Nitric Oxide Induction of Parkin Translocation in PTEN-induced Putative Kinase 1 (PINK1) Deficiency: FUNCTIONAL ROLE OF NEURONAL NITRIC OXIDE SYNTHASE DURING MITOPHAGY*

The failure to trigger mitophagy is implicated in the pathogenesis of familial Parkinson disease that is caused by PINK1 or Parkin mutations. According to the prevailing PINK1-Parkin signaling model, mitophagy is promoted by the mitochondrial translocation of Parkin, an essential PINK1-dependent step that occurs via a previously unknown mechanism. Here we determined that critical concentrations of NO was sufficient to induce the mitochondrial translocation of Parkin even in PINK1 deficiency, with apparent increased interaction of full-length PINK1 accumulated during mitophagy, with neuronal nitric oxide synthase (nNOS). Specifically, optimum levels of NO enabled PINK1-null dopaminergic neuronal cells to regain the mitochondrial translocation of Parkin, which appeared to be significantly suppressed by nNOS-null mutation. Moreover, nNOS-null mutation resulted in the same mitochondrial electron transport chain (ETC) enzyme deficits as PINK1-null mutation. The involvement of mitochondrial nNOS activation in mitophagy was further confirmed by the greatly increased interactions of full-length PINK1 with nNOS, accompanied by mitochondrial accumulation of phospho-nNOS (Ser¹⁴¹²) during mitophagy. Of great interest is that the L347P PINK1 mutant failed to bind to nNOS. The loss of nNOS phosphorylation and Parkin accumulation on PINK1-deficient mitochondria could be reversed in a PINK1-dependent manner. Finally, non-toxic levels of NO treatment aided in the recovery of PINK1-null dopaminergic neuronal cells from mitochondrial ETC enzyme deficits. In summary, we demonstrated the full-length PINK1-dependent recruitment of nNOS, its activation in the induction of Parkin translocation, and the feasibility of NO-based pharmacotherapy for defective mitophagy and ETC enzyme deficits in Parkinson disease.     

Short Mitochondrial ARF Triggers Parkin/PINK1-dependent Mitophagy

Parkinson disease (PD) is a complex neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra. Multiple genes have been associated with PD, including Parkin and PINK1. Recent studies have established that the Parkin and PINK1 proteins function in a common mitochondrial quality control pathway, whereby disruption of the mitochondrial membrane potential leads to PINK1 stabilization at the mitochondrial outer surface. PINK1 accumulation leads to Parkin recruitment from the cytosol, which in turn promotes the degradation of the damaged mitochondria by autophagy (mitophagy). Most studies characterizing PINK1/Parkin mitophagy have relied on high concentrations of chemical uncouplers to trigger mitochondrial depolarization, a stimulus that has been difficult to adapt to neuronal systems and one unlikely to faithfully model the mitochondrial damage that occurs in PD. Here, we report that the short mitochondrial isoform of ARF (smARF), previously identified as an alternate translation product of the tumor suppressor p19ARF, depolarizes mitochondria and promotes mitophagy in a Parkin/PINK1-dependent manner, both in cell lines and in neurons. The work positions smARF upstream of PINK1 and Parkin and demonstrates that mitophagy can be triggered by intrinsic signaling cascades.     

Role of Glucose Metabolism and ATP in Maintaining PINK1 Levels during Parkin-mediated Mitochondrial Damage Responses

