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Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol
Published on: March 25, 2020
Cholesterol impairs the adenine nucleotide translocator-mediated mitochondrial permeability transition through
Anna Colell1, Carmen García-Ruiz, Josep M Lluis
1Liver Unit, Institut de Malalties Digestives, Hospital Clínic y Provincial, Instituto Investigaciones Biomédicas August Pi Suñer, Barcelona, Spain.
Abstract:
Mitochondrial permeability transition (MPT) has been proposed to play a key role in cell death. Downstream MPT events include the release of apoptogenic factors that sets in motion the mitochondrial apoptosome leading to caspase activation. The current work examined the regulation of MPT by membrane fluidity modulated upon cholesterol enrichment. Mitochondria enriched in cholesterol displayed increased microviscosity resulting in impaired MPT induced by atractyloside, a c-conformation stabilizing ligand of the adenine nucleotide translocator (ANT). This effect was dependent on the dose of cholesterol loaded and reversed upon the fluidization of mitochondria by the fatty acid derivative A2C. Mitoplasts derived from cholesterol-enriched mitochondria responded to atractyloside in a similar fashion as intact mitochondria, indicating that a significant amount of cholesterol is still found in the inner membrane. The effects of cholesterol on MPT induced by atractyloside were mirrored by the release of intermembrane proteins, cytochrome c, Smac/Diablo, and apoptosis inducing factor. However, cholesterol loading did not affect the uptake rate of adenine nucleotide hence dissociating the function of ANT as a MPT-mediated protein from its adenine nucleotide exchange function. Thus, these findings indicate that the ability of atractyloside to induce MPT via ANT requires an appropriate membrane fluidity range.
Insights
Cholesterol enrichment of mitochondria impairs mitochondrial permeability transition (MPT) by increasing membrane viscosity. This suggests membrane fluidity is crucial for MPT induction by atractyloside via the adenine nucleotide translocator (ANT).
Area of Science:
- Cell Biology
- Biochemistry
- Mitochondrial Function
Background:
- Mitochondrial permeability transition (MPT) is implicated in programmed cell death.
- MPT involves the release of apoptogenic factors, initiating caspase activation.
- The regulation of MPT by membrane properties is not fully understood.
Purpose of the Study:
- To investigate how cholesterol enrichment and altered membrane fluidity affect MPT.
- To determine the role of membrane microviscosity in MPT induction by atractyloside.
- To differentiate the effects of cholesterol on ANT's role in MPT versus nucleotide exchange.
Main Methods:
- Cholesterol enrichment of isolated mitochondria.
- Measurement of mitochondrial microviscosity.
- Induction of MPT using atractyloside and assessment of protein release.
- Analysis of adenine nucleotide uptake.
- Experiments using mitoplasts to assess inner membrane cholesterol localization.
Main Results:
- Cholesterol enrichment increased mitochondrial microviscosity, inhibiting atractyloside-induced MPT.
- This inhibition was dose-dependent and reversible with A2C treatment.
- Cholesterol affected MPT and release of intermembrane proteins (cytochrome c, Smac/Diablo, AIF) but not adenine nucleotide uptake.
- Mitoplast studies confirmed cholesterol's presence and effect on the inner membrane.
Conclusions:
- MPT induction by atractyloside via ANT is dependent on optimal mitochondrial membrane fluidity.
- Cholesterol modulates MPT by altering membrane biophysical properties, not by directly inhibiting ANT's transport function.
- These findings highlight membrane fluidity as a critical regulatory factor in MPT-mediated cell death pathways.
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