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 mitophagydependent pathways.Parkinson disease is a progressive neurodegenerative disease that is characterized by postural changes, resting tremor, muscle rigidity, and weakness (1, 2). These symptoms are mainly caused by loss of dopaminergic neurons in the substantia nigra, and mitochondrial dysfunction appears to be the primary pathogenic event. Indeed, many of the products of Parkinson disease-related genes such as ␣-synuclein/PARK1/4, PARKIN/ PARK2, PINK1/PARK6, DJ-I/PARK7, and OMI/HTRA2 are physically and functionally linked to mitochondria (3,4).Parkin is a RING domain-containing E3 ubiquitin ligase, and its mutation causes autosomal recessive juvenile Parkinson disease (5). Recent studies have revealed that Parkin is important for mitochondrial quality control through degradation of damaged mitochondria. Narendra et al. (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. Targeting of Parkin to mitochondria requires PTEN-induced putative kinase 1 (Pink1), 3 another Parkinson disease-associated gene product (7-14). Pink1 is an extremely unstable mitochondrial protein, but it is stabilized upon mitochondrial depolarization and subsequently recruits Parkin.Autophagy is a membrane-mediated intracellular degradation process. A portion of cytoplasm is 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 the 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)...

The pathways of mitophagy for quality control and clearance of mitochondria

“…17 Although clearly essential for forming the defining doublemembrane vesicle of the autophagosome, the origin of the membrane component remains controversial. 26 De novo membrane assembly has been proposed; however, various studies have implicated the ER, 26,27 Golgi, 26 plasma membrane, 28 and more recently mitochondria 29 as possible membrane sources for the autophagosome. Yoshi 27 and co-workers observed close association of the isolation membrane with rough ER during mitophagy, implicating ER in particular as a membrane source for mitophagy.…”

Depletion of mitochondria in mammalian cells through enforced mitophagy

“…Importantly, Parkin has been shown to target some mitochondrial proteins for proteasomal degradation independently of mitophagy 37,38 . Therefore, multiple approaches should be used, at least in initial experiments, to verify the method is functional.…”

Peroxisomal protein PEX 13 functions in selective autophagy

“…At an early time point during mitophagy, we observed Parkin colocalization with TOMM20 even after PEX13 knockdown, suggesting that Parkin recruitment to the mitochondria is similar in PEX13‐deficient cells and control cells (Fig EV2B). We confirmed the role of PEX13 in mitophagy using a combination of more selective inhibitors of mitochondrial respiration, oligomycin, and antimycin A (OA) (as CCCP may have direct effects on lysosomal function 19) and by measuring the clearance of mitochondrial double‐stranded DNA (mtDNA) (as the proteasomal system can contribute to the degradation of mitochondrial outer membrane proteins such as TOMM20 but not to mtDNA 20). Our results indicate that four different siRNAs targeting PEX13 block OA‐induced mtDNA clearance as effectively as ATG7 siRNA (Fig 2C and D).…”

“…However, we did observe abnormal mitochondria with disorganized cristae in basal conditions in Pex13 − / − MEFs using electron microscopy (Fig EV2C). In MEFs, endogenous Parkin expression is negligible 20 and Parkin overexpression in primary MEFs does not promote the complete clearance of TOMM20 during CCCP or OA‐induced mitophagy as it does in HeLa cells. Instead, damaged mitochondria in primary MEFs undergo Parkin‐independent partial clearance and compaction around the perinuclear region 16.…”

“…pMXs‐IP‐HA‐Parkin 20 (Addgene #38248) was cotransfected with the helper plasmids pUMVC and pCMV‐VSV‐G 42 (Addgene #8849 and #8454) into HEK293T cells. PEX13 cDNAs containing WT, W313G mutation, and I326T mutation were cloned into pLenti‐C‐Myc‐DDK‐IRES‐Neo vector (Origene), then cotransfected into HEK293 cells with the helper plasmids pCMVΔR8.91 43 and pMDG 44.…”