Figure 1
From: Human Mitochondrial DNA-Protein Complexes Attach to a Cholesterol-Rich Membrane Structure
The majority of the cholesterol in the mitochondrial inner membrane co-fractionates with the membrane associated nucleoid.
Pellet (a) and supernatant (b) preparations from digitonin treated purified mitochondria from HEK293 cells were separated on bottom-up flotation iodixanol gradients and their protein, DNA and lipid contents were analysed. The mtDNA and associated nucleoid proteins (Twinkle, polG, TFAM, SSB) as well as most of the cholesterol migrate in the same fraction in the pellet gradient. Some of the MAM marker protein FACL4 co-fractionates with the nucleoid in fraction 8. mtDNA, mitochondrial DNA; polG, polymerase Gamma; SSB, single-stranded binding protein; TFAM, mitochondrial transcription factor A; PHB, prohibitin, MRPS, mitochondrial ribosomal protein small subunit; MRPL, mitochondrial ribosomal protein large subunit; Clnx, calnexin; coxII, cytochrome oxidase subunit II; TOM20, translocase of outer membrane 20 kDa subunit; VDAC1, voltage-dependent anion channel 1. The lipid data is from the analyses of three replicate gradients (SEM), the protein and DNA profiles are from one representative gradient, while all three gradients were tested with at least the Twinkle, mtSSB, coxII, TFAM and ATAD3 antibodies by Western blot analysis and dot-blot analysis for mtDNA. The antibody probings were done on multiple blots (typically 2–3) with the same fractions to allow for the multiple detections with the many antibodies used.
