Fig. 2. Human Atg4 homologues process from pro-form to form I of LC3, GABARAP and GATE16 at C-termini. (A) Tagged versions of LC3, GABARAP and GATE16. Positions of Myc and HA epitope tags are indicated. The conserved Gly residues are shown in bold; this residue is replaced by Ala in the LC3^G120A mutant. Arrow indicates the cleavage site in yeast Atg8 by Aut2/Atg4 (Kirisako et al., 1999). (B) In vivo analysis of C-terminal hydrolase activities of HsAtg4A and HsAtg4B. The double-tagged mammalian Atg8 family proteins in (A) and the indicated human Atg4 family proteins were co-expressed in Atg4-deficient yeast cells. Total lysate of each transformant was analysed by immunoblot analysis using anti-Myc or anti-HA antibodies. Positions of unprocessed substrates and their processed forms are indicated. (C) Cys74 of HsAtg4B is a putative active site. Enzyme activity of wild type HsAtg4B and HsAtg4B^C74S mutant was examined as in (B) using Myc-LC3-HA as substrate.