Two newly identified sites in the ubiquitin-like protein Atg8 are essential for autophagy

Atg8, a member of a novel ubiquitin-like protein family, is an essential component of the autophagic machinery in yeast. This protein undergoes reversible conjugation to phosphatidylethanolamine through a multistep process in which cleavage of Atg8 by a specific protease is followed by ubiquitin-like conjugation processes. Here, we identify two essential sites in Atg8, one of them involving residues Phe77 and Phe79 and the other, located on the opposite surface of Atg8, residues Tyr49 and Leu50. We show that these two sites are associated with different functions of Atg8: Phe77 and Phe79 seem to be part of the recognition site for Atg4, a cystein protease that acts also as a deubiquitination enzyme, whereas Tyr49 and Leu50 act downstream of the lipidation step. These two newly identified distinct sites that are essential for Atg8 activity provide an explanation for the many protein-protein interactions of this low-molecular-weight protein.