Parkin mediates proteasome-dependent protein degradation and rupture of the outer mitochondrial membrane

Abstract

Upon mitochondrial depolarization, Parkin, a Parkinson disease-related E3 ubiquitin ligase, translocates from the cytosol to mitochondria and promotes their degradation by mitophagy, a selective type of autophagy. Here, we report that in addition to mitophagy, Parkin mediates proteasome-dependent degradation of outer membrane proteins such as Tom20, Tom40, Tom70, and Omp25 of depolarized mitochondria. By contrast, degradation of the inner membrane and matrix proteins largely depends on mitophagy. Furthermore, Parkin induces rupture of the outer membrane of depolarized mitochondria, which also depends on proteasomal activity. Upon induction of mitochondrial depolarization, proteasomes are recruited to mitochondria in the perinuclear region. Neither proteasome-dependent degradation of outer membrane proteins nor outer membrane rupture is required for mitophagy. These results suggest that Parkin regulates degradation of outer and inner mitochondrial membrane proteins differently through proteasome- and mitophagy-dependent pathways.

FIGURE 1.

OMM proteins are degraded mainly in an autophagy-independent manner in depolarized mitochondria. A and B, wild-type (WT) MEFs with and without stable Parkin expression (A) and FIP200 KO and Atg5 KO MEFs with Parkin expression (B) were treated with 20 μm CCCP for different time periods as indicated. Cells were immunostained for C-III core 1 (an IMM/matrix protein) and Tom20 (an OMM protein). Signal color is indicated by the colored text. Scale bars, 10 μm. C, wild-type MEFs (with and without exogenous Parkin expression) and FIP200 KO and Atg5 KO MEFs (with exogenous Parkin expression) expressing either Su9-GFP (a matrix protein) or GFP-Omp25 (an OMM protein) were treated with 20 μm CCCP for different time periods as indicated. The GFP fluorescence was quantified by flow cytometry. Data represent mean ± S.E. of three independent experiments. D, wild-type MEFs (with and without exogenous Parkin expression) and FIP200 KO MEFs (with exogenous Parkin expression) were treated with 20 μm CCCP for different time periods as indicated. The cells were analyzed by SDS-PAGE and subsequent immunoblotting with antibodies against Tom40 and Tom20 (OMM proteins), cytochrome c (CytC) (an intermembrane space (IMS) protein), Tim23 and Tim17 (IMM proteins), and C-III core 1 and Tim44 (IMM/matrix proteins), as well as α-tubulin (a loading control). Asterisks indicate nonspecific immunoreactive bands.

FIGURE 2.

CCCP induces OMM rupture in a Parkin-dependent and autophagy-independent manner. A, WT (panels a–f) and FIP200 KO (panels g–l) MEFs stably expressing Parkin were incubated for 0 h (panels a, b, g, and h), 6 h (panels c, d, i, and j), or 12 h (panels e, f, k, and l) with 20 μm CCCP. These cells were fixed and subjected to conventional electron microscopy analysis. Rectangles show enlarged areas in panels b, d, f, h, j, and l. Stars indicate mitochondria with ruptured OMM. Double stars indicate mitochondria with ruptured OMM and IMM. Mitochondrial clusters are enclosed by isolation membranes (white arrowheads) (panels c, d, e, and f). Two ruptured mitochondria are enclosed by a single isolation membrane (panel f). Arrows indicate residual OMM (panel l). Scale bars, 1 μm (panels a, c, e, g, i, and k) and 500 nm (panels b, d, f, h, j, and l). B, wild-type (panel a) and FIP200 KO (panels b and c) MEFs stably expressing Parkin were incubated for 24 h with 20 μm CCCP and subjected to electron microscopy. Black arrowheads indicate autophagosomes without mitochondria. After 24 h of CCCP treatment, almost all mitochondria disappeared in wild-type cells, but ruptured mitochondria (indicated by stars) accumulated in FIP200 KO cells. Arrows indicate residual OMM (panel c). Scale bars, 1 μm (panels a and b) and 500 nm (panel c). C, wild-type MEFs stably expressing Parkin were incubated with 20 μm CCCP for different time periods as indicated. The number of ruptured and unruptured mitochondria (mt) in the cytoplasm or inside autophagosomes was counted (per mm²) from at least eight randomly selected cells. IM, isolation membranes; AP, autophagosomes.

