Autophagins and ATG8 homologs of Trypanosoma cruzi: a glimpse into autophagy of an early divergent eukaryote

KOSEC, G.; ALVAREZ, V.; TURK, B.; TURK, V.; CAZZULO, J.J.

Glasgow, Escocia.

Congreso; ICOPA XI. 11th International Congress of Parasitology.; 2006

British Society for Parasitology

Autophagy is an evolutionally conserved pathway for the degradation of cellular components directly involved in cytoplasmic component recycling, survival in starvation conditions and cell differentiation. As a general mechanism, cytosol and organelles become enclosed in double-membrane vesicles to form an autophagosome, which then fuses with the lysosome/vacuole and hence cargo material is degraded by the action of lysosomal peptidases. Autophagosome formation involves a set of autophagy (ATG) proteins; among them the ATG4 protease (autophagin) and its natural substrate the ubiquitin-like ATG8 play crucial roles. ATG8 is inserted into the membrane after conjugation to a lipid (phosphatidylethanolamine); this is possible only after cleavage by ATG4, exposing a C-terminal Gly residue. ATG4 is also able to release this lipid enabling ATG8 to recycle. The physiological role proposed for this reaction is membrane organization during autophagy. We found in the Trypanosoma cruzi genome database two genes homologous to ATG4 and two ATG8 homologs. We expressed these genes in an Escherichia coli expression system and established that both proteases cleave both ATG8 homologs in vitro, exposing the evolutionally conserved C-terminal Gly residue. The cleavage was inhibited by irreversible cysteine protease inhibitors. Yeast ATG4 knockouts were transfected with the two autophagins and yeast ATG8 knockouts with their substrates. Both T. cruzi autophagins complemented ATG4 deficiency and reconstituted the autophagy pathway in yeast, whereas only one of the substrates did so in the ATG8-deficient strain. Antibodies raised against ATG8 homologs are being used to follow autophagy in different T. cruzi life processes.