PROJECT SUMMARY Mitochondria are responsible for cellular respiration via the oxidative phosphorylation system (OXPHOS). They are also centers of cellular redox chemistry because respiration is made up of a series of electron transfer reactions. Even though the mitochondrial DNA encodes essential OXPHOS components, the vast majority of the structural subunits and additional bioenergetics factors are encoded in the nucleus, translated in the cytoplasm and utilize sophisticated import machineries (aka translocons) to reach their final destinations. An important and large pool of proteins within the mitochondrial proteome are thiols; cysteine (Cys) containing proteins that perform key functions of protein import and OXPHOS biogenesis and highly relevant in the pathology of a spectrum of diseases. Redox (reduction-oxidation) regulation of these Cys proteins is thus relevant to their protein activity and highly significant for overall mitochondrial function. Thioredoxins are well known to play a role in redox regulation by reducing disulfide bonds in oxidized proteins in order to restore function. Our central hypothesis is that Aim32, a thioredoxin-like [2Fe-2S] ferredoxin mitochondrial protein that belongs to the thioredoxin-superfamily and localizes to the matrix and Inter membrane space (IMS), has an underlying role in the redox regulation of Cys proteins with consequential impacts on OXPHOS biogenesis and protein import, specifically the TIM22 import machinery. However, how these potential substrates of Aim32 are regulated represents a critical barrier to advancing our understanding of how thiol redox modifications allow for rapid adjustment of mitochondrial protein function and overall biogenesis. Furthermore, the identification of pathological variants in subunits of the TIM22 complex and OXPHOS that are implicated in several diseases causing mitochondrial myopathy, makes it imperative to understand mechanisms that regulate OXPHOS and TIM22 biogenesis. Using the budding yeast, Saccharomyces cerevisiae as a model system, the goal of this undergraduate- driven proposal is to evaluate our hypothesis by pursuing three specific aims: 1) Delineate Aim32-mediated mechanisms underlying TIM22 translocon assembly/stability; 2) Identify role of Aim32 in OXPHOS biogenesis; and 3) Determine functional relevance of Aim32 in the IMS and matrix. The proposed work is innovative because we seek to investigate how Aim32 mechanistically regulates TIM22 complex assembly (biogenesis) and bioenergetics (OXPHOS); two fundamental mitochondrial processes that span the mitochondrial IMS and matrix. Additionally, allocating specific functions to Aim32 within these sub-compartments, is equally important. The contribution of this work will be significant in that physiological ramifications of these new-founded roles for Aim32 to mitochondrial and cellular function have not been explored and will be clarified. This research will have a broad impact on public health because ...