PROJECT ABSTRACT Site-one protease (S1P) is a membrane anchored protease in the secretory system and a critical component of cellular signaling pathways including cholesterol biogenesis, the ER stress response, and lysosome biogenesis. S1P begins as an inactive pro-enzyme in the ER and becomes active through autoproteolysis of its inhibitory pro-domains as it folds in the ER and traffics to the Golgi, where it functions as an active enzyme. S1P is best studied in cholesterol metabolism. The spatial control of S1P activity, with the inactive form in the ER and the active form in the Golgi, underpins cholesterol metabolism in human cells. Lipogenic transcription programs that cause the uptake and synthesis of cholesterol are controlled by a family of transcription factor proteins known as Sterol response element binding proteins (SREBP). The SREBP precursors are folded in the ER, where they must be protected from proteolysis by S1P. When cholesterol levels are low, SREBP precursors are transported from the ER to the Golgi, where it they cleaved by S1P to initiate a cascade that results in the liberation of the SREBP transcription factor domain and the upregulation of lipogenesis. Ensuring S1P is active in the Golgi but inactive in the ER is critically important to cells and animals. SPRING (also C12ORF49) is a newly identified co-factor that is critical for the controlled maturation of S1P and understanding their relationship will provide new insights into cholesterol metabolism specifically and protease maturation in the secretory pathway more broadly. In preliminary work, we obtained a high-resolution structure of the soluble S1P-SPRING complex using cryo-electron microscopy (cryo-EM). Structural and biochemical data develop the hypothesis for a proposed mechanism where SPRING matures S1P by competing with and displacing an inhibitory pro-domain. Removing this pro-domain is necessary for S1P to proteolyze external substrates. Experiments with S1P trapped in different maturation stages suggest SPRING binds S1P at an intermediate stage of maturation as S1P traffics from the ER to the Golgi. In this proposal, we will use structural biology, biochemistry, and cellular biology to elucidate how S1P matures in the presence or absence of SPRING and how SPRING controls the enzymatic activity of S1P. In Aim 1, we will obtain cryo-EM reconstructions of S1P in distinct stages of maturation. We will use competition assays to test whether SPRING and the S1P pro-domains compete for binding at the same site of the S1P enzyme. In Aim 2, will use a peptide cleavage assay to determine the substrate specificity of the S1P-SPRING complex and which S1P substrate motifs require SPRING for proteolysis by S1P. In Aim 3, we will determine the functional consequences of disrupting the S1P-SPRING interaction in biochemical and cell-based signaling assays that measure SREBP activity and the ER stress response.