Protein homeostasis ensures the proper levels of proteins to accomplish their tasks. Targeted protein degradation is emerging as a critical mechanism for the regulation of membrane cholesterol and sphingolipids. How the proteins in these pathways, which exist in the ER, are recognized for degradation is poorly understood. Misfolded ER proteins are recognized by conserved ERAD machinery. However, members of multi-protein complexes are not always misfolded apart from their cognate partners and require targeted degradation pathways. The degradation of many sterol and lipid biosynthesis proteins is regulated by their protein-protein interactions or is induced by their metabolic products in negative feedback loops. This proposal will elucidate the molecular and biophysical basis for selective degradation in sterol and sphingolipid metabolism. Cholesterol levels are regulated by the Scap-SREBP system. SREBP2 begins as an integral ER membrane protein. In conditions of low cholesterol, SREBP2 is transported by the cholesterol-sensor Scap to the Golgi. There, SREBP2 is cleaved to release its soluble N-terminal transcription factor domain, which traffics to the nucleus and upregulates genes for cholesterol synthesis and uptake. In my postdoctoral work, I identified a novel degron in the C-terminal regulatory domain of SREBP2. This motif is necessary for the degradation of the SREBP2 precursor in the absence of Scap and for the degradation of the C-terminal SREBP2 product created in the Golgi by the cleavage of SREBP2. This C-terminal SREBP2 product must be cleared to allow Scap recycle and interact with additional SREBP2 precursors. The degradation of SREBP2 is mediated by TRC8, an ER-resident E3 ligase. I developed systems to express and purify Scap-SREBP2 complexes for structural studies. In the K99 period, I will determine the structure of SREBP2-Scap using cutting edge cryo-EM methods and will use cell-based and biophysical methods to characterize the interaction between SREBP2 and TRC8. These studies will reveal SREBP2 is recognized by TRC8 and how this is antagonized by the interaction between SREBP2 and Scap. In the R00 period, I will establish my independent career by determining the mechanisms by which targeted degradation accomplishes the regulation of membrane levels sphingolipids. The ER-resident serine palmitoyltransferase (SPT) complex conducts the rate-limiting step in sphingolipid synthesis. In mammals, SPT’s enzymatic activity is negatively regulated by three highly conserved proteins (ORMDL1-3), which form a direct complex with the SPT. While there is very little biochemical or biophysical insight into how SPT functions, recent studies show that ORMDL activity is regulated through degradation in response to excess sphingolipid metabolites. Moreover, this degradation may be carried out by non-canonical ERAD pathways. I will use functional assays to determine the E3 ligase recognition motifs in the ORMDLs and conduct a biophysical study o...