Accessibility of cholesterol in endoplasmic reticulum membranes and activation of SREBP-2 switch abruptly at a common cholesterol threshold

Recent studies have shown that cooperative interactions in endoplasmic reticulum (ER) membranes between Scap, cholesterol, and Insig result in switch-like control over activation of SREBP-2 transcription factors. This allows cells to rapidly adjust rates of cholesterol synthesis and uptake in response to even slight deviations from physiological set-point levels, thereby ensuring cholesterol homeostasis. In the present study we directly probe for the accessibility of cholesterol in purified ER membranes. Using a soluble cholesterol-binding bacterial toxin, perfringolysin O, we show that cholesterol accessibility increases abruptly at ∼5 mol% ER cholesterol, the same concentration at which SREBP-2 activation is halted. This switch-like change in cholesterol accessibility is observed not only in purified ER membranes but also in liposomes made from ER lipid extracts. The accessibility of cholesterol in membranes is related to its chemical activity. Complex formation between cholesterol and some ER phospholipids can result in sharp changes in cholesterol chemical activity and its accessibility to perfringolysin O or membrane sensors like Scap. The control of the availability of the cholesterol ligand to participate in cooperative Scap/cholesterol/Insig interactions further sharpens the sensitive switch that exerts precise control over cholesterol levels in cell membranes.