Fig 2: PINK1 is stabilized by gemcitabine independently of mitochondrial depolarization. (A) PINK1 immunoblot for the WT and PINK1 KO cell line # 1 following culture with or without gemcitabine for 48 h. Full-length blots are presented in Supplementary Fig. S2A, B. (B) HeLa cells were treated with 100 µM gemcitabine for 48 h or 10 µM CCCP for 3 h and then subjected to subcellular fractionation. All fractions were processed by immunoblotting with anti-PINK1, anti-Tom20 (mitochondria marker), and anti-LDHA (cytoplasm marker) antibodies. PNS, Sup, and pellet represent the post-nuclear supernatant, 10000 × g supernatant fraction, and 10000 × g pellet fraction, respectively. Full-length blots are presented in Supplementary Fig. S2C–E. (C) HeLa cells were cultured with or without 100 µM gemcitabine for 24 h or treated with CCCP. Cells were stained with TMRM and analyzed by fluorescence microscopy. Bars, 10 µm. (D) WT and two PINK1 KO cell lines were transfected with IRES-GFP-NLS empty vector, PINK1 WT or PINK1 KD vectors and cultured with 100 µM gemcitabine for 48 h. Mitophagy was analyzed by fluorescence microscopy as in Fig. 1B. Fluorescence images of PINK1 KO # 1 are shown. Bars, 10 µm. (E) Mitophagy dots shown in D were calculated from at least 20 transfected cells with nuclear GFP signal. Data are shown as the mean ± SEM. The data is a representative of three independent experiments. Similar results were observed in the other two experiments. *p < 0.05; NS, not significant.

Fig 3: p.G411S, but not p.Q456X heterozygotes show persistently impaired PINK1 kinase activity under endogenous conditions over time. Primary human skin fibroblasts were left untreated or stressed with 1 µM of the potassium ionophore valinomycin (VM) for the indicated times to depolarize mitochondrial membranes. Two cell lines per genotype were analysed. PINK1 genotypes of individuals are stated as wild-type (WT), heterozygous (HET) or homozygous (HOMO) along with the respective PINK1 mutation. (A) Representative western blots with total cell lysates were analysed for levels of PINK1, phosphorylated ubiquitin (p-Ser65-Ub), parkin, and its substrate mitofusin 2 (MFN2). Closed and open triangles indicate unmodified MFN2 and its ubiquitinated forms, respectively. Anti-vinculin was used as loading control. (B and C) Quantification of protein levels from western blots of six independent experiments. Shown is the mean per genotype ± SEM. Statistical significance was assessed with two-way ANOVA, Tukey’s post hoc test (*P < 0.05; **P < 0.005; ***P < 0.0005). (B) Shown are levels of PINK1 and p-Ser65-Ub normalized to wild-type at 24 h. Levels of total, unmodified ubiquitin were comparable from all cells and at all times (data not shown). p.Q456X heterozygotes showed significantly reduced levels of PINK1, concomitant with initially decreased levels of p-Ser65-Ub. However, p.G411S showed PINK1 levels similar to wild-type, but significantly reduced levels of p-Ser65-Ub over time. (C) Shown are relative levels of parkin and MFN2 normalized to 0 h valinomycin treatment. Absolute levels of parkin were similar in wild-type and all heterozygous PINK1 mutant cells, but were significantly elevated in p.Q456X homozygotes; however, no difference was seen for MFN2 (data not shown). (D) Quantitative MSD assay in 96-well plate format of p-Ser65-Ub levels from cell lysates. Cells were treated with 1 µM valinomycin (VM) for the indicated times, lysed and p-S65-Ub was captured with phospho-specific ubiquitin antibodies and detected with total ubiquitin antibodies. Shown is the mean per genotype ± SEM normalized to wild-type at 24 h from three independent experiments. Statistical significance was assessed with two-way ANOVA, Tukey’s post-hoc test (*P < 0.05; *** P < 0.0005). (E) Quantitative high content imaging in 96-well plate format of p-Ser65-Ub intensity in primary human fibroblasts before and after valinomycin (VM) treatment. Shown is the mean per genotype ± SEM normalized to wild-type at 0 h and 24 h from three independent experiments. Statistical significance was assessed with two-way ANOVA, Tukey’s post hoc test (*P < 0.05; **P < 0.005; ***P < 0.0005).

