Lithium acetyl coa

Acetylation of TFAM stocks was conducted by mixing TFAM and 5 mM lithium-acetyl-CoA (Sigma) into an acetylation buffer (100 mM HEPES–NaOH pH 8.5, 1 mM EDTA) and incubating for 12 h at 37 °C. The reaction was then buffer exchanged using a 3K MWCO filter (Amicon) into the TFAM storage buffer (Table S1) following manufacturer’s instructions. Bulk phosphorylation of TFAM was conducted by diluting stored TFAM aliquots in a phosphorylation buffer (50 mM Tris–acetate pH 8.0, 100 mM potassium acetate, 5 mM magnesium acetate, 10 nM hPKAc, 0.2 mM ATP, 50 nM bovine serum albumin, and 2 mM DTT) and incubated at 30 °C for 16 h. The reaction was then immediately used for DNA compaction and LC-MS/MS analysis. Phospho-TFAM stocks were buffer exchanged into the TFAM storage buffer using an Amicon 3K MWCO filter to remove ATP and stored.

pUC19 plasmid was compacted by TFAM as above and split into acetylation reactions in which mock-incubated pUC19 plasmid (plasmid DNA in the incubation buffer), TFAM alone, 50 bp:TFAM, or 25 bp:TFAM samples were incubated with 5 mM lithium-acetyl-CoA (Sigma) for 1.5 h or 3 h. Each sample was split to maintain an equal amount of TFAM per sample. After incubation, each acetylation reaction was split for Western blot, EMSA, and SDS-PAGE analyses. For SDS-PAGE analysis, a 4 μl aliquot of 50 bp:TFAM and 2 μl aliquots of the TFAM only and 25 bp:TFAM samples (23 ng TFAM) were boiled and run on a 12.5% SDS-PAGE. For EMSA analysis, 9 μl of each reaction (92 ng TFAM) was loaded onto a 1% agarose gel with 10% glycerol and run at 4 °C for ∼3.5 h in the EMSA running buffer listed before. For Western blot analysis, 16 μl of 50 bp:TFAM reaction and 8 μl of each 25 bp:TFAM reaction and TFAM alone (190 ng TFAM) was boiled and run on 12.5% SDS-PAGE. Protein was transferred onto a nitrocellulose membrane (BioRad) using a Turboblot (BioRad). Detection used anti–acetyl-lysine antibody (Cell Signaling Antibody) according to manufacturer’s details and goat-anti-rabbit horseradish peroxidase-linked secondary antibodies. Image analysis was conducted using ImageQuant software (see SI).