4a) (two belonging to CrAT). the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK. The recent epidemic surge in the rates of obesity and closely related metabolic diseases offers sparked intense research aimed at understanding the cellular and molecular consequences of persistent overnutrition (Ogden et al., 2014). Among many adverse results of chronic positive energy imbalance is a steady decay in mitochondrial performance (Lowell and Shulman, 2005). These organelles are increasingly recognized as a key regulatory hub intended for processes such as nutrient sensing, retrograde signaling, autophagy and cell survival, in addition to their well-established roles in ATP production and cellular bioenergetics (Pagliarini and Rutter, 2013). Accordingly, disease-associated perturbations in mitochondrial quality and function possess broad-ranging clinical and therapeutic implications. While many disease declares are characterized by perturbed expression of multiple genes involved in respiratory function (Mootha et al., 2003), dysregulation at the genomic level does SA 47 not fully explain the changes in mitochondrial bioenergetics frequently associated with obesity and diabetes (Holloszy, 2009). Also contributing to obesity-induced perturbations in mitochondrial performance are several posttranslational modifications (PTMs) that modulate stability, turnover, and/or function of mitochondrial proteins. Recent applications of mass spectrometry has drawn attention to lysine acetylation as a prominent mitochondrial PTM that is increasingly recognized as a marker of cellular energy stress (Dittenhafer-Reed et al., 2015; Hebert et al., 2013; Kendrick et al., 2011; Rardin et al., 2013; Still et al., 2013). Protein acetylation is a reversible modification in which a two-carbon acetyl group is covalently bound to SA 47 the -amino group of a lysine residue (Anderson and Hirschey, 2012). A growing number of reports provide evidence that acetylation of particular lysines can affect mitochondrial protein interactions, function and/or enzymatic activities (Bharathi et al., 2013; Hirschey, 2011; Hirschey et al., 2010; Hirschey et al., 2011; Jing et al., 2011; Still et al., 2013). The strongest evidence that these PTMs can impart adverse physiologic consequences comes from mice missing sirtuin three or more (SIRT3), a NAD+-dependent deacetylase that removes acetyl groups from specific lysine residues (Hebert et al., 2013; Newman et al., 2012; Rardin et al., 2013). SIRT3-deficient mice display different degrees of increased mitochondrial protein acetylation within key metabolic tissues and develop symptoms reminiscent of the metabolic syndrome when challenged by large fat feeding (Dittenhafer-Reed et al., 2015; Hirschey et al., 2011; Lantier et al., 2015). Whereas this field continues to be steadily gaining knowledge about the enzymes and physiological circumstances that regulate mitochondrial protein deacylation, the biological factors that influence the addition of acetyl groups to lysine side chains remain poorly comprehended. One idea gaining increasing traction suggests that unlike acylation reactions in other subcellular compartments, acetylation of mitochondrial proteins occurs mainly through non-enzymatic mechanisms as a consequence of mass action, rather than targeted catalysis (Ghanta et al., 2013; Wagner and Payne, 2013). This model predicts that physiological and nutritional conditions that raise mitochondrial concentrations of acetyl-CoA push these protein modifications by expanding the local pool of acetyl donors. Relevant to this hypothesis is evidence that overfeeding results in incomplete oxidation of carbon fuels, reflected by increased build up of mitochondrial-derived acylcarnitine species that originate from their corresponding acyl-CoA precursors (Koves et al., 2008). Taken with each other, these findings imply that chronic energy surplus results in a mismatch between substrate supply and demand, which in turn raises mitochondrial carbon load (Muoio, 2014). Also related to this general model are recent studies showing that mitochondrial acetyl-CoA balance can be nutritionally SA 47 SA 47 regulatedviathe carnitine-dependent enzyme, carnitine acetyltransferase (CrAT). This enzyme is most abundant in skeletal muscle and heart and localizes to the mitochondrial matrix (Muoio et al., 2012; Noland et al., 2009; Seiler et al., 2015). The freely reversible CrAT reaction interconverts short chain acyl-CoAs and their corresponding carnitine conjugates. Importantly, unlike their acyl-CoA precursors, acylcarnitine metabolites can traverse the inner mitochondrial membrane and thereby permit mitochondrial efflux of excess acyl moieties. These findings raise the intriguing possibility that this acetyl group buffering system plays a key role in mitigating nutrient-induced acetylation of mitochondrial proteins. The goal of the current study was to test the hypothesis that macronutrient load and carnitine-mediated acetyl group buffering directly effect mitochondrial protein acetylation in skeletal muscle. To this end, we used a label-free quantitative mass spectrometry-based acetylproteomics approach to look at the MAM3 consequences of skeletal muscle-specific CrAT deficiency on diet-induced acetylation of mitochondrial proteins..