title: Measurement of Mitochondrial Cholesterol Import Using a Mitochondria-Targeted CYP11A1 Fusion Construct.
abstract: All animal membranes require cholesterol as an essential regulator of biophysical properties and function, but the levels of cholesterol vary widely among different subcellular compartments. Mitochondria, and in particular the inner mitochondrial membrane, have the lowest levels of cholesterol in the cell. Nevertheless, mitochondria need cholesterol for membrane maintenance and biogenesis, as well as oxysterol, steroid, and hepatic bile acid production. Alterations in mitochondrial cholesterol have been associated with a range of pathological conditions, including cancer, hepatosteatosis, cardiac ischemia, Alzheimer's, and Niemann-Pick Type C Disease. The mechanisms of mitochondrial cholesterol import are not fully elucidated yet, and may vary in different cell types and environmental conditions. Measuring cholesterol trafficking to the mitochondrial membranes is technically challenging because of its low abundance; for example, traditional pulse-chase experiments with isotope-labeled cholesterol are not feasible. Here, we describe improvements to a method first developed by the Miller group at the University of California to measure cholesterol trafficking to the inner mitochondrial membrane (IMM) through the conversion of cholesterol to pregnenolone. This method uses a mitochondria-targeted, ectopically expressed fusion construct of CYP11A1, ferredoxin reductase and ferredoxin. Pregnenolone is formed exclusively from cholesterol at the IMM, and can be analyzed with high sensitivity and specificity through ELISA or radioimmunoassay of the medium/buffer to reflect mitochondrial cholesterol import. This assay can be used to investigate the effects of genetic or pharmacological interventions on mitochondrial cholesterol import in cultured cells or isolated mitochondria.
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalhousie University, Sir Charles Tupper Medical Building 9G, 5850 College Street, Halifax, NS, Canada, B3H 4R2.
authors.forename: Barry E
authors.initials: BE
authors.lastname: Kennedy
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalhousie University, Sir Charles Tupper Medical Building 9G, 5850 College Street, Halifax, NS, Canada, B3H 4R2.
authors.forename: Mark
authors.initials: M
authors.lastname: Charman
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalhousie University, Sir Charles Tupper Medical Building 9G, 5850 College Street, Halifax, NS, Canada, B3H 4R2. bkarten@dal.ca.
authors.forename: Barbara
authors.initials: B
authors.lastname: Karten
mesh_terms: D001693:Biological Transport, Active / Q000502:physiology; D002460:Cell Line; D002784:Cholesterol / Q000235:genetics / Q000378:metabolism*; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics / Q000378:metabolism; D005288:Ferredoxins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics; D051336:Mitochondrial Membranes / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D005288:Ferredoxins; D011993:Recombinant Fusion Proteins; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme
keywords: CYP11A1; F2-fusion protein; Mitochondrial cholesterol import; Pregnenolone; Radioimmunoassay