January 1, 2001Journal of Biological Chemistry331 citationsOpen Access

The Human Homolog of Saccharomyces cerevisiae Apg7p Is a Protein-activating Enzyme for Multiple Substrates Including Human Apg12p, GATE-16, GABARAP, and MAP-LC3

ITIsei Tanida ETEmiko Tanida-Miyake TUTakashi Ueno

Key Points

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Abstract

Autophagy is a process that involves the bulk degradation of cytoplasmic components by the lysosomal/vacuolar system. In the yeast, Saccharomyces cerevisiae, an autophagosome is formed in the cytosol. The outer membrane of the autophagosome is fused with the vacuole, releasing the inner membrane structure, an autophagic body, into the vacuole. The autophagic body is subsequently degraded by vacuolar hydrolases. Taking advantage of yeast genetics, apg (autophagy-defective) mutants were isolated that are defective in terms of formation of autophagic bodies under nutrient starvation conditions. One of the APG gene products, Apg12p, is covalently attached to Apg5p via the C-terminal Gly of Apg12p as in the case of ubiquitylation, and this conjugation is essential for autophagy. Apg7p is a novel E1 enzyme essential for the Apg12p-conjugation system. In mammalian cells, the human Apg12p homolog (hApg12p) also conjugates with the human Apg5p homolog. In this study, the unique characteristics of hApg7p are shown. A two-hybrid experiment indicated that hApg12p interacts with hApg7p. Site-directed mutagenesis revealed that Cys572 of hApg7p is an authentic active site cysteine residue essential for the formation of the hApg7p·hApg12p intermediate. Overexpression of hApg7p enhances the formation of the hApg5p·hApg12p conjugate, indicating that hApg7p is an E1-like enzyme essential for the hApg12p conjugation system. Cross-linking experiments and glycerol-gradient centrifugation analysis showed that the mammalian Apg7p homolog forms a homodimer as in yeast Apg7p. Each of three human Apg8p counterparts, i. e. the Golgi-associated ATPase enhancer of 16 kDa, GABAAreceptor-associated protein, and microtubule-associated protein light chain 3, coimmunoprecipitates with hApg7p and conjugates with mutant hApg7pC572S to form a stable intermediate via an ester bond. These results indicate that hApg7p is an authentic protein-activating enzyme for hApg12p and the three Apg8p homologs. Autophagy is a process that involves the bulk degradation of cytoplasmic components by the lysosomal/vacuolar system. In the yeast, Saccharomyces cerevisiae, an autophagosome is formed in the cytosol. The outer membrane of the autophagosome is fused with the vacuole, releasing the inner membrane structure, an autophagic body, into the vacuole. The autophagic body is subsequently degraded by vacuolar hydrolases. Taking advantage of yeast genetics, apg (autophagy-defective) mutants were isolated that are defective in terms of formation of autophagic bodies under nutrient starvation conditions. One of the APG gene products, Apg12p, is covalently attached to Apg5p via the C-terminal Gly of Apg12p as in the case of ubiquitylation, and this conjugation is essential for autophagy. Apg7p is a novel E1 enzyme essential for the Apg12p-conjugation system. In mammalian cells, the human Apg12p homolog (hApg12p) also conjugates with the human Apg5p homolog. In this study, the unique characteristics of hApg7p are shown. A two-hybrid experiment indicated that hApg12p interacts with hApg7p. Site-directed mutagenesis revealed that Cys572 of hApg7p is an authentic active site cysteine residue essential for the formation of the hApg7p·hApg12p intermediate. Overexpression of hApg7p enhances the formation of the hApg5p·hApg12p conjugate, indicating that hApg7p is an E1-like enzyme essential for the hApg12p conjugation system. Cross-linking experiments and glycerol-gradient centrifugation analysis showed that the mammalian Apg7p homolog forms a homodimer as in yeast Apg7p. Each of three human Apg8p counterparts, i. e. the Golgi-associated ATPase enhancer of 16 kDa, GABAAreceptor-associated protein, and microtubule-associated protein light chain 