SCAP, Insig, and Cholesterol Interactions in Mammalian Cells
Cholesterol synthesis in mammalian cells is highly regulated by an end-product feedback mechanism. The transcription of genes necessary for both fatty acid and cholesterol production are controlled by sterol regulatory element binding proteins (SREBPs). The critical regulatory step is the proteolytic release of SREBPs from their inactive membrane bound form. Soon after translation, SREBPs bind SREBP cleavage activating protein (SCAP), a polytopic membrane protein of the endoplasmic reticulum (ER). In sterol depleted situations, SCAP escorts SREBPs to the Golgi, where SREBPs are cleaved and can move freely to the nucleus and activate the numerous enzymes of cellular lipid homeostasis. When cellular sterol levels rise, the SCAP/SREBP complex binds to an ER resident protein named Insig. Upon binding to Insig, the movement of the SCAP/SREBP complex to the Golgi is inhibited, thus halting cholesterol and fatty acid synthesis. The mechanism by which the cell senses sterol levels has been long unknown. Radhakrishnan et al. and Adams et al. demonstrated that SCAP itself binds cholesterol and thus may act directly to sense cellular sterol levels and mediate the end-product feedback control of SREBPs. The goal of this thesis is to elucidate the molecular details of the interactions between SCAP, Insig and cholesterol. My thesis experimentally details the membrane topology of human Insig-1 and shows that it is a polytopic integral membrane protein of the ER with six transmembrane spanning segments. In addition, the amino and carboxy-termini of Insig are both facing the cytosol. Furthermore, crucial residues of Insig that are important for SCAP interaction are identified. My thesis has also defined distinct amino acids of SCAP that are essential for its role as a protein that binds Insig and as a protein that has the ability to bind cholesterol. An aspartic acid in the middle of transmembrane six is necessary for sterol regulated binding to Inisg, while residues in transmembrane segments one and three of SCAP are crucial for cholesterol binding both in vivo and in vitro.