu C. Zhao S. Liu J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). These that MAP1LC3B is not a modifier protein Gly120 is not are to our finding that MAP1LC3B is a modifier protein (9Tanida I. Tanida-Miyake E. Ueno T. Kominami E. J. Biol. Chem. 2001; 276: 1701-1706Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 10Tanida I. Tanida-Miyake E. Komatsu M. Ueno T. Kominami E. J. Biol. Chem. 2002; 277: 13739-13744Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 11Tanida I. Komatsu M. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2003; 300: 637-644Crossref PubMed Scopus (85) Google Scholar, 12Tanida 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 (286) Google Scholar, H. Dang Y. Dai F. Guo Z. Wu J. She X. Pei Y. Chen Y. Ling W. Wu C. Zhao S. Liu J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). To clarify whether the exposure of Gly120 by carboxyl-terminal cleavage of MAP1LC3B is essential for further ubiquitylation-like reactions, we the of Gly120 and Lys122 carboxyl-terminal We whether Gly120 is essential for the formation of E1- and E2-substrate intermediates as well as whether of MAP1LC3B the of MAP1LC3B-PL in to We the of RNA interference of MAP1LC3B mRNA endogenous MAP1LC3B-PL and MAP1LC3B. results indicate that MAP1LC3B is cleaved in vivo to expose Gly120 and is modified to MAP1LC3B-PL at this residue by hAtg7 and and the for and protein was in in the of F. K. in Scholar). was from was from from was from and was from To the system was used to the and mammalian expression mutant and green fluorescent protein mutant been (9Tanida I. Tanida-Miyake E. Ueno T. Kominami E. J. Biol. Chem. 2001; 276: 1701-1706Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 10Tanida I. Tanida-Miyake E. Komatsu M. Ueno T. Kominami E. J. Biol. Chem. 2002; 277: 13739-13744Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). mammalian cell expression of MAP1LC3B-3xFLAG in which three of the were to the carboxyl terminus of the of MAP1LC3B was from I. Tanida-Miyake E. Komatsu M. Ueno T. Kominami E. J. Biol. Chem. 2002; 277: 13739-13744Abstract Full Text Full Text PDF PubMed Scopus (224) Google and into and the was expression of in which Gly120 was to and in which Lys122 was to Ala (see Fig. were into and the were and expression of Myc-MAP1LC3B-His in which MAP1LC3B was to the at its terminus and to at its carboxyl and and were used to a from by the was into the and the was The of was from and into and the was expression of Myc-MAP1LC3BG120A-His, and (see Fig. the were into and the were and used for expression in E. of protein (see Fig. in which MAP1LC3B was to at its terminus and to the at its carboxyl has been 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 (286) Google Scholar). expression of (see Fig. and in which Gly120 was were into and the were and expression of MAP1LC3B without any under the of the (see Fig. 3), the of MAP1LC3B was into and the was expression of and (see Fig. were into and the were and RNA interference of MAP1LC3B the of MAP1LC3B a RNA the we a of and into the to the A of the for MAP1LC3B RNA interference the for that the is the of human and Cell and and HeLa cells were from and in modified containing were with the to the h, the cells were for further was as 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 (286) Google this shows little with human and was from Cell was from was from and was from in vitro assay for cleavage of the carboxyl terminus of has been 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 (286) Google Scholar). The of MAP1LC3B in and Gly120, for showed that hLC3/MAP1LC3B E1- and E2-substrate intermediates with mammalian Atg7 and respectively, and that of hAtg7 with MAP1LC3B the formation of the MAP1LC3B-PL conjugate (9Tanida I. Tanida-Miyake E. Ueno T. Kominami E. J. Biol. Chem. 2001; 276: 1701-1706Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 10Tanida I. Tanida-Miyake E. Komatsu M. Ueno T. Kominami E. J. Biol. Chem. 2002; 277: 13739-13744Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 12Tanida 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 (286) Google Scholar, I. Tanida-Miyake E. T. Komatsu M. H. