Mechanistic Dissection of Insig-1, a Master Regulator of Cholesterol Homeostasis

Insigs are polytopic membrane proteins of the endoplasmic reticulum (ER) that regulate lipid synthesis by controlling the sterol-mediated vesicular transportation of sterol regulatory element binding proteins (SREBPs). SREBPs are ER bound transcription factors that form complexes with Scap. In sterol-depleted cells, Scap escorts SREBPs from the ER to the Golgi apparatus, where SREBPs are proteolytically cleaved to liberate the nuclear fragments that activate genes for cholesterol synthesis and uptake. When sterols overaccumulate in cells, the Scap/SREBP complex is retained in the ER by the anchor proteins called Insigs. In this thesis I describe the formation of a complex between Insig-1 and Scap in a sterol regulated fashion which facilitates the ER retention of Scap. To understand the molecular basis of the interactions between Insig-1 and Scap, I use a site-directed mutagenesis approach to select residues in Insig-1 that are essential for Insig-1/Scap complex formation. This study reveals a functional role for the amino acid Asp-205, which is located at the beginning of the fourth loop of Insig-1. Mutation of this aspartic acid to alanine produces an inactive Insig-1 that no longer binds to Scap, and leads to sterol-resistant processing of SREBPs. Mammalian cells express two Insig proteins differ in their mode of control. Insig-1, but not Insig-2, is an SREBP target gene. Also, Insig-1 protein is degraded more rapidly than Insig-2. Thus, Insig-1 is the focus of the study. I further demonstrate that degradation of Insig-1 is regulated by sterols. When ER cholesterol content is low, Insig-1 is ubiquitinated on lysines 156 and 158 and degraded in proteasomes. Sterol-induced binding of Insig-1 to Scap prevents Insig-1 ubiquitination and degradation. The dynamic change in Insig-1 protein stability, together with its transcriptional control by nuclear SREBPs, creates a new model for the convergent inhibition of SREBP processing and cholesterol supply in animal cells. Taken together, these studies established Insig-1 as the master regulator in the cholesterol homeostasis.