Acetyl-CoA carboxylase (ACC), the first committed enzyme in fatty acid (FA) synthesis, is regulated by phosphorylation/dephosphorylation, transcription, and an unusual mechanism of protein polymerization. Polymerization of ACC increases enzymatic activity and is induced in vitro by supraphysiological concentrations of citrate (>5 mM). Here, we show that MIG12, a 22 kDa cytosolic protein of previously unknown function, binds to ACC and lowers the threshold for citrate activation into the physiological range (<1 mM). In vitro, recombinant MIG12 induced polymerization of ACC (as determined by nondenaturing gels, FPLC, and electron microscopy) and increased ACC activity by >50-fold in the presence of 1 mM citrate. In vivo, overexpression of MIG12 in liver induced ACC polymerization, increased FA synthesis, and produced triglyceride accumulation and fatty liver. Thus, in addition to its regulation by phosphorylation and transcription, ACC is regulated at a tertiary level by MIG12, which facilitates ACC polymerization and enhances enzymatic activity.lipogenesis | SREBPs | steatosis F atty acids (FAs) are the essential components of phospholipids and provide the most important energy depot for the body in the form of triglycerides (TG). Newly synthesized FAs also serve as signaling molecules and modulators of transcription factor activity (1-3). Excess accumulation of FAs in tissues contributes to the pathogenesis of many common diseases, including insulin resistance, nonalcoholic fatty liver disease, diabetes, and cancer (4, 5). Given the central role of FAs in these diseases, the enzymes of FA biosynthesis are potential therapeutic targets.The first committed step in FA biosynthesis is carried out by acetyl-CoA carboxylase (ACC). ACC1 was isolated and characterized by Wakil et al. (6) and functions to carboxylate acetyl-CoA to form malonyl-CoA. Subsequently, a second ACC isoform, ACC2, was identified in mammals that is encoded by a separate gene but carries out the same enzymatic reaction (7,8).ACCs are members of a larger family of carboxylases that require biotin and ATP and use bicarbonate as a carbon donor. The malonyl-CoA produced by ACC can be used by FA synthase (FAS) for the sequential 2-carbon elongation reactions that generate palmitic acid (C16:0) in the cytosol. While both ACC1 and ACC2 produce malonyl-CoA, ACC1 is predominantly cytosolic and generates malonyl-CoA that is used by FAS to synthesize palmitic acid. ACC2 is associated with the mitochondrial membrane (9) and produces malonyl-CoA that serves to allosterically inhibit carnitine palmitoyl transferase I, the protein responsible for transport of long chain FAs into mitochondria for β-oxidation (10).Regulation of ACC1 and ACC2 occurs at multiple levels. ACC activity is acutely regulated by phosphorylation/dephosphorylation. AMP-activated protein kinase phosphorylates ACC and inhibits enzyme activity (11). In the fed state, excess glucose must be converted to FAs for energy storage in the form of TGs. Increased insulin signaling in the fed state res...