Mitochondrial dysfunction has long been associated with Parkinson's disease (PD). Parkin and PINK1, two genes associated with familial PD, have been implicated in the degradation of depolarized mitochondria via autophagy (mitophagy). Here, we describe the involvement of parkin and PINK1 in a vesicular pathway regulating mitochondrial quality control. This pathway is distinct from canonical mitophagy and is triggered by the generation of oxidative stress from within mitochondria. Wild-type but not PD-linked mutant parkin supports the biogenesis of a population of mitochondriaderived vesicles (MDVs), which bud off mitochondria and contain a specific repertoire of cargo proteins. These MDVs require PINK1 expression and ultimately target to lysosomes for degradation. We hypothesize that loss of this parkin-and PINK1-dependent trafficking mechanism impairs the ability of mitochondria to selectively degrade oxidized and damaged proteins leading, over time, to the mitochondrial dysfunction noted in PD.

Despite their importance as signaling hubs, the function of mitochondria-ER contact sites in mitochondrial quality control pathways remains unexplored. Here we describe a mechanism by which Mfn2, a mitochondria-ER tether, gates the autophagic turnover of mitochondria by PINK1 and parkin. Mitochondria-ER appositions are destroyed during mitophagy, and reducing mitochondria-ER contacts increases the rate of mitochondrial degradation. Mechanistically, parkin/PINK1 catalyze a rapid burst of Mfn2 phosphoubiquitination to trigger p97-dependent disassembly of Mfn2 complexes from the outer mitochondrial membrane, dissociating mitochondria from the ER. We additionally demonstrate that a major portion of the facilitatory effect of p97 on mitophagy is epistatic to Mfn2 and promotes the availability of other parkin substrates such as VDAC1. Finally, we reconstitute the action of these factors on Mfn2 and VDAC1 ubiquitination in a cell-free assay. We show that mitochondria-ER tethering suppresses mitophagy and describe a parkin-/PINK1-dependent mechanism that regulates the destruction of mitochondria-ER contact sites.

Antibodies are a key resource in biomedical research yet there are no community-accepted standards to rigorously characterize their quality. Here we develop a procedure to validate pre-existing antibodies. Human cell lines with high expression of a target, determined through a proteomics database, are modified with CRISPR/Cas9 to knockout (KO) the corresponding gene. Commercial antibodies against the target are purchased and tested by immunoblot comparing parental and KO. Validated antibodies are used to definitively identify the most highly expressing cell lines, new KOs are generated if needed, and the lines are screened by immunoprecipitation and immunofluorescence. Selected antibodies are used for more intensive procedures such as immunohistochemistry. The pipeline is easy to implement and scalable. Application to the major ALS disease gene C9ORF72 identified high-quality antibodies revealing C9ORF72 localization to phagosomes/lysosomes. Antibodies that do not recognize C9ORF72 have been used in highly cited papers, raising concern over previously reported C9ORF72 properties.