Mutations in several genes, including PINK1 and Parkin, are known to cause autosomal recessive cases of Parkinson disease in humans. These genes operate in the same pathway and play a crucial role in mitochondrial dynamics and maintenance. PINK1 is required to recruit Parkin to mitochondria and initiate mitophagy upon mitochondrial depolarization. In this study, we show that PINK1-dependent Parkin mitochondrial recruitment in response to global mitochondrial damage by carbonyl cyanide m-chlorophenylhydrazine (CCCP) requires active glucose metabolism. Parkin accumulation on mitochondria and subsequent Parkin-dependent mitophagy is abrogated in glucose-free medium or in the presence of 2-deoxy-d-glucose upon CCCP treatment. The defects in Parkin recruitment correlate with intracellular ATP levels and can be attributed to suppression of PINK1 up-regulation in response to mitochondria depolarization. Low levels of ATP appear to prevent PINK1 translation instead of affecting PINK1 mRNA expression or reducing its stability. Consistent with a requirement of ATP for elevated PINK1 levels and Parkin mitochondrial recruitment, local or individual mitochondrial damage via photoirradiation does not affect Parkin recruitment to damaged mitochondria as long as a pool of functional mitochondria is present in the photoirradiated cells even in glucose-free or 2-deoxy-d-glucose-treated conditions. Thus, our data identify ATP as a key regulator for Parkin mitochondrial translocation and sustaining elevated PINK1 levels during mitophagy. PINK1 functions as an AND gate and a metabolic sensor coupling biogenetics of cells and stress signals to mitochondria dynamics.     

Interaction between RING1 (R1) and the Ubiquitin-like (UBL) Domains Is Critical for the Regulation of Parkin Activity

Parkin is an E3 ligase that contains a ubiquitin-like (UBL) domain in the N terminus and an R1-in-between-ring-RING2 motif in the C terminus. We showed that the UBL domain specifically interacts with the R1 domain and negatively regulates Parkin E3 ligase activity, Parkin-dependent mitophagy, and Parkin translocation to the mitochondria. The binding between the UBL domain and the R1 domain was suppressed by carbonyl cyanide m-chlorophenyl hydrazone treatment or by expression of PTEN-induced putative kinase 1 (PINK1), an upstream kinase that phosphorylates Parkin at the Ser-65 residue of the UBL domain. Moreover, we demonstrated that phosphorylation of the UBL domain at Ser-65 prevents its binding to the R1 domain and promotes Parkin activities. We further showed that mitochondrial translocation of Parkin, which depends on phosphorylation at Ser-65, and interaction between the R1 domain and a mitochondrial outer membrane protein, VDAC1, are suppressed by binding of the UBL domain to the R1 domain. Interestingly, Parkin with missense mutations associated with Parkinson disease (PD) in the UBL domain, such as K27N, R33Q, and A46P, did not translocate to the mitochondria and induce E3 ligase activity by m-chlorophenyl hydrazone treatment, which correlated with the interaction between the R1 domain and the UBL domain with those PD mutations. These findings provide a molecular mechanism of how Parkin recruitment to the mitochondria and Parkin activation as an E3 ubiquitin ligase are regulated by PINK1 and explain the previously unknown mechanism of how Parkin mutations in the UBL domain cause PD pathogenesis.     

Lysine 27 Ubiquitination of the Mitochondrial Transport Protein Miro Is Dependent on Serine 65 of the Parkin Ubiquitin Ligase

Mitochondrial transport plays an important role in matching mitochondrial distribution to localized energy production and calcium buffering requirements. Here, we demonstrate that Miro1, an outer mitochondrial membrane (OMM) protein crucial for the regulation of mitochondrial trafficking and distribution, is a substrate of the PINK1/Parkin mitochondrial quality control system in human dopaminergic neuroblastoma cells. Moreover, Miro1 turnover on damaged mitochondria is altered in Parkinson disease (PD) patient-derived fibroblasts containing a pathogenic mutation in the PARK2 gene (encoding Parkin). By analyzing the kinetics of Miro1 ubiquitination, we further demonstrate that mitochondrial damage triggers rapid (within minutes) and persistent Lys-27-type ubiquitination of Miro1 on the OMM, dependent on PINK1 and Parkin. Proteasomal degradation of Miro1 is then seen on a slower time scale, within 2–3 h of the onset of ubiquitination. We find Miro ubiquitination in dopaminergic neuroblastoma cells is independent of Miro1 phosphorylation at Ser-156 but is dependent on the recently identified Ser-65 residue within Parkin that is phosphorylated by PINK1. Interestingly, we find that Miro1 can stabilize phospho-mutant versions of Parkin on the OMM, suggesting that Miro is also part of a Parkin receptor complex. Moreover, we demonstrate that Ser-65 in Parkin is critical for regulating Miro levels upon mitochondrial damage in rodent cortical neurons. Our results provide new insights into the ubiquitination-dependent regulation of the Miro-mediated mitochondrial transport machinery by PINK1/Parkin and also suggest that disruption of this regulation may be implicated in Parkinson disease pathogenesis.     

