PROJECT SUMMARY Mitochondria are endosymbiotically-derived double membrane-bound organelles which provide cells with energy via oxidative respiration. Mitochondria also serve as a major hub for cellular metabolism and are involved in numerous vital pathways, including cell signaling, innate immune response, and apoptosis. Dysfunction of mitochondria is implicated in aging and many diseases and is a potential causative factor in neurodegenerative diseases. Most of >1,000 mitochondrial proteins are encoded by the nuclear genome and thus are imported from the cytosol shortly after being synthesized as precursors on cytosolic ribosomes. Thus, mitochondrial protein import is an essential process required for biogenesis and functional maintenance of mitochondria. The import process is mainly mediated by two universally conserved membrane complexes, the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complex. The TOM complex mediates the initial translocation of precursor proteins across the outer mitochondrial membrane, and the TIM complex further translocates the precursor proteins across the inner mitochondrial membrane. The TIM complex is also responsible for integration of many integral membrane proteins to the inner membrane. Currently, it is poorly understood how the TOM and TIM complexes mediate these translocation processes. In the current proposal, we aim to address central outstanding questions about protein import mechanisms by the TOM and TIM complexes, using structural, biochemical, and biophysical approaches. These questions include how the translocase complexes specifically recognize their client proteins, how they form a path for protein translocation in the membranes, what are the molecular interactions and forces driving protein translocation, and how the translocase complexes are regulated. In particular, we will perform several cryo-electron microscopy (cryo-EM) studies to visualize the translocase complexes in different functional states, including substrate-engaged states, and gain insights into their mechanisms for substrate engagement and conformational changes. The outcomes of these studies will fundamentally advance our understanding of mitochondrial biology and provide new insights to develop novel approaches to treat mitochondrial-associated diseases, such as neurodegenerative diseases.