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. 2010 Nov 29;191(5):933–942. doi: 10.1083/jcb.201008084

Figure 1.

Figure 1.

Constitutive cleavage of PINK1 is mediated by PARL. (a) HeLa cells were transfected with scrambled control siRNA or PARL siRNA. After 4 h incubation with or without 10 µM CCCP, mitochondria were isolated, and mitochondrial protein extracts were assayed for endogenous levels of PINK1 and PARL by immunoblotting. VDAC1 is a mitochondrial marker. (b) MEFs from PARL WT and KO mice were transfected with PINK1-V5/His for 2 d and treated with DMSO or 10 µM CCCP for 4 h. Exogenous PINK1 levels were assayed by immunoblotting. Tubulin is a loading control. (c) PARL WT and KO MEFs were transfected with PINK1-V5/His as in b and treated with DMSO or 10 µM MG132. After 4 h of treatment, cells were fractionated, and exogenous PINK1 levels in mitochondrial fraction were measured with immunoblotting. VDAC1, mitochondrial loading control. (d) [35S]methionine-labeled PINK1 was incubated for different times with mitochondria isolated from PARL WT or KO MEFs in the presence or absence of 1 µM CCCP. After import, samples were treated with or without 5 µg/ml PK. Radiolabeled PINK1 was detected using digital autoradiography. Asterisks, nonspecific bands. (e) [35S]-PINK1 was imported into PARL KO mitochondria for 60 min as in d, and these mitochondria were incubated in the presence or absence of high PK (100 µg/ml) for 10 min. Hsp70, Htra2/Omi, and Tom20 were identified by immunoblotting as markers for mitochondrial matrix, inter membrane space, and outer membrane, respectively. (f) HeLa cells stably expressing YFP-Parkin were transfected with control (siCtrl) or PARL siRNA (siPARL) for 192 h. After transfection, cells were treated with either DMSO or 10 µM CCCP for 1 h, stained with TMRE, and analyzed by live cell imaging. Bars, 20 µm. (g) PARL WT and KO MEFs transfected with PINK1-YFP and mCherry-Parkin were treated with 10 µM DMSO or CCCP for 3 h. Cells (≥50/treatment) were counted for mitochondrial translocation of Parkin. Counting results were represented as mean ± SEM from four replicates. Blue arrowheads, FL PINK1; orange arrowheads, ΔMTS-PINK1; red arrowheads, 52-kD PINK1.