A dimeric PINK1-containing complex on depolarized mitochondria stimulates Parkin recruitment

Abstract

Parkinsonism typified by sporadic Parkinson disease is a prevalent neurodegenerative disease. Mutations in PINK1 (PTEN-induced putative kinase 1), a mitochondrial Ser/Thr protein kinase, or PARKIN, a ubiquitin-protein ligase, cause familial parkinsonism. The accumulation and autophosphorylation of PINK1 on damaged mitochondria results in the recruitment of Parkin, which ultimately triggers quarantine and/or degradation of the damaged mitochondria by the proteasome and autophagy. However, the molecular mechanism of PINK1 in dissipation of the mitochondrial membrane potential (ΔΨm) has not been fully elucidated. Here we show by fluorescence-based techniques that the PINK1 complex formed following a decrease in ΔΨm is composed of two PINK1 molecules and is correlated with intermolecular phosphorylation of PINK1. Disruption of complex formation by the PINK1 S402A mutation weakened Parkin recruitment onto depolarized mitochondria. The most disease-relevant mutations of PINK1 inhibit the complex formation. Taken together, these results suggest that formation of the complex containing dyadic PINK1 is an important step for Parkin recruitment onto damaged mitochondria.

Keywords: Mitochondria; Parkin; Parkinson Disease; Phosphorylation; Pink1.

FIGURE 1.

A, exogenous PINK1 forms a supermolecular complex following CCCP treatment. HeLa cells expressing non-tagged PINK1 were treated with CCCP (10 μm, 1 h), and the resulting cell lysates were subjected to BN-PAGE and immunoblotted with an anti-PINK1 antibody. The blue arrowhead indicates the supermolecular PINK1 complex (note that this designation will be used hereafter unless otherwise specified). B, mitochondria-rich fractions isolated from cells treated as before were analyzed by BN-PAGE and immunoblotted. C, endogenous PINK1 also forms a supermolecular complex following CCCP treatment. HeLa cells were treated with CCCP (10 μm, 12 h) and subjected to BN-PAGE and immunoblotted. D, the presence of the PINK1 complex following a decrease in ΔΨm was detected using an anti-PINK1 antibody or GFP fluorescence. HeLa cells expressing PINK1-GFP were treated with CCCP (10 μm, 3 h) and subjected to BN-PAGE or CN-PAGE. PINK1 was detected using an anti-PINK1 antibody following blotting to a PVDF membrane (left and middle) or was detected directly in gel by GFP fluorescence using a fluorescence imaging scanner (right). E, dual color imaging of PINK1 using distinct florescent proteins. HeLa cells expressing PINK1-GFP and/or PINK1-mCherry were treated with CCCP (10 μm, 3 h) and subjected to CN-PAGE, and fluorescence signals derived from GFP and mCherry were detected.

FIGURE 2.

Two PINK1 molecules comprise the PINK1 complex on depolarized mitochondria. A, cell extracts from HeLa cells expressing PINK1-GFP, which had been treated with CCCP (10 μm, 3 h), were incubated with an anti-PINK1 (BC100–494) or anti-GFP (A6455, 3E6, or ab6556) antibody and then subjected to CN-PAGE. The red arrowheads and lines indicate a shift in the PINK1 complex following interaction with the antibody. B, a NAMOS assay was used to identify components of the PINK1 complex. HeLa cells expressing PINK1-GFP were treated with CCCP (10 μm, 3 h), and the cellular extracts were incubated with anti-Tom20, anti-Tom22, anti-Tom40, or anti-Tom70 antibodies and then subjected to CN-PAGE with PINK1-GFP fluorescence detected as before. C, a NAMOS assay was used to determine the stoichiometry of PINK1 in the complex. HeLa cells expressing PINK1-GFP and/or PINK1-mCherry treated with CCCP (10 μm, 3 h) were incubated with anti-GFP and/or anti-mCherry antibodies and subjected to CN-PAGE, and GFP or mCherry fluorescence was detected as before. The green asterisks indicate a PINK1-GFP shift caused by interaction between the anti-mCherry antibody and PINK1-mCherry. The red asterisks indicate a PINK1-mCherry shift caused by the anti-GFP antibody. D, HeLa cells expressing PINK1-GFP, PINK1-mCherry, and/or PINK1-V5 treated with CCCP (10 μm, 3 h). These cell extracts were incubated with anti-mCherry, anti-V5, and/or anti-GFP antibodies, and GFP fluorescence was detected as before.

FIGURE 3.

