Autophagy, proteases and the sense of balance

The knowledge of the molecular mechanisms underlying autophagy has considerably improved after the isolation and characterization of autophagy-defective mutants in the yeast Saccharomyces cerevisiae. Two ubiquitin-like conjugation systems are required for yeast autophagy. One of them requires the participation of Atg8 synthesized as a precursor protein, which is cleaved after a Gly residue by a cysteine proteinase called Atg4. The new Gly-terminal residue from Atg8 is activated by Atg7 (an E1-like enzyme) then transferred to Atg3 (an E2-like enzyme) and finally conjugated with membrane-bound phosphatidylethanolamine (PE) through an amide bond. The complex Atg8-PE is also deconjugated by the protease Atg4, facilitating the release of Atg8 from membranes. This modification system, which is essential for the membrane rearrangement dynamics that accompany the initiation and execution of autophagy, is conserved in higher eukaryotes including mammals. We have previously identified and cloned the four human orthologues of the yeast proteinase Atg4, whereas parallel studies have revealed that there are at least six orthologues of yeast Atg8 in mammals (LC3A, LC3B, LC3C, GABARAP, ATG8L/GABARAPL1 and GATE-16/GABARAPL2). Thus, in mammals, the Atg4-Atg8 proteolytic system is composed of four proteinases (autophagins) that may target at least six distinct substrates, contrasting with the simplified yeast system in which one single protease cleaves a sole substrate. Currently, it is unclear why mammals have developed this array of closely related enzymes, as other essential autophagy genes such as Atg3, Atg5 or Atg7 are represented in mammalian cells by a single orthologue. It has been suggested that the multiplication of Atg4 orthologues may reflect a regulatory heterogeneity of functionally redundant proteins or, alternatively, derive from the acquisition of new functions that are not related to autophagy. Our first approach to elucidate this question was based on the generation of autophagin-3/Atg4C-deficient mice, which however presented a minor phenotype. With the generation of autophagin-1/Atg4B-deficient mice, recently reported, we have progressed in our attempt to identify the in vivo physiological and pathological roles of autophagins.