As the proteasome has been found on mitochondria (22), it is possible that it has a more direct role in mitochondrial protein degradation together with Parkin. In this study, we determined the functions of mitophagy and proteasomal degradation in Parkin-dependent degradation of depolarized mitochondria and found that proteins in the outer mitochondrial membrane (OMM) and the intermembrane space can be degraded by the proteasome, whereas those in the inner mitochondrial membrane (IMM) and the mitochondrial matrix are degraded mainly by mitophagy in cultured fibroblasts. and its mutation causes autosomal recessive juvenile Parkinson disease (5). Recent studies have revealed that Parkin is usually important for mitochondrial quality control through degradation of damaged mitochondria. Narendra (6) first demonstrated that Parkin translocates from the cytosol to depolarized mitochondria and triggers elimination of these mitochondria by autophagy, which is known as mitophagy. VU 0361737 Targeting of Parkin to mitochondria requires PTEN-induced putative kinase 1 (Pink1),3 another Parkinson disease-associated gene product (7C14). Pink1 is an extremely unstable mitochondrial protein, but it is usually stabilized upon mitochondrial depolarization and subsequently recruits Parkin. Autophagy VU 0361737 is usually a membrane-mediated intracellular degradation process. A portion of cytoplasm is usually first enclosed by the double-membraned autophagosome, and the autophagosome then fuses with a lysosome to degrade the enclosed materials. Although autophagy has been thought to be mainly non-selective, recent studies have revealed that this autophagosomal membrane can recognize some specific proteins and organelles. Parkin-mediated autophagy of damaged mitochondria is one of the best examples of selective autophagy. However, the precise role of Parkin in the induction of mitophagy has not been fully elucidated. To date, several mitochondrial proteins, voltage-dependent anion channel 1 (VDAC1) (8), mitofusin (a mitochondrial pro-fusion factor) (11, 14, 15), Bcl-2 (16), and Drp1 (17), have been shown to be ubiquitinated by Parkin. Ubiquitination of VDAC1 may recruit the autophagy adaptor p62, which interacts with microtubule-associated protein light chain 3 (LC3) around the autophagosomal membrane (8); however, the requirement of p62 remains controversial (18C21). Ubiquitination of mitofusin may affect mitochondrial fission or fusion, which would facilitate mitophagy (11, 14, 15). Mitochondrial degradation by autophagy has been extensively studied, whereas the involvement of the proteasome in Parkin-mediated mitochondrial degradation is usually less clear. As the proteasome has been found on mitochondria (22), it is possible that it has a more direct role in mitochondrial protein degradation together with Parkin. In this study, we decided the functions of mitophagy and proteasomal degradation in Parkin-dependent degradation of depolarized mitochondria and found that proteins in the outer mitochondrial membrane (OMM) and Rabbit Polyclonal to TRXR2 the intermembrane space can be degraded by the proteasome, whereas those in the inner mitochondrial membrane (IMM) and the mitochondrial matrix are degraded mainly by mitophagy in cultured fibroblasts. Furthermore, we observed that Parkin induces rupture of the OMM, which is also dependent on the proteasome. These results VU 0361737 reveal the novel Parkin-proteasome pathway and also provide new insights into maintenance of mitochondrial morphology. EXPERIMENTAL PROCEDURES Plasmids HA epitope-tagged Parkin (12), enhanced green fluorescent protein (EGFP)-tagged Omp25 (23), and Su9-GFP (24) were subcloned into the pMXs-IP vector (25). Antibodies and Reagents Rabbit polyclonal antibodies against Tom70 (26), Tom40 (27), Tom20 (28), Tim23 (29), Tim17 (29), Tim44 (29), proteasome subunit 7 (30), and LC3 (31) have been previously described. We purchased mouse monoclonal antibodies against cytochrome (BD Biosciences), complex III (C-III) core I (Invitrogen), and -tubulin (DM 1A) (Sigma-Aldrich) and rabbit polyclonal antibodies against Tom20 (Santa Cruz Biotechnology). Alexa Fluor 488-conjugated anti-mouse IgG and Alexa Fluor 568-conjugated anti-rabbit IgG secondary antibodies were purchased from Invitrogen. Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG antibodies were purchased from Jackson ImmunoResearch Laboratories. Puromycin dehydrochloride, bafilomycin A1, actinomycin D, carbonyl cyanide Gold (Invitrogen). Samples were analyzed with a fluorescence microscope (IX81; Olympus) equipped with a charge-coupled device camera (ORCA ER; Hamamatsu Photonics). A 60 PlanAPO oil immersion lens (1.42 NA; Olympus) was used. Images were acquired using MetaMorph image analysis software version 22.214.171.124 (Molecular Devices). Western Blotting MEFs were washed with ice-cold PBS, harvested in cold PBS, and centrifuged at 3,000 rpm for 5 min. Cells were lysed in a lysis buffer (50 mm Tris-HCl, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Complete EDTA-free protease inhibitor; Roche Applied Science)). The lysate was clarified by centrifugation at 15,000 rpm for 5 min at 4 C and was mixed with 6 sample buffer. Samples were subsequently separated by SDS-PAGE and transferred to Immobilon-P transfer membrane (Millipore). Immunoblot analysis was performed with the indicated antibodies and visualized with Immobilon Western (Millipore). The signal intensities were analyzed using an imaging analyzer (LAS-3000mini; Fujifilm) and Multi Gauge software (version 3.0; Fujifilm). Contrast and brightness adjustment was applied using Photoshop 7.0.1 (Adobe). Electron Microscopy MEFs were cultured on collagen-coated plastic coverslips. They were fixed in 2.5% glutaraldehyde in 0.1 m sodium phosphate buffer, pH 7.4 (phosphate buffer) for 2 h. The cells were washed.