Sterol Sensing by Two Luminal Loops in Scap

SREBP cleavage-activating protein (Scap) is an endoplasmic reticulum (ER) membrane protein that controls cholesterol homeostasis by transporting SREBPs from the ER to the Golgi complex. Transport is initiated when COPII proteins bind to Scap and cause the Scap/SREBP complex to enter COPII coated vesicles for transport to the Golgi. In the Golgi complex, two proteases cleave SREBP, thereby releasing its transcriptionally active domain so that it can move to the nucleus and activate transcription of genes involved in cholesterol synthesis and uptake. Scap is not only an escort protein, but also a cholesterol sensor. When cholesterol is abundant in ER membranes, the sterol binds to Scap and triggers a conformational change in the protein that prevents COPII proteins from binding to Scap. The Scap/SREBP complex cannot move to the Golgi and proteolytic cleavage is terminated. This cholesterol feedback inhibition is essential to control cholesterol metabolism in animals. Scap can be divided into two functional regions. The C-terminal cytosolic WD domain interacts with the regulatory domain of SREBPs. The N-terminal membrane attachment domain includes eight transmembrane helices (TM) joined by four small hydrophilic loops and three large loops. One large cytosolic loop (Loop 6) in Scap binds COPII proteins. The other two large loops (Loops 1 and 7) face the ER lumen. Previous studies localized the cholesterol-binding activity to the N-terminal membrane domain of Scap. Studies described in this thesis narrow down the cholesterol binding pocket to the first large luminal loop (Loop 1). Mutational analysis further suggests a direct interaction between luminal Loop 1 and Loop 7 to control Scap transport activity. Scap Loop 1 was purified as a recombinant protein and found to bind [3H]-cholesterol through an in vitro binding assay. The specificity of this binding was determined through competition studies with different unlabeled sterols. Importantly, the binding affinity and specificity of Loop 1 was similar to that of the entire Scap membrane domain. Subsequently, alanine scan mutagenesis was performed on luminal Loop1 and Loop7. Through this approach, two point mutations of Scap (Y234A in Loop 1 and Y640S in Loop 7) were identified that prevent its movement to the Golgi, thus abrogating the processing of SREBPs. Trypsin cleavage assays on the full-length Scap show that Loop 6 of Scap(Y234A) or Scap(Y640S) is always in the configuration that precludes COPII binding, even in sterol-depleted cells. When the Scap TM1-6 segment (containing Loop 1) and the TM7-end segment (containing Loop 7) are expressed in the same cells, the two proteins bind to each other as determined by co-immunoprecipitation. This binding does not occur when Loop 1 contains the Y234A mutation, or Loop 7 contains the Y640S mutation. These data support the model that luminal Loop 1 and luminal Loop 7 must interact in order for Scap movement to occur.