PINK1/Parkin介导的线粒体自噬

线粒体自噬（mitochondrial autophagy, or mitophagy）指的是细胞通过自吞噬作用,降解与清除受损线粒体或者多余线粒体,其对整个线粒体网络的功能完整性和细胞存活具有重要作用。线粒体自噬过程受多种途径调控,PINK1/Parkin通路是其中的一条,其异常与多种疾病的发生密切相关,如心血管疾病、肿瘤和帕金森病等。在去极化线粒体中,磷酸酶及张力蛋白同源物(PTEN)诱导的激酶1（PTEN-induced kinase 1,PINK1）作为受损线粒体的分子传感器,触发线粒体自噬的起始信号,并将Parkin募集至线粒体;Parkin作为线粒体自噬信号的“增强子”,通过对线粒体蛋白质进一步泛素化介导自噬信号的扩大;去泛素化酶和PTEN-long蛋白参与调控该过程,并对维持线粒体稳态具有重要作用。本文主要对PINK1与Parkin蛋白质的分子结构和其介导线粒体自噬发生的分子机制,以及参与调控该途径的关键蛋白质进行综述,为进一步研究以线粒体自噬缺陷为特征的疾病治疗提供理论基础。     

Dual role of USP30 in controlling basal pexophagy and mitophagy

USP30 is an integral protein of the outer mitochondrial membrane that counteracts PINK1 and Parkin‐dependent mitophagy following acute mitochondrial depolarisation. Here, we use two distinct mitophagy reporter systems to reveal tonic suppression by USP30, of a PINK1‐dependent component of basal mitophagy in cells lacking detectable Parkin. We propose that USP30 acts upstream of PINK1 through modulation of PINK1‐substrate availability and thereby determines the potential for mitophagy initiation. We further show that a fraction of endogenous USP30 is independently targeted to peroxisomes where it regulates basal pexophagy in a PINK1‐ and Parkin‐independent manner. Thus, we reveal a critical role of USP30 in the clearance of the two major sources of ROS in mammalian cells and in the regulation of both a PINK1‐dependent and a PINK1‐independent selective autophagy pathway.     

PINK1 Is Dispensable for Mitochondrial Recruitment of Parkin and Activation of Mitophagy in Cardiac Myocytes

Myocyte function and survival relies on the maintenance of a healthy population of mitochondria. The PINK1/Parkin pathway plays an important role in clearing defective mitochondria via autophagy in cells. However, how the PINK1/Parkin pathway regulates mitochondrial quality control and whether it coordinates with other mitophagy pathways are still unclear. Therefore, the objective of this study was to investigate the effect of PINK1-deficiency on mitochondrial quality control in myocytes. Using PINK1-deficient (PINK1-/-) mice, we found that Parkin is recruited to damaged cardiac mitochondria in hearts after treatment with the mitochondrial uncoupler FCCP or after a myocardial infarction even in the absence of PINK1. Parkin recruitment to depolarized mitochondria correlates with increased ubiquitination of mitochondrial proteins and activation of mitophagy in PINK1-/- myocytes. In addition, induction of mitophagy by the atypical BH3-only protein BNIP3 is unaffected by lack of PINK1. Overall, these data suggest that Parkin recruitment to depolarized cardiac mitochondria and subsequent activation of mitophagy is independent of PINK1. Moreover, alternative mechanisms of Parkin activation and pathways of mitophagy remain functional in PINK1-/- myocytes and could compensate for the PINK1 deficiency.     

