A., K. is not caused by mitochondrial loss. Our findings suggest that PINK1 and PARKIN play crucial functions in selective cell death in which damaged mitochondria are retained, impartial of mitochondrial autophagy. are causal genes for autosomal recessive early-onset parkinsonism (1). PINK1 is a unique Ser/Thr kinase localized around the outer membrane of damaged mitochondria, where it is subsequently autophosphorylated, followed by the formation of a larger protein complex that contains a translocase of the outer membrane (TOM)4 complex (2,C4). PINK1 localized on damaged mitochondria selectively recruits PARKIN (5, 6), and phosphorylates PARKIN to uncover latent ligase activity (7). PINK1 and phosphorylated PARKIN share a cooperative role to modify mitochondrial outer membrane proteins with phospho-ubiquitin chains, and mitochondria decorated by poly-ubiquitin chains are eliminated by selective mitochondrial autophagy (1), thereby maintaining mitochondrial quality. Programmed cell death serves fundamental functions in tissue development and homeostasis and is associated with several human pathologies, including neurodegradation, autoimmune diseases, and malignancy (8). Apoptosis, the best studied form of programmed cell death, is characterized by cell shrinkage, blebbing, nuclear fragmentation, and chromatin condensation, and it requires caspase activation (9). Many studies have revealed caspase-independent but genetically regulated forms of cell death that are classified according to their unique morphologic features and specific inhibitors (10). PARKIN prevents cells from dying in response to proapoptotic stimuli (11, 12). The cytoprotective effects of PARKIN are relatively accepted because loss of PARKIN function prospects to progressive degeneration of dopaminergic neurons, giving rise to Parkinson disease. The functions of PINK1 and PARKIN in programmed cell death caused by non-apoptotic triggers, however, remain CCNB2 poorly characterized. Recently, we reported that removal of a conventional mitochondrial targeting sequence corresponding to the N-terminal 34-amino acid residues allows PINK1 to translocate to the outer membrane in an unconventional signal-dependent manner and induces the autophosphorylation of PINK1 and translocation of PARKIN without mitochondrial depolarization (13). The truncated PINK1 is usually constitutively active, can recruit PARKIN to the mitochondria, and promotes subsequent events, even when the mitochondria are energized. Here, we utilized both a chemical uncoupler to depolarize mitochondria and a constitutively active form of PINK1 to reveal that PINK1 activation caused cell death that did not involve caspase activation or mitochondrial permeability transition (MPT), and we induced definite morphologic features, such as plasma membrane rupture. Cell death was induced with a 12-h delay after depriving mitochondria of membrane potential, which differs from the time profile of selective autophagy of mitochondria. Importantly, autophagic activity was dispensable for the cell death induced by PINK1 activation, and mitochondria were still retained in the lifeless cells. Proteasomal activity, however, was crucial for the PINK1-induced cell death. Our findings suggest that PINK1 Timosaponin b-II and PARKIN regulate not only mitochondrial clearance but also proteasome-dependent cell death with different durations of mitochondrial depolarization. Results PARKIN-dependent Cell Death in Response to Mitochondrial Depolarization In normal culture conditions with a high glucose concentration, HeLa cell morphology is not significantly altered by treatment with the protonophore CCCP, because malignancy cells mainly utilize glycolysis to produce ATP (14). In contrast to HeLa cells lacking endogenous PARKIN expression (Fig. 1control HeLa and HeLa cell lines stably expressing exogenous PARKIN (GFP-PARKIN or HA-PARKIN) were cultured for 48 h in the indicated combinations of DMSO, Timosaponin b-II 10 m CCCP, and 100 m Z-VAD-fmk and then stained with PI. HA-PARKIN-expressing HeLa cells were transfected with GFP to visualize the cell shape, cultured for 30 h with Timosaponin b-II DMSO (10 mm. total cell lysates prepared from SH-SY5Y, HEK293, control HeLa, and PARKIN-expressing HeLa cell lines were analyzed by immunoblotting with antibodies to PARKIN and actin as a loading control. indicates endogenous PARKIN. control HeLa and HeLa cell lines expressing exogenous PARKIN were cultured for 40 h as explained in control HeLa and HA-PARKIN-expressing HeLa cells were cultured for the indicated occasions in the presence of Timosaponin b-II CCCP and stained with PI. HA-PARKIN-expressing HeLa cells cultured for 48 h with either DMSO (and and and and show high magnification images of the corresponding cell in the indicates a mitochondrion. 10 m (and and SH-SY5Y and HEK293 cells were cultured as explained in and then stained with PI. SH-SY5Y and HEK293 cells were cultured for the indicated occasions in the presence of CCCP and stained with PI. Data in represent the mean S.E. of three impartial experiments ( 100 individual cells were counted). *, 0.05; **, 0.005; ***, 0.0005 (Student’s test). Electron microscopic.