Quantification of Mitochondrial Acetylation Dynamics Highlights Prominent Sites of Metabolic Regulation*

Lysine acetylation is rapidly becoming established as a key post-translational modification for regulating mitochondrial metabolism. Nonetheless, distinguishing regulatory sites from among the thousands identified by mass spectrometry and elucidating how these modifications alter enzyme function remain primary challenges. Here, we performed multiplexed quantitative mass spectrometry to measure changes in the mouse liver mitochondrial acetylproteome in response to acute and chronic alterations in nutritional status, and integrated these data sets with our compendium of predicted Sirt3 targets. These analyses highlight a subset of mitochondrial proteins with dynamic acetylation sites, including acetyl-CoA acetyltransferase 1 (Acat1), an enzyme central to multiple metabolic pathways. We performed in vitro biochemistry and molecular modeling to demonstrate that acetylation of Acat1 decreases its activity by disrupting the binding of coenzyme A. Collectively, our data reveal an important new target of regulatory acetylation and provide a foundation for investigating the role of select mitochondrial protein acetylation sites in mediating acute and chronic metabolic transitions.