Abstract
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.
Highlights
Functions as an endoplasmic reticulum (ER) cholesterol sensor is essential in mediating ER-Golgi transport of SREBP
To ensure that this result was not due to reaction kinetics, we extended the time of His6-perfringolysin O (PFO)(C459A)-liposome incubations from 1 h to 24 h and observed the same step-like response at the same switch-point cholesterol concentration
Cooperative interactions between Scap1⁄7cholesterol1⁄7Insig in ER membranes result in switch-like control over SREBP-2 activation in response to small changes in ER cholesterol concentration [1, 3, 4]
Summary
Functions as an ER cholesterol sensor is essential in mediating ER-Golgi transport of SREBP. A recent study, using highly purified ER membranes, showed that the feedback response is switch-like, resulting in almost complete inactivation or activation of SREBP when the cholesterol concentration in ER membranes increases or decreases by as little as 2 mol % from the physiological set-point of ϳ5 mol % [4]. This switchlike response was proposed to arise because of cholesterol-mediated cooperative interactions between Scap and Insig. Such acute control of accessibility of the cholesterol ligand may enhance the cooperativity of Scap1⁄7cholesterol1⁄7Insig complex formation, further sharpening the switch-like response that controls cholesterol composition of cell membranes
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