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The generation of mitochondrial-derived vesicles (MDVs) is implicated in a plethora of vital cell functions, from mitochondrial quality control to peroxisomal biogenesis. The discovery of distinct subtypes of MDVs has revealed the selective inclusion of mitochondrial cargo in response to varying stimuli. However, the true scope and variety of MDVs is currently unclear, and unbiased approaches have yet to be used to understand their biology. Furthermore, as mitochondrial dysfunction has been implicated in many neurodegenerative diseases, it is essential to understand MDV pathways in the nervous system. To address this, we sought to identify the cargo in brain MDVs. We used an in vitro budding assay and proteomic approach to identify proteins selectively enriched in MDVs. 72 proteins were identified as MDV enriched, of which 31% were OXPHOS proteins. Interestingly, the OXPHOS proteins localized to specific modules of the respiratory complexes, hinting at the inclusion of sub-assemblies in MDVs. Small TIM chaperones were also highly enriched in MDVs, linking mitochondrial chaperone-mediated protein transport to MDV formation. As the two Parkinson's disease genes PINK1 and Parkin have been previously implicated in MDV biogenesis in response to oxidative stress, we compared the MDV proteomes from the brains of wild-type mice with those of PINK1 -/and Parkin -/mice. No significant difference was found, suggesting that PINK1-and Parkin-dependent MDVs make up a small proportion of all MDVs in the brain. Our findings demonstrate a previously uncovered landscape of MDV complexity and provide a foundation from which to discover further novel MDV functions..

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Amyotrophic lateral sclerosis (ALS) is a fatal disease, characterized by the selective loss of motor neurons leading to paralysis. Mutations in the gene encoding superoxide dismutase 1 (SOD1) are the second most common cause of familial ALS, and considerable evidence suggests that these mutations result in an increase in toxicity due to protein misfolding. We previously demonstrated in the SOD1G93A rat model that misfolded SOD1 exists as distinct conformers and forms deposits on mitochondrial subpopulations. Here, using SOD1G93A rats and conformation-restricted antibodies specific for misfolded SOD1 (B8H10 and AMF7-63), we identified the interactomes of the mitochondrial pools of misfolded SOD1. This strategy identified binding proteins that uniquely interacted with either AMF7-63 or B8H10-reactive SOD1 conformers as well as a high proportion of interactors common to both conformers. Of this latter set, we identified the E3 ubiquitin ligase TNF receptor–associated factor 6 (TRAF6) as a SOD1 interactor, and we determined that exposure of the SOD1 functional loops facilitates this interaction. Of note, this conformational change was not universally fulfilled by all SOD1 variants and differentiated TRAF6 interacting from TRAF6 noninteracting SOD1 variants. Functionally, TRAF6 stimulated polyubiquitination and aggregation of the interacting SOD1 variants. TRAF6 E3 ubiquitin ligase activity was required for the former but was dispensable for the latter, indicating that TRAF6-mediated polyubiquitination and aggregation of the SOD1 variants are independent events. We propose that the interaction between misfolded SOD1 and TRAF6 may be relevant to the etiology of ALS.