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2002; PubMed Scopus Google Scholar). We showed that the carboxyl terminus of MAP1LC3B is cleaved in vitro by hAtg4B and that of hAtg4B MAP1LC3B lipidation in vivo 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 (286) Google and that the carboxyl-terminal Gly120 of rat LC3 is essential for its modification (3Kabeya 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 (5510) Google Scholar). Since there is significant MAP1LC3B and rat LC3 we that carboxyl-terminal cleavage of MAP1LC3B expose Gly120. a recombinant Myc-MAP1LC3B-His in which MAP1LC3B was to the at its terminus and to the at its carboxyl it was reported that the carboxyl terminus of human MAP1LC3B is not cleaved and that it is modified at not at Gly120 (13He H. Dang Y. Dai F. Guo Z. Wu J. She X. Pei Y. Chen Y. Ling W. Wu C. Zhao S. Liu J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Gly120 is not the and reactions not To any of an the LC3 we carboxyl-terminal cleavage of MAP1LC3B a of mutant MAP1LC3B-3xFLAG in which MAP1LC3B was to three of the at its carboxyl terminus MAP1LC3B-3xFLAG was expressed in HEK293 cell proteins were and MAP1LC3B in the was by with to MAP1LC3B and MAP1LC3B-PL were by not by of mutant in which Gly120 of MAP1LC3B was to resulted in a with that was by with both and We the of MAP1LC3B Lys122 the cleavage of in which Lys122 of MAP1LC3B was to resulted in to MAP1LC3B and both of which were by with not with that Lys122 has the carboxyl-terminal cleavage of MAP1LC3B. We similar results for and to the protein at the carboxyl terminus and expressed in HeLa and HEK293 cells not These results were with our from (9Tanida I. Tanida-Miyake E. Ueno T. Kominami E. J. Biol. Chem. 2001; 276: 1701-1706Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 10Tanida I. Tanida-Miyake E. Komatsu M. Ueno T. Kominami E. J. Biol. Chem. 2002; 277: 13739-13744Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 12Tanida 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 (286) Google Scholar, I. Tanida-Miyake E. T. Komatsu M. H. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2002; PubMed Scopus Google Scholar). To clarify in a manner whether the carboxyl terminus of Myc-MAP1LC3B-His is cleaved in we a of mutant Myc-MAP1LC3B-His proteins When Myc-MAP1LC3B-His protein was expressed in HEK293 cells, to the and of were by with not with results were when and were expressed in HEK293 cells and of mutant resulted in a with Myc-MAP1LC3B-His that was by both and of Gly120 to Ala showed that at the carboxyl terminus the of mutant These results indicate that cleavage of the carboxyl terminus of MAP1LC3B in HEK293 cells Gly120, not Gly120 for the of MAP1LC3B by hAtg4B in further the of Gly120 for carboxyl-terminal cleavage an in vitro assay for cleavage of MAP1LC3B by hAtg4B We three recombinant which MAP1LC3B was to at its terminus and to the at its carboxyl and which Gly120 was When was with at for and a to the which was by with a and this was not by When and were used as for were by both and for and both showed little in In reaction was for These results indicate that Gly120 of MAP1LC3B is essential for its carboxyl-terminal cleavage by in Gly120, of MAP1LC3B for of E1- and showed that when the mammalian mutant is expressed with the proteins form a I. Tanida-Miyake E. T. Komatsu M. H. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2002; PubMed Scopus Google Scholar). In mutant which the for the reaction was to intermediates with the Atg8 human and GABARAP, with in the proteins essential for the reaction (11Tanida I. Komatsu M. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2003; 300: 637-644Crossref PubMed Scopus (85) Google Scholar). It was not however, whether Gly120 of MAP1LC3B is essential for the formation of the To any of an MAP1LC3B in its and reactions with hAtg7 and we a of mutant MAP1LC3B proteins without any and expressed proteins with in HEK293 cells cell proteins were and MAP1LC3B and hAtg7 were by with their When MAP1LC3B and were both the and the proteins were intermediates were when was expressed with in which the carboxyl-terminal were to expose Gly120, and When was however, little was and there was little when was expressed with in which the carboxyl-terminal were with results were when a of mutant and proteins were expressed of MAP1LC3B not These indicate that MAP1LC3B can with hAtg7 carboxyl-terminal cleavage of the and that exposure of Gly120 is essential for the reaction mediated