Protein Composition and Subcellular Localization of the De Novo Lipogenic Metabolon
McKean, William Bennion, Jr.
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Fatty acids are the major components of triglycerides, phospholipids, and sphingolipids. Production of palmitate, the most abundant saturated fatty acid, involves the stepwise actions of three enzymes: ATP citrate lyase, acetyl-CoA carboxylase, and fatty acid synthase. Canonically each enzyme catalyzes discrete reactions, and it is thought that they localize diffusely in cellular cytoplasm separate from one another. If true, transfer of metabolic intermediates must occur through passive diffusion from one lipogenic enzyme to another. Such a model proposes an extremely inefficient and potentially hazardous method of palmitate production. We demonstrated that two related proteins - designated MIG12 and Spot 14 - modulate fatty acid synthesis and triglyceride production by regulating the polymerization and activity of acetyl-CoA carboxylase. To better characterize the relationship between these three proteins, biochemical properties of purified recombinant MIG12, Spot 14, and MIG12:Spot 14 heterodimer were assayed in combination with acetyl-CoA carboxylase. We found that Spot 14 abrogates the ability of MIG12 to polymerize and activate acetyl-CoA carboxylase. Co-immunoprecipitation studies using Spot 14 in rat liver revealed Spot 14 exists in a complex with fatty acid synthase and acetyl-CoA carboxylase. MIG12 and Spot 14 co-immunoprecipitation also revealed that ATP citrate lyase was in association with the complex, suggesting that these proteins can function as scaffolds for the three enzymes required for palmitate synthesis. Studies of the subcellular localization of these lipogenic proteins corroborated a functional interaction between these proteins. Confocal images of MIG12 and acetyl-CoA carboxylase in primary hepatocytes show filamentous structures that are immunofluorescent along junctions between the endoplasmic reticulum and mitochondria. Under high carbohydrate dietary conditions in which lipogenesis is stimulated, these structures expand to include fatty acid synthase, ATP citrate lyase, and Spot 14. They also co-localize around lipid droplets - storage organelles for excess triglycerides. Finally, the structural integrity of this lipogenic complex is shown to require microtubules. Blockade of microtubule formation inhibits proper formation of acetyl-CoA carboxylase structure and decreases total fatty acid synthesis in cells. Combined, these findings support the existence of a functional metabolon complex which facilitates the efficient channeling of fatty acid synthesis intermediates through an enzyme cascade that results in the production of palmitate at functionally relevant locations within the cell.