3, coimmunoprecipitates with hApg7p and conjugates with mutant hApg7pC572S to form a stable intermediate via an ester bond. These results indicate that hApg7p is an authentic protein-activating enzyme for hApg12p and the three Apg8p homologs. yeast autophagy mutant and wild-type genes expression products from the APG gene cytoplasm-to-vacuole targeting γ-aminobutyric acid receptor-associated protein GAL4 activation domain GAL4 DNA binding domain Golgi-associated ATPase enhancer of 16 kDa green fluorescent protein human murine microtubule-associated protein light chain 3 N-ethylmaleimide-sensitive fusion protein soluble NSF attachment protein receptor polyacrylamide gel electrophoresis expressed sequence tag human embryonic kidney cells ubiquitin-activating enzyme ubiquitin-conjugating enzyme. Tes, 2-2-hydroxy-1, 1-bis (hydroxymethyl) ethylaminoethanesulfonic acid endoplasmic reticulum Post-translational modifications regulate the functions and localization of target proteins, resulting in many significant intracellular events. One unique modification is the covalent attachment of modifier proteins, ubiquitin, ubiquitin-related proteins (SUMO-1/Smt3p and NEDD-8/RUB1), and Apg12p (for reviews, see Refs. 1Varshavsky A. Genes Cells. 1997; 2: 13-28Crossref PubMed Scopus (271) Google Scholar, 2Bonifacino J. S. Weissman A. M. Annu. Rev. Cell Dev. Biol. 1998; 14: 19-57Crossref PubMed Scopus (536) Google Scholar, 3Ciechanover A. EMBO J. 1998; 17: 7151-7160Crossref PubMed Scopus (1196) Google Scholar, 4Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6902) Google Scholar, 5Klionsky D. J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Crossref PubMed Scopus (391) Google Scholar, 6Kim J. Klionsky D. J. Annu. Rev. Biochem. 2000; 69: 303-342Crossref PubMed Scopus (320) Google Scholar). The enzymatic processes of these modifications have been intensively studied in ubiquitylation. Ubiquitin forms conjugates with a target protein via a three step mechanism. First, ubiquitin is activated at its C-terminal Gly by the ubiquitin-activating enzyme (UBA1, E1 enzyme) to form a conjugate with the active site Cys in the E1 enzyme via a thiol ester bond. Next, ubiquitin is transferred from the E1 enzyme to one of several ubiquitin-conjugating enzymes (UBCs, E2 enzymes). In the last step, ubiquitin is attached to a Lys within the target protein via an isopeptide bond. This step is often catalyzed by a member of the ubiquitin-protein ligase family, an E3 enzyme. The reaction mechanism is basically common for each modifier protein. Autophagy is a process of bulk degradation of cytoplasmic components by the lysosomal/vacuolar system (5Klionsky D. J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Crossref PubMed Scopus (391) Google Scholar, 7Seglen P. O. Bohley P. Experientia. 1992; 48: 158-172Crossref PubMed Scopus (369) Google Scholar, 8Dunn W. A. J. Trends in Cell Biol. 1994; 4: 139-143Abstract Full Text PDF PubMed Scopus (444) Google Scholar). In the initial step of macroautophagy, a cup-shaped membrane sac surrounds cytosolic components to form an autophagosome (9Baba M. Takeshige K. Baba N. Ohsumi Y. J. Cell Biol. 1994; 124: 903-913Crossref PubMed Scopus (403) Google Scholar). The outer membrane of the autophagosome fuses with a lysosome/vacuole (10Baba M. Ohsumi M. Ohsumi Y. Cell Struct. Funct. 1995; 20: 465-471Crossref PubMed Scopus (128) Google Scholar). Taking advantage of yeast genetics, apg 1 andaut mutants were isolated as autophagy-defective mutants in the yeast, Saccharomyces cerevisiae (12Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1409) Google Scholar, 13Thumm M. Egner R. Koch B. Schlumpberger M. Straub M. Veenhuis M. Wolf D. H. FEBS Lett. 1994; 349: 275-280Crossref PubMed Scopus (481) Google Scholar). Surprisingly, most of the apg mutants overlap genetically withcvt mutants, which have a defect in the cytoplasm-to-vacuole targeting of aminopeptidase I, indicating that these genes function in a unique transport system under vegetative growth conditions in addition to starvation conditions (14Harding T. M. Hefner-Gravink A. Thumm