Fig 4: PINK1 stability is regulated by PDE2A2 activity.a Representative western blot and (b) summary quantification of PINK1 levels in HEK293 whole-cell lysates treated with forskolin 25 µM, BAY-60 1 µM or ISO 100 nM, as indicated, followed by CCCP 10 µM treatment for 2 h, as indicated. N = 3. c Representative western blot and (d) summary quantification of PINK1 levels in HEK293 cells treated with CCCP 10 µM and forskolin 25 µM, as indicated, in the absence or presence of PDE2A2-DN-RFP. N = 4. All values represent mean ± s.e.m. and are expressed as relative to the level of PINK1 detected in cell treated with CCCP only. One-way (d) and two-way (b) ANOVA and Tukey’s multiple comparison test were used *P < 0.05; **P < 0.01; ***P < 0.005.

Fig 5: USP30 restricts basal pexophagy Representative images of hTERT-RPE1 cells treated with non-targeting (NT1) or USP30 siRNA oligos (D1, D3). After 24 h, cells were transfected with Keima-SKL and imaged 48 h later. Graphs show the quantification of Keima-SKL “red” puncta (dots) per cell. Average ± SD, n = 4 independent experiments, 40 cells per experiment. Left: one-way ANOVA and Dunnett's multiple comparison's test, **P < 0.01; right: two-way ANOVA and Bonferroni's multiple comparison test, ***P < 0.001, **P < 0.01, *P < 0.05.Quantification of Keima-SKL as in (A) for hTERT-RPE1 cells treated with siRNA targeting USP30 (D1), ATG7 or both. Average ± SD, n = 3 independent experiments, 40 cells per experiment; one-way ANOVA and Dunnett's multiple comparison's test, *P < 0.05.Representative images of hTERT-RPE1 cells treated as in (A) except Keima-SKL was cotransfected with siRNA-resistant USP30-GFP (U30) and USP30C77S-GFP (C77S). Three independent experiments were analysed for the first three conditions, two of which also included the C77S rescue. Graph shows the average ± SD or range; 20 cells per experiment. One-way ANOVA and Dunnett's multiple comparison's test, *P < 0.05.Representative images of Keima-SKL “red” puncta in USP30 KO or WT hTERT-RPE1 cells. Keima-SKL was either transfected on its own or together with USP30-GFP and USP30C77S-GFP for 48 h. Three independent experiments were analysed for the first three conditions, of which two experiments also included the C77S rescue. Graph shows the average ± SD or range; 20 cells per experiment. One-way ANOVA and Dunnett's multiple comparison's test, **P < 0.01.Representative images of mt-Keima “red”-puncta in USP30 KO and WT hTERT-RPE1 clones, transfected on its own or together with USP30-GFP and USP30C77S-GFP for 48 h with mt-Keima. Graph shows the average from three independent experiments ± SD; 20 cells per experiment. One-way ANOVA, *P < 0.05.hTERT-RPE1 USP30 WT and KO cells (each two independent clones) were treated for 6 or 24 h with 100 nM folimycin or DMSO as a control. Lysates were separated by SDS–page and analysed by Western blot as indicated.Quantification of Keima-SKL “red” puncta in hTERT-RPE1 cells transfected with siRNA targeting USP30 (D1), PINK1 and PARKIN or non-targeting oligos (NT1). After 24 h, cells were transfected with the Keima-SKL for another 48 h prior to image capture. Average ± SD, n = 3 independent experiments; 20 cells per experiment. One-way ANOVA and Dunnett's multiple comparison's test, **P < 0.01, overlaid on top of the data points.Summary illustration: USP30 opposes both basal pexophagy- and PINK1-dependent basal mitophagy. Scale bars (A, C, D, E) 10 µm. Source data are available online for this figure.