Parkin-catalyzed Ubiquitin-Ester Transfer Is Triggered by PINK1-dependent Phosphorylation

PINK1 and PARKIN are causal genes for autosomal recessive familial Parkinsonism. PINK1 is a mitochondrial Ser/Thr kinase, whereas Parkin functions as an E3 ubiquitin ligase. Under steady-state conditions, Parkin localizes to the cytoplasm where its E3 activity is repressed. A decrease in mitochondrial membrane potential triggers Parkin E3 activity and recruits it to depolarized mitochondria for ubiquitylation of mitochondrial substrates. The molecular basis for how the E3 activity of Parkin is re-established by mitochondrial damage has yet to be determined. Here we provide in vitro biochemical evidence for ubiquitin-thioester formation on Cys-431 of recombinant Parkin. We also report that Parkin forms a ubiquitin-ester following a decrease in mitochondrial membrane potential in cells, and that this event is essential for substrate ubiquitylation. Importantly, the Parkin RING2 domain acts as a transthiolation or acyl-transferring domain rather than an E2-recruiting domain. Furthermore, formation of the ubiquitin-ester depends on PINK1 phosphorylation of Parkin Ser-65. A phosphorylation-deficient mutation completely inhibited formation of the Parkin ubiquitin-ester intermediate, whereas phosphorylation mimics, such as Ser to Glu substitution, enabled partial formation of the intermediate irrespective of Ser-65 phosphorylation. We propose that PINK1-dependent phosphorylation of Parkin leads to the ubiquitin-ester transfer reaction of the RING2 domain, and that this is an essential step in Parkin activation.     

Cytosolic cleaved PINK1 represses Parkin translocation to mitochondria and mitophagy

PINK1 is a mitochondrial kinase proposed to have a role in the pathogenesis of Parkinson''s disease through the regulation of mitophagy. Here, we show that the PINK1 main cleavage product, PINK1_52, after being generated inside mitochondria, can exit these organelles and localize to the cytosol, where it is not only destined for degradation by the proteasome but binds to Parkin. The interaction of cytosolic PINK1 with Parkin represses Parkin translocation to the mitochondria and subsequent mitophagy. Our work therefore highlights the existence of two cellular pools of PINK1 that have different effects on Parkin translocation and mitophagy.     

A Ubl/ubiquitin switch in the activation of Parkin

Mutations in Parkin and PINK1 cause an inherited early‐onset form of Parkinson's disease. The two proteins function together in a mitochondrial quality control pathway whereby PINK1 accumulates on damaged mitochondria and activates Parkin to induce mitophagy. How PINK1 kinase activity releases the auto‐inhibited ubiquitin ligase activity of Parkin remains unclear. Here, we identify a binding switch between phospho‐ubiquitin (pUb) and the ubiquitin‐like domain (Ubl) of Parkin as a key element. By mutagenesis and SAXS, we show that pUb binds to RING1 of Parkin at a site formed by His302 and Arg305. pUb binding promotes disengagement of the Ubl from RING1 and subsequent Parkin phosphorylation. A crystal structure of Parkin Δ86–130 at 2.54 Å resolution allowed the design of mutations that specifically release the Ubl domain from RING1. These mutations mimic pUb binding and promote Parkin phosphorylation. Measurements of the E2 ubiquitin‐conjugating enzyme UbcH7 binding to Parkin and Parkin E3 ligase activity suggest that Parkin phosphorylation regulates E3 ligase activity downstream of pUb binding.     