M. Klionsky D. J. J. Biol. Chem. 1996; 271: 17621-17624Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 15Baba M. Osumi M. Scott S. V. Klionsky D. J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (279) Google Scholar, 16Scott S. V. Hefner-Gravink A. Morano K. A. Noda T. Ohsumi Y. Klionsky D. J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12304-12308Crossref PubMed Scopus (214) Google Scholar). A novel modifier protein, Apg12p, was discovered as an APG gene product (11Mizushima 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 (1284) Google Scholar). Apg12p shows little homology to ubiquitin, but it is covalently attached to Apg5p via the C-terminal Gly of Apg12p as in the case of ubiquitylation. In this conjugation reaction, Apg7p and Apg10p function as E1- and E2-like enzymes for Apg12p, respectively (11Mizushima 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 (1284) Google Scholar, 17McGrath J. P. Jentsch S. Varshavsky A. EMBO J. 1991; 10: 227-236Crossref PubMed Scopus (189) Google Scholar, 18Tanida I. Mizushima N. Kiyooka M. Ohsumi M. Ueno T. Ohsumi Y. Kominami E. Mol. Biol. Cell. 1999; 10: 1367-1379Crossref PubMed Scopus (325) Google Scholar, 19Shintani T. Mizushima N. Ogawa Y. Matsuura A. Noda T. Ohsumi Y. EMBO J. 1999; 18: 5234-5241Crossref PubMed Scopus (235) Google Scholar). After the formation of the Apg12p· Apg5p conjugate, Apg16p attaches to Apg5p forming an Apg12p·Apg5p·Apg16p complex for autophagy (20Mizushima N. Noda T. Ohsumi Y. EMBO J. 1999; 15: 3888-3896Crossref Scopus (340) Google Scholar). Unlike other modifier-conjugation systems, the unique character of the Apg12p-conjugation system is that it plays indispensable roles in the formation of membrane structures, including autophagosomes and Cvt-vesicles. Apg7p, an authentic E1-like enzyme essential for Apg12p, plays an indispensable role in the initial step of the conjugation system, whereas the enzyme shows slight homology to other E1 enzymes (18Tanida I. Mizushima N. Kiyooka M. Ohsumi M. Ueno T. Ohsumi Y. Kominami E. Mol. Biol. Cell. 1999; 10: 1367-1379Crossref PubMed Scopus (325) Google Scholar). Apg7p interacts with Apg8p/Aut7p and Aut1p/Apg3p in addition to Apg12p (21Uetz P. Giot L. Cagney G. Mansfield T. A. Judson R. S. Knight J. R. Lockshon D. Narayan V. Srinivasan M. Pochart P. Qureshi-Emili A. Li Y. Godwin B. Conover D. Kalbfleisch T. Vijayadamodar G. Yang M. Johnston M. Fields S. Rothberg J. M. Nature. 2000; 403: 623-627Crossref PubMed Scopus (3915) Google Scholar, 22Kirisako T. Ichimura Y. Okada H. Kabeya Y. Mizushima N. Yoshimori T. Ohsumi M. Takao T. Noda T. Ohsumi Y. J. Cell Biol. 2000; 151: 263-275Crossref PubMed Scopus (736) Google Scholar). 2M. Komatsu, I. Tanida, T. Ueno, M. Ohsumi, Y. Ohsumi, and E. Kominami, submitted manuscript. The dimerization of Apg7p via the C-terminal region is essential for these interactions, suggesting that Apg7p forms multimeric complexes with these proteins. 2 Apg8p/Aut7p is localized on autophagosomes and Cvt-vesicles (23Kirisako T. Baba M. Ishihara N. Miyazawa K. Ohsumi M. Yoshimori T. Noda T. Ohsumi Y. J. Cell Biol. 1999; 147: 435-446Crossref PubMed Scopus (713) Google Scholar). The AUT1/APG3 gene is a multicopy suppressor of the apg8/aut7 mutant (24Lang T. Schaeffeler E. Bernreuther D. Bredschneider M. Wolf D. H. Thumm M. EMBO J. 1998; 17: 3597-3607Crossref PubMed Scopus (230) Google Scholar). Apg8p/Aut7p also interacts with two ER-to-Golgi v-SNAREs (Bet1p and Sec22p) and vacuolar t- and v-SNAREs (Vam3p and Nyv1p, Ref. 25Legesse-Miller A. Sagiv Y. Gluzman R. Elazar Z. J. Biol. Chem. 2000; 275: 32966-32973Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Furthermore, more recent findings suggest that Apg8p/Aut7p, Aut1p/Apg3p, and Apg7p comprise a second protein-conjugation system indispensable for autophagy and Cvt pathways (22Kirisako T. Ichimura Y. Okada H. Kabeya Y. Mizushima N. Yoshimori T. Ohsumi M. Takao T. Noda T. Ohsumi Y. J. Cell Biol. 2000; 151: 263-275Crossref PubMed Scopus (736) Google Scholar, 26Ichimura 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 (1516) Google Scholar). The