Project Summary: Peroxisomes are ubiquitous, membrane-bound organelles that encapsulate specialized metabolic reactions, typically including those that produce hydrogen peroxide as a byproduct. In humans, where peroxisomes breakdown very long chain fatty acids and synthesize precursors of bile acids and myelin sheath lipids, defects in peroxisomes cause Peroxisome Biogenesis Disorders (PBDs), characterized by neuronal degeneration, liver dysfunction, and decreased lifespan. There are currently no treatments for PBDs, and our understanding of peroxisomes in human health is hindered by our limited understanding of the biochemical mechanisms of peroxisome molecular membrane biology. De novo biogenesis of peroxisomes requires approximately 30 dedicated Pex proteins. During this process, the peroxisome membrane and membrane proteins traffic through the endoplasmic reticulum, while the matrix proteins are imported fully folded from the cytosol. The majority of PBDs are caused by mutations in Pex1 and Pex6, two AAA+-ATPase motor proteins that perform an uncharacterized task crucial for peroxisome matrix protein import. A complete understanding of Pex1 and Pex6 architecture, substrates, processing mechanism, and function at the peroxisome would reveal new mechanistic details of peroxisome formation, novel therapeutic strategies, and expand our understanding of the role of peroxisomes in disease. As a Miller Fellow in Dr. Andreas Martin's lab, I used in vitro biochemistry and electron microscopy to determine the architecture of the active Pex1/Pex6 complex and its interaction and regulation by its membrane tether protein Pex15. This represents the first structural and biochemical characterization of Pex1/Pex6 and a unique molecular handle to dissect the energy-dependent steps of peroxisome assembly. In this proposal I will expand my research to understand the function of Pex1/Pex6 in the context of the cell and peroxisome dysfunction in the context of cellular homeostasis. In Aim 1, with the mentorship of Dr. Martin, an expert on AAA+-ATPase mechanisms, I propose to identify the substrates of Pex1/Pex6 and determine their processing mechanism. In Aim 2, with mentorship from Dr. Schekman, an expert in biochemical dissections of membrane trafficking, I will determine Pex1/Pex6 function in peroxisome matrix protein import using a novel cell-free reconstitution. Finally, I propose to determine the cellular response to induced peroxisome dysfunction and identify novel proteins required for peroxisome maintenance. With the support of my mentors and the greater research environment at UC Berkeley, I will receive training in mass spectrometry, cryo-electron microscopy, cell-free reconstitutions, fluorescence microscopy, and high throughput screening. These skills will help me bridge my background in biochemistry with my fascination with organelle biology to build a successful independent research program investigating the biochemical mechanisms of peroxisome biology.