PROJECT SUMMARY Sphingolipids not only are essential structural components of eukaryotic membranes, but also function as signaling molecules and mediate many biological processes. Defects in sphingolipid metabolism are often associated with cancers and neurodegenerative diseases. Despite the importance of bioactive sphingolipids, molecular mechanisms of their biogenesis and homeostasis remain poorly understood. In humans, the first and rate-limiting step of sphingolipid biosynthesis is catalyzed by the serine palmitoyltransferase complex, which consists of catalytic component SPTLCs and regulatory components ssSPT and ORMDL. This key enzyme complex also senses cellular sphingolipid levels to adjust sphingolipid production accordingly, making it a critical feedback regulator in maintaining sphingolipid homeostasis. Human mutations in the complex lead to hereditary sensory neuropathy and early-onset amyotrophic lateral sclerosis. However, the mechanism and regulation of the complex are unclear. We propose to elucidate 1) the substrate recognition and catalytic reaction of the complex, 2) the role of regulatory components, and 3) the sphingolipid-sensing and feedback mechanism. To accomplish these aims, we will combine high-resolution structural biology, biochemistry, and biophysics to provide structural and mechanistic insights into sphingolipid biogenesis and homeostasis governed by the serine palmitoyltransferase complex. Our proposed studies will greatly advance our understanding of sphingolipid biology by uncovering the molecular basis of an essential step in sphingolipid biogenesis and a mechanism of sphingolipid homeostasis.