PINK1-Mediated Phosphorylation of Parkin Boosts Parkin Activity in Drosophila

Two genes linked to early onset Parkinson''s disease, PINK1 and Parkin, encode a protein kinase and a ubiquitin-ligase, respectively. Both enzymes have been suggested to support mitochondrial quality control. We have reported that Parkin is phosphorylated at Ser65 within the ubiquitin-like domain by PINK1 in mammalian cultured cells. However, it remains unclear whether Parkin phosphorylation is involved in mitochondrial maintenance and activity of dopaminergic neurons in vivo. Here, we examined the effects of Parkin phosphorylation in Drosophila, in which the phosphorylation residue is conserved at Ser94. Morphological changes of mitochondria caused by the ectopic expression of wild-type Parkin in muscle tissue and brain dopaminergic neurons disappeared in the absence of PINK1. In contrast, phosphomimetic Parkin accelerated mitochondrial fragmentation or aggregation and the degradation of mitochondrial proteins regardless of PINK1 activity, suggesting that the phosphorylation of Parkin boosts its ubiquitin-ligase activity. A non-phosphorylated form of Parkin fully rescued the muscular mitochondrial degeneration due to the loss of PINK1 activity, whereas the introduction of the non-phosphorylated Parkin mutant in Parkin-null flies led to the emergence of abnormally fused mitochondria in the muscle tissue. Manipulating the Parkin phosphorylation status affected spontaneous dopamine release in the nerve terminals of dopaminergic neurons, the survivability of dopaminergic neurons and flight activity. Our data reveal that Parkin phosphorylation regulates not only mitochondrial function but also the neuronal activity of dopaminergic neurons in vivo, suggesting that the appropriate regulation of Parkin phosphorylation is important for muscular and dopaminergic functions.     

The ubiquitin signal and autophagy: an orchestrated dance leading to mitochondrial degradation

The quality of mitochondria, essential organelles that produce ATP and regulate numerous metabolic pathways, must be strictly monitored to maintain cell homeostasis. The loss of mitochondrial quality control systems is acknowledged as a determinant for many types of neurodegenerative diseases including Parkinson's disease (PD). The two gene products mutated in the autosomal recessive forms of familial early‐onset PD, Parkin and PINK1, have been identified as essential proteins in the clearance of damaged mitochondria via an autophagic pathway termed mitophagy. Recently, significant progress has been made in understanding how the mitochondrial serine/threonine kinase PINK1 and the E3 ligase Parkin work together through a novel stepwise cascade to identify and eliminate damaged mitochondria, a process that relies on the orchestrated crosstalk between ubiquitin/phosphorylation signaling and autophagy. In this review, we highlight our current understanding of the detailed molecular mechanisms governing Parkin‐/PINK1‐mediated mitophagy and the evidences connecting Parkin/PINK1 function and mitochondrial clearance in neurons.     

Cannabidiol activates PINK1-Parkin-dependent mitophagy and mitochondrial-derived vesicles

The PINK1/Parkin pathway plays an important role in maintaining a healthy pool of mitochondria. Activation of this pathway can lead to apoptosis, mitophagy, or mitochondrial-derived vesicle formation, depending on the nature of mitochondrial damage. The signaling by which PINK/Parkin activation leads to these different mitochondrial outcomes remains understudied. Here we present evidence that cannabidiol (CBD) activates the PINK1-Parkin pathway in a unique manner. CBD stimulates PINK1-dependent Parkin mitochondrial recruitment similarly to other well-studied Parkin activators but with a distinctive shift in the temporal dynamics and mitochondrial fates. The mitochondrial permeability transition pore inhibitor cyclosporine A exclusively diminished the CBD-induced PINK1/Parkin activation and its associated mitochondrial effects. Unexpectedly, CBD treatment also induced elevated production of mitochondrial-derived vesicles (MDV), a potential quality control mechanism that may help repair partial damaged mitochondria. Our results suggest that CBD may engage the PINK1-Parkin pathway to produce MDV and repair mitochondrial lesions via mitochondrial permeability transition pore opening. This work uncovered a novel link between CBD and PINK1/Parkin-dependent MDV production in mitochondrial health regulation.     

A Dimeric PINK1-containing Complex on Depolarized Mitochondria Stimulates Parkin Recruitment

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.     

