The mitochondrial lysine acetylome of Arabidopsis

• More than 100 mitochondrial proteins of Arabidopsis are lysine-acetylated.
• All proteins from the TCA cycle were found to be lysine-acetylated.
• Lysine-acetylated proteins can be detected in native ETC protein complexes.
• Mitochondrially-encoded proteins of complex V are lysine-acetylated.
• Lysine acetylation can occur non-enzymatically inside mitochondria.

Posttranslational modifications are essential regulators of protein functions as they can modify enzyme activities or protein–molecule interactions by changing the charge state or chemical properties of their target amino acid. The acetyl moiety of the central energy metabolite acetyl-CoA can be transferred to the ε-amino group of lysine, a process known as lysine acetylation which is implicated in the regulation of key metabolic enzymes in various organisms. Since plant mitochondria are of great importance for plant growth and development and as they house key enzymes of oxidative phosphorylation and photorespiration, it is essential to investigate the occurrence of lysine acetylation in this organelle. Here we characterised the plant mitochondrial acetylome of Arabidopsis mitochondria by LC-MS/MS analysis. In total 120 lysine-acetylated mitochondrial proteins containing 243 acetylated sites were identified. These proteins were mapped into functional categories showing that many proteins with essential functions from the tricaboxylic cycle and the respiratory chain are lysine-acetylated, as well as proteins involved in photorespiration, amino acid and protein metabolism, and redox regulation. Immuno-detection of mitochondrial proteins revealed that many lysine-acetylated proteins reside in native protein complexes. Furthermore, in vitro experiments demonstrated that lysine acetylation can occur non-enzymatically in Arabidopsis mitochondria at physiological matrix pH.