Broad activation of the ubiquitin-proteasome system by Parkin is critical for mitophagy

Abstract

Parkin, an E3 ubiquitin ligase implicated in Parkinson's disease, promotes degradation of dysfunctional mitochondria by autophagy. Using proteomic and cellular approaches, we show that upon translocation to mitochondria, Parkin activates the ubiquitin-proteasome system (UPS) for widespread degradation of outer membrane proteins. This is evidenced by an increase in K48-linked polyubiquitin on mitochondria, recruitment of the 26S proteasome and rapid degradation of multiple outer membrane proteins. The degradation of proteins by the UPS occurs independently of the autophagy pathway, and inhibition of the 26S proteasome completely abrogates Parkin-mediated mitophagy in HeLa, SH-SY5Y and mouse cells. Although the mitofusins Mfn1 and Mfn2 are rapid degradation targets of Parkin, we find that degradation of additional targets is essential for mitophagy. These results indicate that remodeling of the mitochondrial outer membrane proteome is important for mitophagy, and reveal a causal link between the UPS and autophagy, the major pathways for degradation of intracellular substrates.

Figure 1.

Parkin mediates extensive proteolysis of outer membrane proteins via the UPS. (A) Parkin- and CCCP-dependent proteolysis of mitochondrial outer membrane proteins. HeLa S3 cells or clonal Parkin-expressing HeLa S3 cells were treated with vehicle or 20 μm CCCP to dissipate the mitochondrial membrane potential. Total cell lysates at the indicated time points were analyzed by immunoblotting for the indicated proteins. Outer membrane proteins: Mfn1, Mfn2, Tom70, VDAC1, Bak, Fis1, Tom20. Intermembrane space protein: cytochrome c. Inner membrane protein: Opa1. Matrix proteins: Hsp60, Sod2, F1β. Loading control: actin. (B) Inhibition of outer membrane protein degradation by the proteasome inhibitor MG132. Immunoblot analysis of Mfn1, Mfn2, VDAC1, Tom20 and actin (loading control) levels after CCCP with or without treatment with the proteasome inhibitor MG132 (10 μm). (C) Inhibition of outer membrane protein degradation by the proteasome inhibitor epoxomicin. Same as (B), except epoxomicin (2 μm), a more specific proteasome inhibitor, was used.

Figure 2.

Proteolysis of the outer membrane occurs independently of the autophagy pathway. (A) CCCP-dependent degradation of mitochondrial outer membrane proteins in Atg3-null MEFs expressing Parkin. Atg3-null MEFs expressing EGFP-Parkin were treated with dimethyl sulfoxide (DMSO) (vehicle) or 20 μm CCCP for the indicated time. Total cell lysates were isolated and immunoblotted against the indicated proteins. (B) Inhibition of outer membrane protein degradation by the proteasome inhibitor epoxomicin. Atg3-null MEFs expressing EGFP-Parkin were treated with DMSO (vehicle) or the proteasome inhibitor epoxomicin (2 μm) for 2 h prior to treatment with 20 μm CCCP for the indicated times. Total cell lysates were isolated and immunoblotted against the indicated proteins.

Figure 3.

Parkin activation results in mitochondrial K48-linked and K63-linked polyubiquitination and proteasome recruitment. (A) The SILAC ratio for K48-linked and K63-linked polyubiquitination obtained from mass spectrometric analysis of mitochondria isolated after 2 h of CCCP treatment. Both modifications yield unique diglycine signature peptides that can be quantified (41). Mitochondria were isolated under conditions where the activity of the 26S proteasome was not inhibited. (B) Accumulation of K48- and K63-linked polyubiquitinated proteins in mitochondria of CCCP-treated cells. Immunoblot analysis of mitochondria isolated from HeLa S3 or Parkin-expressing HeLa S3 cells, with or without CCCP treatment. Blots were probed with the following antibodies: anti-ubiquitin, anti-K48-linked polyubiquitin, anti-K63-linked polyubiquitin and anti-F₁β (loading control). Purified polyubiquitin chains of the K48-linked or K63-linked type were used as controls to verify the specificity of the antibodies used. Cells were pretreated with the proteasome inhibitor MG132 (10 μm) together with CCCP, and mitochondria were isolated in the presence of N-ethylmaleimide (10 mm) to prevent deubiquitination. (C) Analysis of proteasome localization. HeLa cells expressing Parkin were treated with DMSO (vehicle) or 20 μm CCCP for 4 h. Formalin-fixed cells were stained for the β subunit PSMB5 of the proteasome (green), Hsp60 (red) and nuclei [4′,6-diamidino-2-phenylindole (DAPI), blue]. (D) Same as (C), except cells were immunostained for PSMA2 (green), an α subunit of the proteasome.

Figure 4.

