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Most mitochondrial proteins are encoded by the nuclear genome, synthesized in the cytosol, and imported into the organelle. Mitochondrial protein import is therefore vital for the maintenance of mitochondrial function and cell survival. Alterations in this process are suspected to contribute to various diseases, including neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease. Our understanding of the cytosolic signaling mechanisms and posttranslational modifications regulating the mitochondrial import process is still in its infancy and hampered by the lack of tools for its dynamic assessment in cells. We recently engineered an inducible molecular biosensor for monitoring one of the main mitochondrial import routes, the so-called presequence pathway, using a quantitative luminescence-based readout. Here, we provide basic guidelines for using this probe in common cell types of general use in the scientific community: HEK293T cells, human fibroblasts, and mouse primary neurons. These guidelines can serve as a starting point for the development of more elaborated protocols for the dynamic investigation of the presequence import pathway and its regulation in relevant physiological and pathological conditions.

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 SOD1 G93A rat model that misfolded SOD1 exists as distinct conformers and forms deposits on mitochondrial subpopulations. Here, using SOD1 G93A 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., (© 2020 Semmler et al.)