PROJECT SUMMARY (See instructions): Mammalian cells tightly control the membrane lipid composition of their organelles, and the key homeostatic machinery for many lipids resides within the endoplasmic reticulum (ER). Proteins in the ER must respond to changes in lipid levels, but how proteins respond to changes in lipid composition remains largely unknown. Protein degradation is a key part of feedback loops that regulate cholesterol and sphingolipid biosynthetic pathways, yet how protein homeostasis is influence by the membrane itself is unknown. Resolving this basic knowledge gap will provide insights into membrane protein biology and potentially uncover new directions for treating metabolic diseases. To better understand how the ER membrane influences protein function and how this feeds back into lipid metabolism, we focus on the activity ER resident ubiquitin E3 ligases and their substrates. E3 ligases label substrate proteins with ubiquitin, which causes the substrate proteins to be degraded by the proteasome system. We will study how these enzymes and their substrate proteins are regulated in the ER membrane and how their activity, in turn, regulates lipid levels. In the first aim, we will study how membrane cholesterol levels influence the activity and substrate recognition of two ER-localized E3 ligases called MARCH6 and TRC8. We will use cellular and biochemical assays to determine the lipid binding specificity and activity relationships for these two enzymes. We will use cryo-electron microscopy to elucidate how these enzymes recognize sterols and how membrane lipids influence the ability of these enzymes to form active complexes with substrate proteins. Through these efforts we will discover biophysical principles that may apply to a broad range of membrane bound mammalian E3 ligases. In the second aim, we will begin to uncover how targeted protein degradation regulates sphingolipid metabolism. We will use molecular dissection techniques along with candidate and discovery-based approaches to elucidate the molecular determinants of the regulated degradation of regulatory proteins called ORMDLs that control sphingolipid biosynthesis. Together, these aims will provide the first insights into substrate recognition of mammalian membrane E3 ligases and a new understanding of how these membrane sensors function in cells.