Measurement of Mitochondrial Cholesterol Import Using a Mitochondria-Targeted CYP11A1 Fusion Construct

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First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1583)

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.

Key words

Mitochondrial cholesterol import Pregnenolone F2-fusion protein CYP11A1 Radioimmunoassay 
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Notes

Acknowledgments

We thank Dr. Walter Miller (UCSF San Francisco, CA) for generously sharing the original F2-fusion expression vector and the critical reading of the manuscript. Our work on mitochondrial cholesterol import was supported by the Canadian Institutes of Health Research, the Dalhousie Medical Research Foundation, and the Nova Scotia Health Research Foundation.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Barry E. Kennedy
    • 1
  • Mark Charman
    • 1
  • Barbara Karten
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyDalhousie UniversityHalifaxCanada

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