second modifier is Apg8p/Aut7p, and Apg7p and Aut1p/Apg3p are corresponding E1- and E2-like enzymes. These results suggest that Apg7p, because it is involved in two distinct conjugation systems, is a key enzyme for membrane formation and the targeting of autophagosomes and Cvt vesicles. In mammalian cells, several homologs of yeast APG gene products have been reported. hApg12p conjugates with hApg5p (first identified as an apoptosis-specific protein), suggesting that the Apg12p conjugation system exists even in human cells (27Hammond E. M. Brunet C. L. Johnson G. D. Parkhill J. Milner A. E. Brady G. Gregory C. D. Grand R. J. FEBS Lett. 1998; 425: 391-395Crossref PubMed Scopus (79) Google Scholar, 28Mizushima N. Sugita H. Yoshimori T. Ohsumi Y. J. Biol. Chem. 1998; 273: 33889-33892Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). There are three candidates for mammalian Apg8p/Aut7p homologs, GATE-16 (Golgi-associated ATPase enhancer of 16 kDa), GABARAP (GABA receptor-associated protein), and MAP-LC3 (microtubule-associated protein light chain 3) (25Legesse-Miller A. Sagiv Y. Gluzman R. Elazar Z. J. Biol. Chem. 2000; 275: 32966-32973Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 29Mann S. S. Hammarback J. A. J. Biol. Chem. 1994; 269: 11492-11497Abstract Full Text PDF PubMed Google Scholar, 30Mann S. S. Hammarback J. A. J. Neurosci. Res. 1996; 43: 535-544Crossref PubMed Scopus (48) Google Scholar, 31Wang H. Bedford F. K. Brandon N. J. Moss S. J. Olsen R. W. Nature. 1999; 397: 69-72Crossref PubMed Scopus (490) Google Scholar, 32Paz Y. Elazar Z. Fass D. J. Biol. Chem. 2000; 275: 25445-25450Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 33Sagiv Y. Legesse-Miller A. Porat A. Elazar Z. EMBO J. 2000; 19: 1494-1504Crossref PubMed Scopus (207) Google Scholar). GATE-16 was first identified as a ganglioside expression factor, but was recently characterized as a soluble transport factor. GATE-16 interacts with NSF and the Golgi v-SNARE GOS-28 (33Sagiv Y. Legesse-Miller A. Porat A. Elazar Z. EMBO J. 2000; 19: 1494-1504Crossref PubMed Scopus (207) Google Scholar). The mRNA of GATE-16 is expressed ubiquitously but at significantly higher levels in The of yeast Apg8p/Aut7p with two ER-to-Golgi v-SNAREs was as a GATE-16 homolog (25Legesse-Miller A. Sagiv Y. Gluzman R. Elazar Z. J. Biol. Chem. 2000; 275: 32966-32973Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). GABARAP interacts with and suggesting in cells H. Bedford F. K. Brandon N. J. Moss S. J. Olsen R. W. Nature. 1999; 397: 69-72Crossref PubMed Scopus (490) Google Scholar, M. S. H. H. Olsen R. W. H. Proc. Natl. Acad. Sci. 2000; PubMed Scopus Google Scholar). MAP-LC3 with and is a of the which is of light and 3 and S. S. Hammarback J. A. J. Biol. Chem. 1994; 269: 11492-11497Abstract Full Text PDF PubMed Google S. S. Hammarback J. A. J. Neurosci. Res. 1996; 43: 535-544Crossref PubMed Scopus (48) Google Scholar). MAP-LC3 is localized on suggesting that MAP-LC3 is also a Apg8p/Aut7p homolog Y. Mizushima N. Ueno T. A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: PubMed Scopus Google Scholar). These results suggest that mammalian Apg8p/Aut7p homologs have functions in mammalian cells, in Apg7p, are several in the and the sequence of hApg7p been to to which is essential for W. A. J. Mol. Biol. Cell. 1999; 10: PubMed Scopus Google Scholar). as is that hApg7p is an E1-like enzyme for There is a which of and GABARAP is an authentic for In this that hApg7p conjugates with hApg12p and three Apg8p/Aut7p homologs as hApg7p in mammalian were in an on from to for at were a and this cells, the for and protein were in in the of as R. J. A. K. in Scholar). and were as R. J. A. K. in Scholar, S. A. in Scholar). The yeast for two experiments was in yeast and as S. A. in Scholar). was by protein the The chain reaction was with a system The DNA sequence was with an DNA DNA was into mammalian cells with to the enzymes were from and were by the and were from P. P. J. 1996; PubMed Google Scholar). was from was from and were and was from on the DNA sequence of the human homolog an of the