Project Summary/Abstract To adapt to metabolic cues and survive stresses like nutrient starvation, cells store high- energy lipids in specialized organelles called lipid droplets (LDs) that emerge from the endoplasmic reticulum (ER), the metabolic center of cells. Recent studies reveal that LDs serve many roles in cell physiology, but how they are functionally ear-marked for specific tasks is unclear. Indeed, the ER network itself executes numerous cellular functions, but how this functional diversity is mechanistically achieved is unclear. The purpose of this grant is to dissect the molecular mechanisms by which LDs and the ER network achieve functional diversity at the sub-organelle level. Capitalizing on published and preliminary data, we find that LDs exhibit unique surface proteomes that provide functional specificity for LDs within single cells. Furthermore, we find that inter-organelle contact sites define ER sub-domains with specific roles in the compartmentalization of mevalonate metabolism. Here, I outline three directions that enable the mechanistic dissection of LD and ER functional compartmentalization: 1) In direction 1, we will leverage yeast genetics, biochemistry, and metabolic dissection to dissect how yeast ER-lysosome contacts (called nucleus-vacuole junctions, NVJs) serve as ER sub-domains and “metabolic platforms” that spatially compartmentalize mevalonate metabolism, and promote metabolic remodeling during glucose starvation. 2) In direction 2, we will capitalize on a large-scale screen to dissect how LDs are labeled with specific proteins to enable unique roles within cells. 3) In direction 3, we will utilize state-of-the-art cryo-FIB-SEM imaging to dissect how lipid phase transitions within LDs selectively remodel the LD surface proteome, and ultimately influence LD function and LD inter-organelle contact sites. Collectively, these directions will provide insights into new cellular organizational principles that enable to LDs and the ER network to achieve a functional division-of- labor. They will also characterize lipid phase transition properties of sterols, which promote disease pathologies in obesity, heart disease, and atherosclerosis.