PROJECT SUMMARY Mitochondrial biogenesis relies on efficient protein import as most mitochondrial proteins are imported via protein import pathways after synthesis in the cytosol. The mitochondrial intermembrane space (IMS) assembly (MIA) pathway that specifically imports proteins into the IMS is unique in that oxidative folding drives import and folding of target proteins. Specifically, a series of well-studied thiol-disulfide exchange reactions carried out by the two main components of the MIA pathway, namely Erv1 and Mia40 dictate vectorial translocation into the mitochondrial IMS. Studies have shown that several non-classical substrates, which do not possess the twin CX3C or CX9C motifs, utilize this pathway and importantly, connect the MIA pathway with other vital processes in the IMS, unrelated to oxidative folding. Hence, the MIA pathway is highly relevant in pathology of a spectrum of diseases such as, myopathies, neuropathies, Huntington?s disease, ALS and cancer. However, several unanswered questions remain. With the growing spectrum of substrates of this pathway, there is a need to understand the underlying molecular mechanisms. The MIA pathway must adapt to redox changes via interactions with antioxidant enzymes involved in reductive reactions in the IMS (thioredoxin 1, peroxiredoxin, and glutaredoxin 2). However, the role of these redox-balancing systems with the MIA machinery is not well known, and more notably, other reductive mechanisms may exist in the IMS. Finally, because MIA pathway is operational under anaerobic conditions, there must be additional electron acceptors. The goal of this undergraduate-driven proposal is to investigate the function of the newly identified Erv1-interacting protein, Aim32p in the experimental model, the budding yeast Saccharomyces cerevisiae. Preliminary studies strongly suggest that Aim32p is important for protein translocation across multiple translocons, stabilizes several native protein complexes, and belongs to a class of proteins, termed as thioredoxin-like ferredoxins (Fds); functions of which are unknown but range from electron shuttling to redox sensing. Because of its unique placement in the IMS, it is imperative to examine mechanisms by which Aim32p could affect multiple important mitochondrial processes of import, electron transfer, and have a regulatory role in redox. Three specific proposal aims that utilize a combination of biochemical and bioinformatic approaches will be undertaken: In Aim 1 role of Aim32p within the MIA pathway will be explored. In Aim 2, biochemical studies to validate if Aim32p is a Fe-S protein, identification of key cysteine residues, and pathways crucial for its cellular stress response, will be performed. Finally, Aim 3 will elucidate the Aim32p interaction network. Upon successful completion, this work will provide exciting new information on the function of a multi- faceted mitochondrial protein and advance our fundamental knowledge of the process of protein translocation. This research will have a broad impact on public health because these mechanistic studies will provide key insights into how defects in mitochondrial biogenesis lead to disease.