We are excited, if presented with the opportunity, by the prospect of continuing over 3 decades of research into the mechanisms of peroxisome (PO) biogenesis. Our current work relies on yeast models, which have provided, and will continue to reveal, deep insights into this conserved and important biological process in humans, while also enhancing the diagnosis and understanding of the myriad of PO biogenesis disorders (PBDs). Our past work has centered on PO homeostasis, which balances the biogenesis and turnover processes, but we focus here on the birth of POs in cells that have no pre-existing POs, because previous genetic screens were done in cells that generate POs by redundant pathways involving both growth and division of pre-existing POs and de novo PO biogenesis. In doing so, redundant and essential genes were not identified, leaving a serious gap in our understanding. We remedy this using a novel, innovative, high-throughput screen (HTS) for PO biogenesis mutants in cells that are incapable of producing POs via growth and division. We show, using a pilot mini-screen, that unlike previous screens, our strategy is yielding putative hits in many steps of PO biogenesis, while identifying both known and novel genes, for the first time, including those involved in PO dynamics. This promising HTS platform, which we validate by proof-of-concept experiments, has provided a treasure trove of genes that now need deep investigation to understand how they function. Notably, many of the new genes we identified interact with known players we and others have been investigating, and a significant number are present at membrane contact sites (MCSs) between POs and other organelles. This presents a second fascinating avenue of pursuit, wherein we will probe the importance of metabolite (especially lipids) transport functions of the ER-PO MCSs. Our analyses will judiciously explore two yeast models to address evolutionary conservation and derive common, conserved principles. This leads naturally to the third, poorly-explored arena of interorganellar communication, for which we have found a novel, experimentally-tractable link, which is the requirement of mitochondrial redox and oxidative phosphorylation (OXPHOS) for PO proliferation (i.e. increase in PO number/cell). Thus, each of three new avenues of pursuit is expected to open new doors that will exceed what we alone can understand, but our long-term vision is to create interesting, new, community opportunities for future exploration in PO biology, knowing that it will be relevant, albeit indirectly, to patients with PBDs and the alleviation of their suffering. This proposal represents an expansion of scope by synergizing our molecular and cell biology efforts with the technical and intellectual prowess of the Aitchison laboratory (SCRI) and is backed by a significant track record of advances made by both PIs in PO biology. The Aims of our proposal are: Aim 1: Identify and perform functional studies on th...