The regulatory domain of squalene monooxygenase contains a re-entrant loop and senses cholesterol via a conformational change [Lipids]

October 3rd, 2015 by Howe, V., Chua, N. K., Stevenson, J., Brown, A. J.

Squalene monooxygenase (SM) is an important control point in cholesterol synthesis beyond 3 hydroxy 3 methylglutaryl CoA reductase (HMGCR). Although it is known to associate with the endoplasmic reticulum (ER), its topology has not been determined. We have elucidated the membrane topology of the sterol-responsive domain of SM, comprising the first 100 amino acids fused to GFP (SM N100-GFP) by determining the accessibility of 16 introduced cysteines to the cysteine-reactive, membrane-impermeable reagent PEG-maleimide (mPEG). We have identified a region integrally associated with the ER membrane that is likely to interact with cholesterol or respond to cholesterol-induced membrane effects. By comparing cysteine accessibility with and without cholesterol treatment, we further present evidence to suggest that cholesterol induces a conformational change in SM N100-GFP. This change is likely to lead to its targeted degradation by the ubiquitin-proteasome system (UPS), since degradation is blunted by treatment with the chemical chaperone glycerol, which retains SM N100-GFP in its native conformation. Furthermore, degradation can be disrupted by insertion of two N-terminal myc tags, implicating the N-terminus in this process. Together, this information provides new molecular insights into the regulation of this critical control point in cholesterol synthesis.