Peroxisomes are essential subcellular organelles that play crucial roles in the oxidation of fatty-acids and homeostasis of glutathione, as well as reactive oxygen and nitrogen species (ROS and RNS, respectively). They play critical roles in the regulation of intracellular redox states and antiviral signaling, as well as cellular differentiation and metabolism, and their impairment causes many debilitating, and often fatal, human peroxisome biogenesis disorders (PBDs). Their biogenesis is orchestrated by 36 PEX genes, encoding peroxins, involved in the biogenesis of peroxisomal membrane and matrix proteins, as well as in the control of organelle size, number and inheritance. Their biogenesis has been studied in many organisms from yeast to plants and mammals, and more than 15 peroxins and their modes of action are conserved from yeast to man. While much has been learned about the biogenesis of peroxisomal matrix and membrane proteins to pre-existing peroxisomes, far less is known about how this organelle is generated de novo from other endogenous membranes. Such an ability to generate new peroxisomes de novo is obviously relevant under conditions where peroxisome biogenesis is impaired (e.g. human PBDs), or under conditions of stress (e.g. ROS) when peroxisomes are turned over by autophagy (pexophagy). Indeed, any disorders associated with imbalanced peroxisome homeostasis can be corrected, in principle, by manipulating either peroxisome biogenesis or its turnover, as we have shown. Such a global understanding of the mechanisms involved in peroxisome homeostasis is the long-term interest of my lab. Over almost 3 decades, we exploited the yeast, Pichia pastoris, to provide many major insights into our knowledge of peroxisome biogenesis and turnover. This proposal focuses on gleaning a deeper understanding of the proteins involved in the intra-ER sorting and budding of peroxisomal membrane proteins (PMPs) to a pre- peroxisomal exit site on the ER (pER) from where at least two type of pre-peroxisomal vesicles (ppVs) bud to ultimately generate peroxisomes, either by fusion with pre-existing peroxisomes or anew when peroxisomes are absent. Budding of ppVs is conserved between yeast and mammals and several proteins we will study have counterparts involved in human health. There are also reports of ppVs derived from mitochondria contributing to peroxisome biogenesis. Our approach is based on the use of novel genetic and biochemical strategies, including ppV purification and characterization, in vitro budding reactions and the use of innovative techniques to follow what these novel proteins do, where they act, who they interact with and how they function. The Aims are: Aim 1 - Isolation and characterization of the ATPase/s and other proteins involved in ppV budding. Aim 2 – How do Pex25 and Pex36 act in stimulating intra-ER sorting and budding of Pex2 and other RING- domain peroxins? Aim 3 – Does ppV budding occur from yeast mitochondria and what is its ...