Apg7p/Cvt2p: A novel protein-activating enzyme essential for autophagy

Ln the yeast Saccharomyces cerevisiae, the Apg12p-Apg5p conjugating system is essential for autophagy. Apg7p is required for the conjugation reaction, because Ag12p is unable to form a conjugate with Apg5p in the apg7/cvt2 mu tant. Apg7p shows a significant similarity to a ubiquitin-activating enzyme , Uba1p. In this article, we investigated the function of Apg7p as an Apg12 p-activating enzyme. Hemagglutinin-tagged Apg12p was coimmunoprecipitated w ith c-myc-tagged Apg7p. A two-hybrid experiment confirmed the interaction T he coimmunoprecipitation was sensitive to a thiol-reducing reagent. Further more, a thioester conjugate of Apg7p was detected in a lysate of cells over expressing both Apg7p and Apg12p. These results indicated that Apg12p inter acts with Apg7p via a thioester bond. Mutational analyses of Apg7p suggeste d that Cys(507) Of Apg7p is an active site cysteine and that both the ATP-b inding domain and the cysteine residue are essential for the conjugation of Apg7p with Apg12p to form the Apg12-Apg5p conjugate. Cells expressing muta nt Apg7ps, Apg7p,or Apg7p(C507A) showed defects in autophagy and cytoplasm- to-vacuole targeting of aminopeptidase I. These results indicated that Apg7 p functions as a novel protein-activating enzyme necessary for Apg12p-Apg5p conjugation.