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J Biol Chem. 2015 Nov 13;290(46):27533-44. doi: 10.1074/jbc.M115.675181. Epub 2015 Oct 3.

The Regulatory Domain of Squalene Monooxygenase Contains a Re-entrant Loop and Senses Cholesterol via a Conformational Change.

Author information

1
From the School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney 2052, Australia and.
2
Department of Nutritional Sciences and Toxicology, University of California, Berkeley, California 94720.
3
From the School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney 2052, Australia and aj.brown@unsw.edu.au.

Abstract

Squalene monooxygenase (SM) is an important control point in cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl-CoA reductase. Although it is known to associate with the endoplasmic reticulum, 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. We have identified a region integrally associated with the endoplasmic reticulum 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 because 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.

KEYWORDS:

cholesterol; cholesterol regulation; conformational change; membrane; membrane topology; protein turnover; squalene monooxygenase

PMID:
26434806
PMCID:
PMC4646006
DOI:
10.1074/jbc.M115.675181
[Indexed for MEDLINE]
Free PMC Article

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