human by chain reaction with W. A. J. Mol. Biol. Cell. 1999; 10: PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google the DNA into the site of and the for an expression as of the was into the and a DNA an of the and chain reaction was with for with a human as a and the was into and The isolated DNA were into to fusion proteins and Cys572 within hApg7p was by by the in mutagenesis system with an to the The expression for mutant was as in the case of and was A a corresponding to of hApg7p was in The was by on the of murine Apg12p homolog were with a fusion protein. The to was by on The was from The from the for was to for homologs in the human and on the DNA sequence of of the in by chain reaction with Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). The DNA was into and the DNA sequence was The DNA of were and the of the significant homology to and murine in cells, the DNA was into cells were in cells were on After for at the cells were with a of of DNA and of 1 of each was The was for an 1 cells were with 1 of and in of The was by for at in the were by and transferred to a membrane analysis was with and and the were by an system were isolated from a and in of The was at 1 and the was as the was an in and at for of were from the of the Apg7p was from each with and to because hApg7p with Apg7p. kDa, kDa, kDa, and kDa, were as analysis was as by P. J. 1996; PubMed Google Scholar). In yeast, S. cerevisiae, Apg7p is a protein-activating enzyme for Apg12p (18Tanida I. Mizushima N. Kiyooka M. Ohsumi M. Ueno T. Ohsumi Y. Kominami E. Mol. Biol. Cell. 1999; 10: 1367-1379Crossref PubMed Scopus (325) Google Scholar). hApg7p is a protein-activating enzyme essential for the conjugation system, hApg12p with hApg7p. first the hApg7p and hApg12p by a two-hybrid experiment yeast expression of hApg7p and hApg12p and expressed fusion proteins in a yeast The and on whereas and and and These results indicate that hApg12p interacts with hApg7p. hApg7p forms an mutagenesis of a active site cysteine residue within hApg7p. on a homology yeast and human Apg7p, that the active site cysteine residue within hApg7p Cys572 an active site cysteine residue within an is to formed of a thiol ester bond. Cys572 within hApg7p to by mutagenesis and expressed the mutant hApg7pC572S and hApg12p in cells Cell proteins were and by hApg7p was by with hApg7p and mutant hApg7pC572S were expressed in cells and hApg7p of hApg7pC572S and were expressed in cells, a higher with a stable intermediate in addition to the of kDa for hApg7p This higher was also by with in the of These results indicate that hApg12p is an authentic for hApg7p. hApg7p is an E1-like enzyme in the system, it is that the of hApg7p the conjugation of hApg12p with this expressed hApg7p and in cells, the cells with and Cys and a hApg12p was with and the were by and was was with In cells and a corresponding to the conjugate was in addition to with The formation of the conjugate was by a second The of mutant hApg7pC572S the conjugation These results indicate that hApg7p is an authentic protein-activating enzyme essential for the human Apg12p-conjugation system. have that yeast Apg7p forms a homodimer via the C-terminal the homology yeast and human Apg7p, it is that hApg7p also form a this a A hApg7p was and with a After the was by and hApg7p was by with with the hApg7p was in the as a corresponding to kDa After the of this was and a at kDa Apg7p in by the of the with Apg7p. The cytosolic of a was and to a Apg7p was with The resulting were by and Apg7p was identified by with Apg7p was in and with a of A two-hybrid experiment also indicated that hApg7p interacts with that mammalian Apg7p forms a homodimer to yeast Apg7p. findings have indicated that yeast Apg7p also functions as an enzyme for Apg8p and is essential for Apg8p targeting to (22Kirisako T. Ichimura Y. Okada H. Kabeya Y. Mizushima N. Yoshimori T. Ohsumi M. Takao T. Noda T. Ohsumi Y. J. Cell Biol. 2000; 151: 263-275Crossref PubMed Scopus (736) Google Scholar, 26Ichimura 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 (1516) Google that are