by whereas Lys122 little to We showed that MAP1LC3B an E2-substrate with in which the was to in the of hAtg7 I. Tanida-Miyake E. Komatsu M. Ueno T. Kominami E. J. Biol. Chem. 2002; 277: 13739-13744Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). It was however, whether Gly120 of MAP1LC3B is essential for the formation of the To determine whether Gly120 is essential for formation of this we expressed a of mutant MAP1LC3B proteins with hAtg7 and mutant of cell proteins and were by When was expressed with hAtg7 and intermediates were by similar to the results with MAP1LC3B and and In when was expressed with hAtg7 and little E2-substrate was by and These results indicate that MAP1LC3B can with hAtg3 and that the exposure of Gly120 is essential for this whereas Lys122 little to In contrast to a that the carboxyl terminus of Myc-MAP1LC3B-His is not cleaved (13He H. Dang Y. Dai F. Guo Z. Wu J. She X. Pei Y. Chen Y. Ling W. Wu C. Zhao S. Liu J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), we found that the carboxyl terminus of Myc-MAP1LC3B-His was cleaved in vivo the Gly120 of Myc-MAP1LC3B-His is exposed carboxyl-terminal Myc-MAP1LC3B-His form E1- and E2-substrate intermediates with and We whether Myc-MAP1LC3B-His can with and shown for MAP1LC3B Myc-MAP1LC3B-His intermediates, whereas Lys122 was not essential for this reaction of MAP1LC3B the MAP1LC3B-PL and the into the shown that, in HeLa cells, endogenous MAP1LC3B-PL is a form of endogenous MAP1LC3B 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 (286) Google Scholar), and the protein fractionated into the 100,000 × g pellet (11Tanida I. Komatsu M. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2003; 300: 637-644Crossref PubMed Scopus (85) Google Scholar, 12Tanida 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 (286) Google Scholar). recombinant MAP1LC3B is by a ubiquitylation-like reaction its carboxyl-terminal of MAP1LC3B in in the and of MAP1LC3B. We whether the of recombinant MAP1LC3B-PL is similar to that of endogenous MAP1LC3B-PL When we expressed MAP1LC3B under the of the in HeLa cells, recombinant and endogenous proteins showed similar and 3 and by at 100,000 × g for h, a to MAP1LC3B-PL was in the pellet and that recombinant MAP1LC3B-PL is a similar to the endogenous and that this does not a degradation of in with our I. Tanida-Miyake E. Komatsu M. Ueno T. Kominami E. J. Biol. Chem. 2002; 277: 13739-13744Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 11Tanida I. Komatsu M. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2003; 300: 637-644Crossref PubMed Scopus (85) Google Scholar, 12Tanida 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 (286) Google Scholar, I. T. T. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2002; PubMed Scopus Google Scholar, T. Tanida I. Tanida-Miyake E. Minematsu-Ikeguchi N. M. Ohsumi M. Ueno T. Kominami E. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). results were when and were expressed in HeLa cells not We shown that in which MAP1LC3B was to the at its is as in HEK293 cells (11Tanida I. Komatsu M. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2003; 300: 637-644Crossref PubMed Scopus (85) Google Scholar, I. T. T. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2002; PubMed Scopus Google and that a form of is fractionated into the pellet in HEK293 cells (11Tanida I. Komatsu M. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2003; 300: 637-644Crossref PubMed Scopus (85) Google Scholar). To the that the lipidation of is to HEK293 cells, we whether is fractionated into the pellet in HeLa When was expressed in HeLa cells, showed with the and was fractionated into the pellet in a manner similar to that of the endogenous protein in with our in HEK293 cells (11Tanida I. Komatsu M. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2003; 300: 637-644Crossref PubMed Scopus (85) Google Scholar, I. T. T. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2002; PubMed Scopus Google Scholar). We shown that in which MAP1LC3B was to at its E1- and E2-substrate intermediates with mammalian Atg7 and I. Tanida-Miyake E. Komatsu M. Ueno T. Kominami E. J. Biol. Chem. 2002; 277: 13739-13744Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, I. T. T. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2002; PubMed Scopus Google Scholar). it is whether be as and whether is fractionated into the When was expressed in HeLa cells and with were These were of the with the is the that was in the pellet, along with the of the endogenous and recombinant proteins and These results indicate that, the endogenous recombinant MAP1LC3B is in RNA of hLC3/MAP1LC3B of LC3 in HEK293 MAP1LC3B and MAP1LC3B-PL were by with this significant with To determine whether this is the we used a RNA under the of a human to with MAP1LC3B In the of this by and to endogenous MAP1LC3B and MAP1LC3B-PL be the shows little to the of and and a found identical When we HEK293 cells with the cells for h, and the cell with we found that the of both endogenous MAP1LC3B and MAP1LC3B-PL were with cells Since RNA interference mRNA containing identical results indicate that endogenous MAP1LC3B-PL and MAP1LC3B are from authentic hLC3/MAP1LC3B mRNA and not from We shown that the carboxyl terminus of MAP1LC3B is cleaved in vivo and in vitro and that its Gly120 is essential for the carboxyl termini of and are cleaved. In contrast to (13He H. Dang Y. Dai F. Guo Z. Wu J. She X. Pei Y. Chen Y. Ling W. Wu C. Zhao S. Liu J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), reported that Myc-MAP1LC3B-His protein is modified at Lys122 without any carboxyl-terminal we shown that Gly120 of MAP1LC3B is essential for the cleavage of Myc-MAP1LC3B-His as well as both the and of Myc-MAP1LC3B-His were as by with not with Myc-MAP1LC3B-His and E1- and E2-substrate intermediates with and respectively, as shown for and 3 and 10Tanida I. Tanida-Miyake E. Komatsu M. Ueno T. Kominami E. J. Biol. Chem. 2002; 277: 13739-13744Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). RNA interference of MAP1LC3B mRNA showed that endogenous not and In this endogenous MAP1LC3B as to the and and the endogenous form of MAP1LC3B in human cell T. Ueno, and E. Kominami, These results that the carboxyl terminus of Myc-MAP1LC3B-His is cleaved to expose Gly120 for further lipidation in a manner similar to that with endogenous MAP1LC3B. the of hAtg4B cysteine that cleaves MAP1LC3B at its carboxyl exposing hAtg7 enzyme for and hAtg3 enzyme for shown and (9Tanida I. Tanida-Miyake E. Ueno T. Kominami E. J. Biol. Chem. 2001; 276: 1701-1706Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 10Tanida I. Tanida-Miyake E. Komatsu M. Ueno T. Kominami E. J. Biol. Chem. 2002; 277: 13739-13744Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 11Tanida I. Komatsu M. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2003; 300: 637-644Crossref PubMed Scopus (85) Google Scholar, 12Tanida 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 (286) Google Scholar, I. Tanida-Miyake E. T. Komatsu M. H. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2002; PubMed Scopus Google Scholar, I. T. T. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2002; PubMed Scopus Google Scholar, T. Tanida I. Tanida-Miyake E. Minematsu-Ikeguchi N. M. Ohsumi M. Ueno T. Kominami E. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, K. Tanida I. I. Ueno T. H. T. Kominami E. Y. J. 2003; PubMed Scopus Google Scholar), it is that the carboxyl-terminal cleavage of recombinant to ubiquitylation-like modification in a manner similar to that shown for endogenous MAP1LC3B. and and MAP1LC3B can form E1- and E2-substrate intermediates with and respectively, whereas mutant that MAP1LC3B is cleaved at to expose Gly120. We similar results a of Myc-MAP1LC3B-His Since Gly120 is exposed carboxyl-terminal it is not that Lys122 to the and reactions in little of the MAP1LC3B carboxyl-terminal residues When MAP1LC3B was in HeLa cells, the of recombinant MAP1LC3B and MAP1LC3B-PL was similar to that of the endogenous and recombinant MAP1LC3B-PL proteins were fractionated into the These indicate that the carboxyl terminus of MAP1LC3B is cleaved to expose Gly120 and that this and not is essential for further ubiquitylation-like mediated by hAtg7 and MAP1LC3B is modified to hLC3/MAP1LC3B is an authentic modifier, as is rat LC3. Lys122 of MAP1LC3B is modified to carboxyl-terminal it not to the modifier of MAP1LC3B to its as a autophagosomal We Y. Ohsumi for and T. Yoshimori of for significant and and J. Ezaki, and M. Komatsu for We N. Minematsu-Ikeguchi for

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