The Complex I Subunit NDUFA10 Selectively Rescues Drosophila pink1 Mutants through a Mechanism Independent of Mitophagy

Mutations in PINK1, a mitochondrially targeted serine/threonine kinase, cause autosomal recessive Parkinson''s disease (PD). Substantial evidence indicates that PINK1 acts with another PD gene, parkin, to regulate mitochondrial morphology and mitophagy. However, loss of PINK1 also causes complex I (CI) deficiency, and has recently been suggested to regulate CI through phosphorylation of NDUFA10/ND42 subunit. To further explore the mechanisms by which PINK1 and Parkin influence mitochondrial integrity, we conducted a screen in Drosophila cells for genes that either phenocopy or suppress mitochondrial hyperfusion caused by pink1 RNAi. Among the genes recovered from this screen was ND42. In Drosophila pink1 mutants, transgenic overexpression of ND42 or its co-chaperone sicily was sufficient to restore CI activity and partially rescue several phenotypes including flight and climbing deficits and mitochondrial disruption in flight muscles. Here, the restoration of CI activity and partial rescue of locomotion does not appear to have a specific requirement for phosphorylation of ND42 at Ser-250. In contrast to pink1 mutants, overexpression of ND42 or sicily failed to rescue any Drosophila parkin mutant phenotypes. We also find that knockdown of the human homologue, NDUFA10, only minimally affecting CCCP-induced mitophagy, and overexpression of NDUFA10 fails to restore Parkin mitochondrial-translocation upon PINK1 loss. These results indicate that the in vivo rescue is due to restoring CI activity rather than promoting mitophagy. Our findings support the emerging view that PINK1 plays a role in regulating CI activity separate from its role with Parkin in mitophagy.     

PINK1 controls mitochondrial localization of Parkin through direct phosphorylation

PTEN-induced putative kinase 1 (PINK1) and Parkin, encoded by their respective genes associated with Parkinson’s disease (PD), are linked in a common pathway involved in the protection of mitochondrial integrity and function. However, the mechanism of their interaction at the biochemical level has not been investigated yet. Using both mammalian and Drosophila systems, we here demonstrate that the PINK1 kinase activity is required for its function in mitochondria. PINK1 regulates the localization of Parkin to the mitochondria in its kinase activity-dependent manner. In detail, Parkin phosphorylation by PINK1 on its linker region promotes its mitochondrial translocation, and the RING1 domain of Parkin is critical for this occurrence. These results demonstrate the biochemical relationship between PINK1, Parkin, and the mitochondria and thereby suggest the possible mechanism of PINK-Parkin-associated PD pathogenesis.     

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

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.     

PINK1/parkin通路调控线粒体自噬机制的研究进展

线粒体自噬指细胞通过自噬机制选择性除去损伤或多余的线粒体。真核生物通过线粒体自噬调控线粒体质量,维持供能细胞器的功能。大量研究表明,帕金森病相关基因PINK1和parkin可通过线粒体自噬参与并维持线粒体功能。PINK1与parkin能协同特异性识别损伤的线粒体,PINK1作为线粒体质量调控的探测器被活化,此过程中泛素化酶和去泛素化酶对维持parkin活性及线粒体自噬的效率有重要作用。本文主要总结PINK1/parkin通路在线粒体自噬中的功能与作用。     

Voltage-dependent Anion Channels (VDACs) Recruit Parkin to Defective Mitochondria to Promote Mitochondrial Autophagy

Mutations in the ubiquitin ligase Parkin and the serine/threonine kinase PINK1 can cause Parkinson disease. Both proteins function in the elimination of defective mitochondria by autophagy. In this process, activation of PINK1 mediates translocation of Parkin from the cytosol to mitochondria by an unknown mechanism. To better understand how Parkin is targeted to defective mitochondria, we purified affinity-tagged Parkin from mitochondria and identified Parkin-associated proteins by mass spectrometry. The three most abundant interacting proteins were the voltage-dependent anion channels 1, 2, and 3 (VDACs 1, 2, and 3), pore-forming proteins in the outer mitochondrial membrane. We demonstrate that Parkin specifically interacts with VDACs when the function of mitochondria is disrupted by treating cells with the proton uncoupler carbonyl cyanide p-chlorophenylhydrazone. In the absence of all three VDACs, the recruitment of Parkin to defective mitochondria and subsequent mitophagy are impaired. Each VDAC is sufficient to support Parkin recruitment and mitophagy, suggesting that VDACs can function redundantly. We hypothesize that VDACs serve as mitochondrial docking sites to recruit Parkin from the cytosol to defective mitochondria.