Radhakrishnan, Arun2022-01-032022-01-032021-122021-09-29December 2https://hdl.handle.net/2152.5/9651Page xii is misnumbered as page xi.For cells to properly respond to environmental changes, cellular interiors must be exquisitely organized both spatially and temporally. In particular, metabolism must be spatially coordinated so metabolites are appropriately shunted into either storage or growth. Despite our understanding of how membrane-bound organelles organize metabolic processes, little is known about how metabolic regulation occurs at sub-organelle length scales. At these length scales, physical interactions between the endoplasmic reticulum (ER) and other organelles at ER-membrane-contact-sites (ER-MCSs) are now recognized as sub-organelle hubs for the regulation of metabolic processes. Our work uses the nucleus-vacuole-junction (NVJ) in S. cerevisiae (yeast) as a model ER-MCS to further an understanding about potential general functions of ER-MCSs. We have noted that the NVJ, a physical connection between the nuclear-ER and the vacuole, is a hub for lipid metabolic enzymes and regulators. When yeast are exposed to low glucose conditions, the NVJ recruits several metabolic proteins, including the enzyme Hmg1. Hmg1 catalyzes the conversion of HMG-CoA to mevalonate and is the rate-limiting enzyme in sterol biogenesis. We noted that Hmg1 is less catalytically active when Nvj1, the protein that recruits Hmg1 to the NVJ, is genetically ablated, or when Nvj1 lacks a minimal motif required to recruit Hmg1. Hmg1 NVJ partitioning is accompanied by its assembly into high molecular weight species, which may underlie its increase in enzymatic efficiency. Indeed, artificial tetramerization of Hmg1 overcomes the deficiencies of an Nvj1 knock-out. During Hmg1 partitioning, mevalonate is preferentially shunted into synthesis of sterol-esters (SEs), which are storage lipids found in large cytoplasmic organelles, lipid droplets (LDs). Coordinately, glucose starvation promotes the degradation of triglycerides (TAGs), the other major lipid species contained in LDs. We found that the SE/TAG imbalance in LDs during glucose starvation leads to a phase separation of SEs from a liquid to liquidcrystalline state. Upon SE phase separation, the proteome of LDs is considerably changed. Collectively, our studies of the NVJ have identified a novel function for an ER-MCS and connected it to a lipid metabolic circuit that controls the proteome of LDs.application/pdfenEndoplasmic ReticulumGlucoseLipid MetabolismLipidsMevalonic AcidSaccharomyces cerevisiaeSaccharomyces cerevisiae ProteinsThesis2022-01-031290684165