Sullivan 2015 Abstract MiPschool Greenville 2015

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Lipid microdomains containing supercomplexes are enriched with multiple phospholipids including cardiolipin: A detergent extraction study.

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Sullivan EM Fix AM, Brown DA, Shaikh SR (2015)

Event: MiPschool Greenville 2015

It is hypothesized that cardiolipin molecules in the inner mitochondrial membrane form spatially distinct lipid microdomains that facilitate the formation of supercomplexes and allow for efficient electron transport and ATP production. Cardiolipin-enriched microdomains from mitochondria can potentially be isolated biochemically in the presence of 1% Triton-X 100 [1]. Previous studies show that the amount and type of detergent are critical variables in the isolation of lipid microdomains in other organelles. Therefore, we first investigated the influence of different detergents at different concentrations on the isolation of biochemical cardiolipin- enriched microdomains [2]. Next, we determined if supercomplexes were associated with the detergent resistant cardiolipin-enriched microdomains.

Biochemical isolation of mitochondrial microdomains was adapted from a previous protocol [1]. Briefly, isolated mitochondria were subjected to a range of detergents (Triton X-100, NP-40 or digitonin) at varying concentrations (0.01%, 0.05% or 0.50%). Next, mitochondria were centrifuged at 20,000 x g, which separated the detergent resistant membranes (DRM) microdomains from the non-microdomain fraction, known as detergent soluble membranes (DSM). Both DSM and DRM fractions were subjected to lipid extraction; the lipids were separated via thin layer chromatography (TLC), visualized by phosphoric charring and quantified via densitometry. Blue native PAGE (BN-PAGE) determined whether the DSM or DRM fraction contained supercomplexes. BN-PAGE protocol was adapted from the NativePAGE system from Life Technologies. Briefly, DSM and DRM fractions underwent solubilization with digitonin. Samples were centrifuged at 16,875 x g and the supernatants containing supercomplexes were collected. Protein was loaded onto the native gel as directed and ran until completion. Gels were fixed, and then destained overnight until the desired background was reached.

Analysis of TLC plates strikingly revealed that DRM fractions were not just enriched in cardiolipin but also contained phosphatidylcholine and phosphatidylethanolamine. Increasing the concentration of each detergent resulted in a decreased amount of phosphatidylethanolamine, cardiolipin and phosphatidylcholine in the DRMs. This decrease in phospholipids in the DRMs was paralleled by an increase in lipid content of the DSMs with one exception. No decrease in lipids was detected in the DRMs from 0.01% digitonin to 0.05% digitonin. Preliminary data with BN-PAGE results from each detergent at the 0.05% concentration showed supercomplexes to only be present in the DRM fraction.

Taken together, our results reveal no differences in the type of detergent used to isolate DRMs. These results also suggest that lipid microdomains formed in the mitochondria are not simply enriched in cardiolipin and likely contain several other lipid species. Although more precise analytical tools may be required for more in-depth mitochondrial membrane analysis, the supercomplexes housed in the inner mitochondrial membrane were associated with DRMs implying supercomplexes may rely on distinct lipid microdomains for optimal function.


O2k-Network Lab: US NC Greenville Brown DA, US NC Greenville Neufer PD, US VA Blacksburg Brown DA


Labels: MiParea: mt-Membrane 



Preparation: Isolated mitochondria  Enzyme: Supercomplex 



Event: Poster 


Affiliations

1-Dept Biochem Mol Biol; 2-Dept Physiol, East Carolina Univ, NC, USA. - sullivanel13@students.ecu.edu

References and acknowledgements

  1. Mitsopoulos P, Madrenas J (2013) Quantification of multimolecular complexes and supercomplexes in compartment-selective membrane microdomains. Methods in Cell Biology 117:411-31.
  2. Sorice M, Manganelli V, Matarrese P, Tinari A, Misasi R, Malorni R, Garofalo T (2009) Cardiolipin-enriched raft-like microdomains are essential activating platforms for apoptotic signals on mitochondria. FEBS Letters 583:2447-50.