at three Apg8p homologs, GATE-16 GABARAP and and MAP-LC3 in mammalian A of the human that are human MAP-LC3 homologs and was isolated from a human by of the to the from the The acid sequence of shows with its The C-terminal of the three proteins significant homology to yeast the significant homology human and yeast these proteins also for Apg7p. these Apg8p homologs with hApg7p as first a and were expressed as fusion proteins with hApg7p in Cell hApg7p and a fusion protein were The fusion proteins were The were by and hApg7p was by hApg7p with and but with The results indicate that and with hApg7p. the formation of stable conjugates of mutant hApg7pC572S with and via bond. hApg7pC572S was with in cells, and the were by under conditions. The homologs were by with A corresponding to a stable hApg7pC572S intermediate was in hApg7pC572S and a but in cells wild-type hApg7p and homologs, indicating that a stable conjugate is formed hApg7pC572S and the Apg8p homologs These results indicate that three proteins, and are authentic for hApg7p. In this study, showed that the human Apg7p homolog is an authentic E1-like enzyme for the hApg12p conjugation system and that and are for hApg7p. GATE-16 as a soluble transport interacts with NSF and is localized in the and is expressed in the in (33Sagiv Y. Legesse-Miller A. Porat A. Elazar Z. EMBO J. 2000; 19: 1494-1504Crossref PubMed Scopus (207) Google Scholar). GABARAP is receptor-associated protein that with the receptor in and interacts with H. Bedford F. K. Brandon N. J. Moss S. J. Olsen R. W. Nature. 1999; 397: 69-72Crossref PubMed Scopus (490) Google Scholar, 32Paz Y. Elazar Z. Fass D. J. Biol. Chem. 2000; 275: 25445-25450Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, M. S. H. H. Olsen R. W. H. Proc. Natl. Acad. Sci. 2000; PubMed Scopus Google Scholar). MAP-LC3 is localized on Y. Mizushima N. Ueno T. A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: PubMed Scopus Google Scholar). the functions and intracellular of the three Apg8p homologs, it is that three human Apg8p homologs are for hApg7p. yeast Apg7p plays an indispensable role in autophagy and the Cvt transport of aminopeptidase I, mammalian Apg7p also essential for autophagy and other forms of membrane common the formation of cup-shaped membrane MAP-LC3 is localized on in as in yeast Apg8p Y. Mizushima N. Ueno T. A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: PubMed Scopus Google at two MAP-LC3 and roles in autophagy in mammalian been of a mammalian the expression of GATE-16 and GABARAP in and cells, a other membrane transport pathways in which GATE-16 and GABARAP function as protein also in these is to hApg7p the and the the There with the hApg7p homodimer to form multimeric candidates to hApg7p by a two-hybrid experiment a human and of Apg7p with in several the target proteins of and MAP-LC3 There is that these proteins conjugate with other have targeting protein with which MAP-LC3 forms a Kabeya Y. Mizushima N. Ueno T. A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: PubMed Scopus Google that MAP-LC3 is to several forms in mammalian cells, mechanism. analysis of is more and more that the plays an role in at and and and of a of have that degradation via autophagy (for a see Ref. L. M. J. Dev. 2000; 10: PubMed Scopus Google Scholar). Furthermore, and of vacuolar in and have indicated that autophagic processes an indispensable role in mammalian bodies I. J. K. T. S. T. M. S. J. Y. A. N. S. E. E. I. S. M. Nature. 2000; PubMed Scopus Google Scholar, Y. G. A. D. R. J. K. P. Nature. 2000; PubMed Scopus Google Scholar). In autophagy is activated by in L. A. M. Mol. Cell. Neurosci. 1999; 14: PubMed Scopus Google Scholar). In of the of hApg12p and human Apg8p homologs, and and the of hApg7p with these it is that mammalian Apg7p plays an essential role in of and in addition to autophagy. are the functions of hApg7p These to the of the functions of mammalian Apg7p. Y. Ohsumi, T. T. N. Y. K. Kirisako for D. J. Klionsky of and M. for and P. of for and and K. J. and D. for

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