Figure 6. PINK1 precursor retrotranslocates from mitochondria to the cytosol after cleavage by PARL. (A) In vitro synthesized 35S-labeled proteins were incubated with mitochondria with or without valinomycin (Val). The mitochondrial pellet (pel) and the supernatant (sup) fractions were separated by centrifugation. 10% and 20% represent the amount of input of translation product. (B) PINK1 import samples were treated with PK and then the pel and sup were separated. The samples were subjected to radioimaging (low and high exposures) for PINK1 or immunoblotting (IB). (C) PINK1 import into parl−/− mitochondria. Blue, green and red arrowheads indicate the full-length, MPP-cleaved and PARL-cleaved PINK1, respectively. (D) Microscopic analysis of Parl+/+ and parl−/− MEF cells transiently expressing PINK1F104M-YFP. Cells were immunostained with anti-TOMM20. Scale bars: 10 μm. Right panel shows quantification of localization of PINK1-YFP wild-type (F104F), F104M and 104Δ mutants in Parl+/+ and parl−/− MEF cells. Dual means YFP signal both in mitochondria and in the cytosol. ND, not determined because of the low expression level. (E) Schematic representation of YFP-fusion proteins with N-terminal 155 residues of PINK1 or HTRA2. Arrows indicate the cleavage site by PARL for PINK1-YFP variants and by an IMS protease for HTRA2(1-155)-YFP. (F) Microscopic analysis of HeLa cells transiently expressing PINK1(1-155)-YFP, PINK1(1-155)F104M-YFP and HTRA2(1-155)-YFP. Cells were immunostained with anti-TOMM20. Scale bars: 10 μm. (G) The transfected cells as in (F) were treated with or without MG132 for 3 h. Total cell lysates were analyzed by immunoblotting with anti-GFP and anti-TOMM20 antibodies. Blue and red arrowheads represent precursor proteins and cleaved proteins, respectively.