Autophagy is an important catabolic process with roles in cell survival and cell 
death. It sequesters cytosol and organelles within double-membrane 
autophagosomes that deliver their contents to lysosomes for degradation. 
Autophagosome biogenesis is coordinated by the autophagy-related protein 4 
(Atg4) family of C54 endopeptidases (Atg4A-Atg4D). These enzymes prime and then 
later delipidate the autophagosome marker, Atg8. Here, we show that one family 
member, Atg4D, is cleaved by caspase-3 in vitro and in apoptotic cells. Atg4D is 
a poor priming and delipidation enzyme in vitro, but truncated DeltaN63 Atg4D 
displays increased activity against the Atg8 paralogue, gamma-aminobutyric acid 
receptor-associated protein-like 1 (GABARAP-L1). In living cells, DeltaN63 Atg4D 
stimulates the delipidation of GABARAP-L1, whereas siRNA silencing of the gene 
expressing Atg4D abrogates GABARAP-L1 autophagosome formation and sensitises 
cells to starvation and staurosporine-induced cell death. Interestingly, Atg4D 
overexpression induces apoptosis, which is preceded by the caspase-independent 
recruitment of Atg4D to mitochondria and is facilitated by a putative C-terminal 
Bcl-2 homology 3 (BH3) domain. Atg4D also acquires affinity for damaged 
mitochondria in cells treated with hydrogen peroxide. These data suggest that 
Atg4D is an autophagy regulator that links mitochondrial dysfunction with 
apoptosis.