FIGURE 3.

OMM protein degradation is inhibited by proteasome inhibition. A, WT and FIP200 KO MEFs (with and without exogenous Parkin expression) were treated with 20 μm CCCP for 12 h in the presence or absence of 0.2 μm bafilomycin A₁ (Baf) or 5 μm lactacystin (Lac). Cells were immunostained for C-III core 1 and Tom20. Signal color is indicated by the colored text. Scale bars, 10 μm. B, degradation of GFP-Omp25 and Su9-GFP was analyzed by flow cytometry as in Fig. 1C. Wild-type and autophagy-deficient (FIP200 KO and Atg5 KO) MEFs were treated with 20 μm CCCP for 12 h in the presence or absence of 0.2 μm bafilomycin A₁ or 5 μm lactacystin. Data represent mean ± S.E. of three independent experiments. C, wild-type and FIP200 KO MEFs (with and without exogenous Parkin expression) were treated with 20 μm CCCP in the presence or absence of 0.2 μm bafilomycin A₁ or 5 μm lactacystin for 12 h. The cells were analyzed by immunoblotting using different antibodies as indicated. IMS, intermembrane space; CytC, cytochrome c.

FIGURE 4.

Proteasome inhibition suppresses OMM rupture but not mitophagy. A, wild-type (panels a–g) and FIP200 KO (panels h–k) MEFs stably expressing Parkin were incubated with 20 μm CCCP for 12 h with (panels c–g, j, and k) or without (panels a, b, h, and i) 5 μm lactacystin (Lac) and then subjected to electron microscopy. Rectangles show enlarged areas in panels b, d, i, and k. Autophagosomes without mitochondria (black arrowheads) and with mitochondria (white arrowheads) are shown. Single stars indicate mitochondria with ruptured OMM (panels b and i). In panel g, double stars indicate a degrading mitochondrion inside an autophagic vacuole. In panels b and i, arrows indicate residual OMM. Scale bars, 1 μm (panels a, c, h, and j) and 500 nm (panels b, d, e–g, i, and k). B, the ratio of ruptured and unruptured mitochondria (mt) in isolation membrane (IM)/autophagosome (AP)-like structures or in the cytoplasm was calculated from at least eight randomly selected cells. Lac, lactacystin. C, the number of IM/AP-like structures with (white columns) and without (black columns) mitochondria was counted from at least eight randomly selected cells. For bafilomycin A₁ (Baf) treatment, cells were incubated with 20 μm CCCP and 0.2 μm bafilomycin A₁ for 12 h. Selected images of wild-type cells are shown in supplemental Fig. S4.

FIGURE 5.

Proteasomes are recruited to depolarized mitochondria. A, wild-type and FIP200 KO MEFs stably expressing Parkin were treated with 20 μm CCCP for different periods of time as indicated in the presence or absence of 5 μm lactacystin (Lac). The cells were immunostained for C-III core 1 and α7 subunit of the proteasome. Signal color is indicated by the colored text. Scale bars, 10 μm. B, wild-type MEFs stably expressing Parkin were treated with 20 μm CCCP for 12 h and subjected to immunoelectron microscopy using anti-proteasomal α7 subunit antibody. Scale bars, 1 μm (panel a) and 500 nm (panel b).

FIGURE 6.

Proposed model of mitochondrial protein degradation and OMM rupture. Reduction in mitochondrial membrane potential (ΔΨm) causes fragmentation of mitochondria. Parkin is recruited to the OMM, resulting in clustering of mitochondria at the perinuclear region. Some OMM proteins (X) are ubiquitinated (Ub) directly or indirectly by Parkin and then degraded by the proteasome, which may eventually cause OMM rupture. Both unruptured and ruptured mitochondria are enclosed by autophagosomes and degraded by mitophagy. The step blocked by lactacystin is indicated.