Abstract
Mammalian cells carefully control their cholesterol levels by employing multiple feedback mechanisms to regulate synthesis of cholesterol and uptake of cholesterol from circulating lipoproteins. Most of a cell’s cholesterol (~80% of total) is in the plasma membrane (PM), but the protein machinery that regulates cellular cholesterol resides in the endoplasmic reticulum (ER) membrane, which contains a very small fraction (~1% of total) of a cell’s cholesterol. How does the ER communicate with PM to monitor cholesterol levels in that membrane? Here, we describe a tool, ALOD4, that helps us answer this question. ALOD4 traps cholesterol at the PM, leading to depletion of ER cholesterol without altering total cell cholesterol. The effects of ALOD4 are reversible. This tool has been used to show that the ER is able to continuously sample cholesterol from PM, providing ER with information about levels of PM cholesterol.
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References
Brown MS, Goldstein JL (1997) The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89:331–340
Brown MS, Goldstein JL (2009) Cholesterol feedback: from Schoenheimer’s bottle to Scap’s MELADL. J Lipid Res 50(Suppl):S15–S27
Lange Y, Steck TL (2016) Active membrane cholesterol as a physiological effector. Chem Phys Lipids 199:74–93
Lange Y, Steck TL (1997) Quantitation of the pool of cholesterol associated with acyl-CoA:cholesterol acyltransferase in human fibroblasts. J Biol Chem 272:13103–13108
Das A, Goldstein JL, Anderson DD et al (2013) Use of mutant 125I-perfringolysin O to probe transport and organization of cholesterol in membranes of animal cells. Proc Natl Acad Sci U S A 110:10580–10585
Das A, Brown MS, Anderson DD et al (2014) Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis. elife 3:e02882
Infante RE, Radhakrishnan A (2017) Continuous transport of a small fraction of plasma membrane cholesterol to endoplasmic reticulum regulates total cellular cholesterol. elife 6:e25466
Gay A, Rye D, Radhakrishnan A (2015) Switch-like responses of two cholesterol sensors do not require protein oligomerization in membranes. Biophys J 108:1459–1469
Chakrabarti RS, Ingham SA, Kozlitina J et al (2017) Variability of cholesterol accessibility in human red blood cells measured using a bacterial cholesterol-binding toxin. elife 6:e23355
Rong S, Cortes VA, Rashid S et al (2017) Expression of SREBP-1c requires SREBP-2-mediated generation of a sterol ligand for LXR in livers of mice. elife 6:e25015
Yang J, Brown MS, Ho YK et al (1995) Three different rearrangements in a single intron truncate sterol regulatory element binding protein-2 and produce sterol-resistant phenotype in three cell lines. Role of introns in protein evolution. J Biol Chem 270:12152–12161
Ikeda Y, DeMartino GN, Brown MS et al (2009) Regulated endoplasmic reticulum-assisted degradation of a polytopic protein: p97 recruits proteasomes to Insig-1 before extraction from membranes. J Biol Chem 284:34889–34900
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Endapally, S., Infante, R.E., Radhakrishnan, A. (2019). Monitoring and Modulating Intracellular Cholesterol Trafficking Using ALOD4, a Cholesterol-Binding Protein. In: Drin, G. (eds) Intracellular Lipid Transport. Methods in Molecular Biology, vol 1949. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9136-5_12
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DOI: https://doi.org/10.1007/978-1-4939-9136-5_12
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Publisher Name: Humana Press, New York, NY
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