Human Apg3p/Aut1p homologue is an authentic E2 enzyme for multiple substrates, GATE-16, GABARAP, and MAP-LC3, and facilitates the conjugation of hApg12p to hApg5p.
Autophagy is a process of bulk degradation of cytoplasmic components by the lysosome/vacuole and has a significant relationship to several neurodegenerative disorders and myopathies in mammals. One of APG gene products essential for autophagy in yeast, Apg3p, is a protein-conjugating enzyme for Apg8p lipidation (Ichimura, Y., Kirisako, T., Takao, T., Satomi, Y., Shimonishi, Y., Ishihara, N., Mizushima, N., Tanida, I., Kominami, E., Ohsumi, M., Noda, T., and Ohsumi, Y. (2000) Nature 408, 488-492). In this study, the cloning of a human Apg3p homologue (hApg3p) as an E2 enzyme essential for human Apg8p homologues (i.e. GATE-16, GABARAP, and MAP-LC3) is shown, and its unique characteristics are described. The predicted amino acid sequence of the isolated clone shows 34.1% identity and 48.1% similarity to yeast Apg3p. Site-directed mutagenesis revealed that Cys(264) of hApg3p is an authentic active-site cysteine residue essential for the formation of hApg3p small middle dothApg8p homologue intermediates. Overexpression of hApg7p enhances the formation of a stable E2-substrate complex between hApg3p(C264S) and each of the hApg8p homologues, and MAP-LC3 is preferred as the substrate over the other two Apg8p homologues. These results indicate that hApg3p is an E2-like enzyme essential for three human Apg8p homologues. Co-immunoprecipitation of hApg7p with hApg3p indicates that hApg3p forms an E1.E2 complex with hApg7p as in the case of yeast Apg3p and Apg7p. Furthermore, hApg3p coimmunoprecipitates with hApg12p, and the overexpression of hApg3p facilitates the formation of the GFPhApg12p.thApg5p conjugate, suggesting that hApg3p cross-talks with the hApg12p conjugation system.
Tanida
Isei
I
Department of Biochemistry, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.
Tanida-Miyake
Emiko
E
Komatsu
Masaaki
M
Ueno
Takashi
T
Kominami
Eiki
E
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D000071187:Autophagy-Related Protein 5; D000071193:Autophagy-Related Protein 7; D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry*; D002460:Cell Line; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D008025:Ligases / Q000737:chemistry*; D008164:Luminescent Proteins / Q000378:metabolism; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins / Q000737:chemistry*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases
C102268:ATG5 protein, S cerevisiae; C114830:ATG7 protein, S cerevisiae; D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D000071187:Autophagy-Related Protein 5; D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; C116741:GABARAP protein, human; C498698:GABARAPL2 protein, human; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D000071193:Autophagy-Related Protein 7; C104235:ATG3 protein, S cerevisiae; C455621:ATG3 protein, human; D003545:Cysteine