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. 2019:1949:153-163.
doi: 10.1007/978-1-4939-9136-5_12.

Monitoring and Modulating Intracellular Cholesterol Trafficking Using ALOD4, a Cholesterol-Binding Protein

Shreya Endapally  1 , Rodney E Infante  2 , Arun Radhakrishnan  3
Affiliations

Monitoring and Modulating Intracellular Cholesterol Trafficking Using ALOD4, a Cholesterol-Binding Protein

Shreya Endapally et al. Methods Mol Biol. 2019.

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.

Keywords: Anthrolysin O; Cholesterol trafficking; Endoplasmic reticulum; Plasma membrane.

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Figures

Fig. 1
Fig. 1
Biochemical characterization of ALOD4. Gel filtration chromatography of purified proteins. Recombinant ALOD4 was purified and labeled with Alexa Fluor 488 (fALOD4-488) or Alexa Fluor 647 (fALOD4-647) fluorescent dyes as described in Subheading 3. Buffer B (1 mL) containing 0.8 mg of ALOD4, fALOD4-488, or fALOD4-647 was loaded onto a Tricorn 10/300 Superdex 200 column and chromatographed at a flow rate of 0.5 mL/min. Absorbance at 280 nm (A280) was monitored continuously to identify ALOD4(blue), fALOD4-488(green), or fALOD4-647(red) proteins. Maximum A280 values for each protein (ALOD4: 390 mAU, fALOD4-488: 231 mAU, and fALOD4-647: 279 mAU) are normalized to 1. (Inset) 3 μg of each protein was subjected to 15% SDS/PAGE and stained with Coomassie (left) or imaged with the 600 nm filter (middle) or the 700 nm filter (right) on a LICOR instrument. Coom, Coomassie. Figure adapted from [7]
Fig. 2
Fig. 2
ALOD4 triggers activation of SREBP transcription factors. Immunoblot analysis of CHO-K1 cells after incubation with ALOD4 or ALOD4(Mut) proteins. On day 0, CHO-K1 cells were set up in medium A at a density of 3 × 104 cells/well of 48-well plates. On day 2, medium was removed, cells were washed with 500 μL of PBS and then incubated with 200 μL of medium C with indicated concentrations of ALOD4 or ALOD4(Mut). After incubation for 1 h at 37 °C, the medium was collected, and cells were harvested as described in Subheading 3. Equal aliquots of cells and media (10% of total) were subjected to immunoblot analysis or Coomassie staining as described in Subheading 3. P precursor form of SREBP2, N cleaved nuclear form of SREBP2. Figure adapted from [7]
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