Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment

Abstract

Mutations in phosphatase and tensin homologue-induced kinase 1 (PINK1) cause recessively inherited Parkinson's disease (PD), a neurodegenerative disorder linked to mitochondrial dysfunction. In healthy mitochondria, PINK1 is rapidly degraded in a process involving both mitochondrial proteases and the proteasome. However, when mitochondrial import is compromised by depolarization, PINK1 accumulates on the mitochondrial surface where it recruits the PD-linked E3 ubiquitin ligase Parkin from the cytosol, which in turn mediates the autophagic destruction of the dysfunctional organelles. Using an unbiased RNA-mediated interference (RNAi)-based screen, we identified four mitochondrial proteases, mitochondrial processing peptidase (MPP), presenilin-associated rhomboid-like protease (PARL), m-AAA and ClpXP, involved in PINK1 degradation. We find that PINK1 turnover is particularly sensitive to even modest reductions in MPP levels. Moreover, PINK1 cleavage by MPP is coupled to import such that reducing MPP activity induces PINK1 accumulation at the mitochondrial surface, leading to Parkin recruitment and mitophagy. These results highlight a new role for MPP in PINK1 import and mitochondrial quality control via the PINK1–Parkin pathway.

Figure 1

PINK1 processing is regulated by MPP, PARL, m-AAA and ClpXP. (A) Immunoblots of mitochondrial fractions from siRNA-transfected HEK293T cells±CCCP. (B) Immunoblots against indicated proteases in siRNA-transfected cells. (C) Quantification of full-length PINK1 (n=4) and knockdown efficiency (n=2) compared with non-targeting (NT) siRNA from experiments performed as in B. ^*P<0.05; ^**P<0.01; ^***P<0.001. CCCP, carbonyl cyanide m-chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; PARL, presenilin-associated rhomboid-like protease; PINK1, phosphatase and tensin homologue-induced kinase 1; siRNA, short interfering RNA.

Figure 2

Modest effects of transient MPPβ, PARL and AFG3L2 knockdown on mitochondrial integrity. (A) Live confocal micrographs of siRNA-transfected HEK293T cells±CCCP stained with MitoTracker (MTR) Green and Deep Red (DR); Scale bar, 5 μm. (B) MTR DR/Green ratio from images as in A. N=3; ^*P<0.05 compared with NT siRNA. (C) Mitochondrial interconnectivity from images as in A, N=3. (D) Oxygen consumption in state 3 respiration in mitochondrial fractions from siRNA-transfected cells. Antimycin A with NT siRNA was used as a control. A.U., arbitrary unit; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non-targeting; PARL, presenilin-associated rhomboid-like protease; siRNA, short interfering RNA.

Figure 3

The levels of mature 52-kDa PINK1 are regulated by MPPβ, PARL, AFG3L2, ClpP and the proteasome. (A) Immunoblots of mitochondrial (M) and cytosolic (C) fractions from siRNA-transfected HEK293T cells±CCCP for 3.5 h, ±MG132 for the final hour. (B) Quantification of 52-kDa PINK1 from A (with MG132), n=3; ^*P<0.05; ^**P<0.001 compared with NT siRNA. (C) Mitochondria from siRNA-transfected cells treated with 10 μM CCCP or dimethylsulphoxide were incubated with or without respiration substrate (Resp. mix) for 30 min at 37°C. CCCP, carbonyl cyanide m-chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non-targeting; PARL, presenilin-associated rhomboid-like protease; PINK1, phosphatase and tensin homologue-induced kinase 1; siRNA, short interfering RNA.

Figure 4

Knockdown of MPPβ leads to full-length PINK1 accumulation at the outer mitochondrial membrane. (A) Mitochondria from CCCP-treated or siRNA-transfected HEK293T cells were incubated with increasing concentrations of proteinase K (PK). (B) Mitochondria from CCCP-treated or MPPβ knockdown cells were incubated ±0.1 M Na₂CO₃ before supernatant (S) and pellet (P) were separated by centrifugation. CCCP, carbonyl cyanide m-chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non-targeting; PARL, presenilin-associated rhomboid-like protease; PINK1, phosphatase and tensin homologue-induced kinase 1; siRNA, short interfering RNA; WB, western blot.

Figure 5

MPPβ knockdown induces Parkin recruitment and mitophagy. (A) Live confocal micrographs of siRNA-, GFP-Parkin- and OCT-DsRed-transfected HEK293T cells ±CCCP; Scale bars, 20 μm (low magnification) and 5 μm (high magnification). (B) Percentage of transfected cells displaying a punctate mitochondrial pattern of GFP-Parkin distribution, n=3; ^*P<0.05; ^**P<0.01 compared with NT siRNA. (C) Immunoblots of endogenous Parkin in mitochondrial fractions from siRNA-transfected cells ±CCCP. (D) Mitochondrial mass measured by flow cytometry of MTR Green fluorescence in siRNA-transfected cells treated ±CCCP for 24 h, n=3. A.U., arbitrary unit; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; GFP, green fluorescent protein; MPP, mitochondrial processing peptidase; MTR, MitoTracker; NT, non-targeting; PARL, presenilin-associated rhomboid-like protease; PINK1, phosphatase and tensin homologue-induced kinase 1; siRNA, short interfering RNA.