The mitochondrial rhomboid protease Parl governs apoptosis, morphology, 
metabolism and might be implicated in Parkinson's disease, but the structural 
basis of its activity and complex regulation remain unknown. We report the 
discovery of γ-cleavage, a proteolytic event on the loop connecting the first 
transmembrane helix (TMH) of Parl to the 6-TMH catalytic rhomboid domain of the 
protease. This cleavage disrupts the '1+6' structure that defines every 
mitochondrial rhomboid and generates a new form of Parl, PROD 
(Parl-rhomboid-domain). Structure-function analysis of Parl suggests that 
γ-cleavage could be implicated in eliminating Parl proteolytic activity, and 
structural modeling of PROD reveals structural conservation with the bacterial 
rhomboid GlpG. However, unlike bacterial rhomboids, which employ a diad-based 
mechanism of catalysis, Parl appears to use a conserved mitochondrial 
rhomboid-specific Asp residue on TMH-5 in a triad-based mechanism of catalysis. 
This work provides unexpected insights into the structural determinants 
regulating Parl stability and activity in vivo, and reveals a complex cascade of 
proteolytic events controlling the function of the protease in the 
mitochondrion.