The PINK1 supermolecular complex is composed of phosphorylated PINK1. A–D, HeLa cells expressing non-tagged PINK1 were treated with CCCP (10 μm, 1 h), and lysates were subjected to Phos-tag SDS-PAGE (A and B) or two-dimensional electrophoresis consisting of BN-PAGE and Phos-tag SDS-PAGE (C and D). The same sample as that in Fig. 1A was used. Total cell lysates were electrophoresed (shown in the leftmost panel) as controls in C and D. Red arrowheads, phosphorylated PINK1 (pPINK1); black arrowheads, non-phosphorylated full-length PINK1 (FL) or the cleaved form (Δ1).

FIGURE 4.

Most of the pathogenic PINK1 mutants prevent formation of the 850-kDa complex. A, HeLa cells expressing PINK1-GFP with KD or various pathogenic mutations were treated with CCCP (10 μm, 3 h) and subjected to CN-PAGE with GFP fluorescence detected as before. The blue arrowheads indicate a PINK1 complex similar to the complex containing WT PINK1-GFP. B, the same samples were subjected to conventional PAGE and immunoblotted using an anti-PINK1 antibody. The black arrowheads indicate the position of the full-length (FL) or cleaved form (Δ1) of PINK1. C, components of the TOM machinery are included in mutant PINK1 (C92F, G309D, and G409V)-containing complexes.

FIGURE 5.

A, the proposed mechanisms described are illustrated. A reduction in ΔΨm results in the accumulation of PINK1 and cross-phosphorylation of the two PINK1 molecules. Reestablishment of ΔΨm following CCCP washout results in the reimport and degradation of PINK1. B, HeLa cells expressing PINK1-FLAG WT, KD, or various pathogenic mutants were treated with CCCP (10 μm, 1 h) and fractionated. Mitochondria-rich fractions were incubated with 50 μg/μl proteinase K (PK) and subjected to SDS-PAGE using precast 4–12% BisTris gels and immunoblotted using an anti-PINK1, anti-Tom20, anti-Tom40, anti-Tim23, or anti-HSP60 antibody. C, HeLa cells expressing PINK1-FLAG WT, KD, or pathogenic mutants were treated with CCCP (10 μm, 3 h). The CCCP was subsequently washed out for 1 h, and SDS-PAGE was performed using precast 4–12% BisTris gels and then immunoblotted using anti-PINK1 and anti-actin antibodies.

FIGURE 6.

Formation of the PINK1 complex is correlated with the intermolecular phosphorylation of PINK1. HeLa cells expressing the indicated PINK1 mutants were treated with CCCP (10 μm, 1 h), subjected to ±Phos-tag SDS-PAGE using hand-made 7.5% Tris-glycine gels, and immunoblotted using either an anti-PINK1 or anti-FLAG antibody. The red and blue arrowheads indicate phosphorylated PINK1-FLAG (see “Results”). FL, pFL, and Δ1, the full-length, phosphorylated full-length, and cleaved forms of PINK1, respectively.

FIGURE 7.

PINK1 Ser-402 is essential for PINK1 complex formation and its dysfunction specifically affects Parkin recruitment onto depolarized mitochondria. A, HeLa cells expressing PINK1-GFP WT, S228A, or S402A mutants were treated with CCCP (10 μm, 3 h) and subjected to CN-PAGE, and GFP fluorescence was detected as before. B, HeLa cells expressing non-tagged PINK1 WT, S228A, or S402A mutants were treated with CCCP (10 μm, 3 h), subjected to SDS-PAGE using 7.5% Tris-glycine gels with or without Phos-tag, and then immunoblotted using an anti-PINK1 antibody. C, HeLa cells stably expressing GFP-Parkin were pretreated with PINK1 siRNA and then transfected with CMV d1 promoter-driven PINK1 WT or a control vector. DsRed was used as transfection marker. After 42 h, these cells were treated with CCCP (10 μm, 1.5 h) and subjected to microscopic observation. Bar, 20 μm. D, HeLa cells stably expressing GFP-Parkin were pretreated with PINK1 siRNA and transfected with CMV d1 promoter-driven PINK1 WT, S228A, S402A, or a control vector, and then the number of cells with Parkin-positive mitochondria was determined in >100 transfected (DsRed-positive) cells. Bars, mean ± S.D. (error bars) of three experiments. Statistical significance was calculated using Student's t test. NS, not significant. E, PINK1 knockdown HeLa cells stably expressing HA-Parkin were transfected with CMV d1 promoter-driven PINK1 WT, S228A, and S402A. Cells were treated with CCCP for 16 h, and then the number of Tom20-immunoreactive cells was determined using >100 transfected (GFP-positive) cells. Bars, mean ± S.D. values of three experiments. Statistical significance was calculated using a Student's t test. NS, not significant. F, representative images from E demonstrating the Tom20 signal loss. Bar, 10 μm.