Apg7p/Cvt2p: A novel protein-activating enzyme essential for autophagy.
In the yeast Saccharomyces cerevisiae, the Apg12p-Apg5p conjugating system is essential for autophagy. Apg7p is required for the conjugation reaction, because Apg12p is unable to form a conjugate with Apg5p in the apg7/cvt2 mutant. Apg7p shows a significant similarity to a ubiquitin-activating enzyme, Uba1p. In this article, we investigated the function of Apg7p as an Apg12p-activating enzyme. Hemagglutinin-tagged Apg12p was coimmunoprecipitated with c-myc-tagged Apg7p. A two-hybrid experiment confirmed the interaction. The coimmunoprecipitation was sensitive to a thiol-reducing reagent. Furthermore, a thioester conjugate of Apg7p was detected in a lysate of cells overexpressing both Apg7p and Apg12p. These results indicated that Apg12p interacts with Apg7p via a thioester bond. Mutational analyses of Apg7p suggested that Cys507 of Apg7p is an active site cysteine and that both the ATP-binding domain and the cysteine residue are essential for the conjugation of Apg7p with Apg12p to form the Apg12p-Apg5p conjugate. Cells expressing mutant Apg7ps, Apg7pG333A, or Apg7pC507A showed defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I. These results indicated that Apg7p functions as a novel protein-activating enzyme necessary for Apg12p-Apg5p conjugation.
Tanida
I
I
Department of Biochemistry, Juntendo University School of Medicine, Tokyo 113-8421, Japan.
Mizushima
N
N
Kiyooka
M
M
Ohsumi
M
M
Ueno
T
T
Ohsumi
Y
Y
Kominami
E
E
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism; D001343:Autophagy / Q000502:physiology*; D000071187:Autophagy-Related Protein 5; D000071193:Autophagy-Related Protein 7; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D044767:Ubiquitin-Protein Ligases; D014617:Vacuoles / Q000378:metabolism
C102268:ATG5 protein, S cerevisiae; C114830:ATG7 protein, S cerevisiae; D000071187:Autophagy-Related Protein 5; D005656:Fungal Proteins; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D044767:Ubiquitin-Protein Ligases; D000626:Aminopeptidases; C019689:APE1 protein, S cerevisiae; D000071193:Autophagy-Related Protein 7; D003545:Cysteine