Degradation of Tom20 occurs prior to mitophagy and does not require the autophagy pathway. (A) Degradation of Tom20 induced by CCCP treatment. After 4 and 24 h of CCCP (20 μm) or vehicle treatment, HeLa cells expressing Parkin were stained for Hsp60 (green), Tom20 (red) and nuclei (DAPI, blue). In the second row, the insets show enlarged views of the boxed area. Arrowheads mark examples of dispersed mitochondria that are positive for Hsp60 but negative for Tom20. In the fourth row, the asterisk indicates a cell with Tom20-negative/Hsp60-positive mitochondria. (B) Loss of Tom20 in both dispersed mitochondria and within patches of the mitochondrial aggregate. HeLa cells expressing Parkin were treated with CCCP for 4 h and stained for Hsp60 (green), Tom20 (red) and nuclei (DAPI, blue). The lower three panels correspond to the boxed area in the top panel. The filled arrowhead marks a patch in the perinuclear mitochondrial aggregate that is positive for Hsp60, but negative for Tom20. Unfilled arrowheads mark dispersed mitochondria that are Tom20 negative/Hsp60 positive. (C) Quantitation of the 24 h time point in (A). Cells that were Tom20 negative were scored for Hsp60 immunoreactivity. Error bars indicate standard deviations from three independent experiments; 100 cells were scored per experiment. (D) Tom20 degradation in Atg3-null MEFs. Wild-type and Atg3-null MEFs expressing EGFP-Parkin were treated with 20 μm CCCP for 24 h and immunostained for Tom20 or Hsp60. Cells were scored for complete loss of Tom20 or Hsp60. Representative images of Atg3-null cells are shown on the right. The left image shows an example of a Tom20-negative cell (asterisk). No Hsp60-negative cells were ever found (right image). Error bars represent standard deviations from three independent experiments; 100 cells were analyzed per experiment. The P-value was calculated using the t-test. All scale bars are 10 microns.

Figure 5.

Activation of the ubiquitin–proteasome pathway is essential for mitophagy. (A) Co-localization of dispersed mitochondria and LC3B in CCCP-treated cells. HeLa cells expressing Parkin and EGFP-LC3B were treated with 100 nm bafilomycin A1 and CCCP for 4 h, and stained for Hsp60 (red), EGFP-LC3B (green) and nuclei (DAPI, blue). Enlarged views of the boxed area are shown in the right column. Filled arrowheads mark examples of co-localization between dispersed mitochondria and EGFP-LC3B. The unfilled arrowhead marks an example of a dispersed mitochondrion that does not co-localize with EGFP-LC3B. Quantitation of this experiment is shown in the graph below. Error bars represent standard deviations from three independent experiments. Twenty cells were analyzed for each replicate, and ∼2400 dispersed mitochondria were manually assessed in total. In (B)–(D), Parkin-expressing HeLa cells were treated with CCCP in the presence or absence the proteasome inhibitor MG132 (10 μm). Cells were immunostained for Hsp60 (green), Tom20 (red) and nuclei (DAPI, blue). (B) MG132 inhibits Tom20 loss after 4 h of persistent CCCP treatment. Inset shows an enlarged view of the boxed area, highlighting the presence of dispersed, Tom20-negative mitochondria when MG132 is not present. The graph on the right shows quantification of this experiment. Each cell was scored into one of the five indicated bins, depending on the number of dispersed mitochondria that are Tom20 negative, but Hsp60 positive. Error bars indicate standard deviations from three independent experiments; 100 cells were scored per experiment. (C) MG132 preserves mitochondrial morphology at 12 h after a 100 min pulse treatment with CCCP. The graph on the right shows quantification of mitochondrial morphology. Cells were scored as having tubular mitochondria, fragmented mitochondria or no mitochondria. Error bars indicate standard deviations from three independent experiments; 500 cells were scored per experiment. (D) MG132 or epoxomicin abrogates CCCP-induced mitophagy. The images show cells 24 h after a 100 min pulse treatment with CCCP in the presence or absence of MG132 (10 μm). The graph on the right shows quantification of this experiment and a related one with epoxomicin (2 μm). Cells without mitochondria were defined by the complete lack of both Tom20 and Hsp60 signal. Error bars indicate standard deviations from three independent experiments. 1000 cells were scored for each MG132 experiment, and 200 cells were scored for each epoxomicin experiments. Scale bars equal 10 μm for (A)–(D). (E) Degradation of Tom20 in human neuroblastoma SH-SY5Y cells expressing exogenous Parkin. Cells were treated with DMSO (vehicle) or CCCP (20 μm) for 4 h. Cells were scored into one of the indicated five bins, depending on the number of dispersed mitochondria lacking Tom20, as described for (B). Error bars indicate standard deviations in three independent experiments; 100 cells were scored per experiment. (F) Epoxomicin abrogates CCCP-induced mitophagy in SH-SY5Y cells expressing Parkin. Cells were treated as in (D) in the presence or absence of epoxomicin (2 μm) and stained for Hsp60 and nuclei (DAPI). Cells without mitochondria were identified by complete loss of Hsp60 signal around the DAPI-stained nucleus. Error bars represent standard deviations from three independent experiments; 200 cells were analyzed per experiment.

Figure 6.

Parkin-mediated mitophagy in Mfn-null cells is blocked by the proteasome inhibitor epoxomicin. EGFP-Parkin was expressed in wild-type (WT) and Mfn1/Mfn2-null MEFs containing matrix-targeted TagRFP-T. Cultures were treated with the indicated drugs, and EGFP-positive cells were scored for the presence of mitochondria. Error bars indicate standard deviations from three experiments; 200 cells were scored per experiment. The P-values were calculated using the t-test.