(A) Eukaryotic cells have maintained two distinct fatty acid synthesis pathways—the canonical cytoplasmic FASI and the mitochondrial FASII, which is homologous to the prokaryotic fatty acid synthesis pathway. FASII, which is dependent on ACP, is comprised of a set of monofunctional enzymes that catalyze the various steps in fatty acid synthesis. In order to facilitate mitochondrial fatty acid synthesis, apo-ACP must first be converted to holo-ACP by a 4’-phosphopanthetheine transferase (Ppt2), which catalyzes the covalent attachment of the 4-phosphopantetheine prosthetic group (4-PP) to an absolutely conserved Ser residue on ACP. The 4-PP contains a terminal thiol that serves as the attachment site to enable Acp1 to scaffold de novo fatty acid synthesis. The enzymes of FASII use malonyl-coA to initiate the fatty acid chain and acetyl-coA for acyl chain elongation. The canonical product of FASII is octanoate, which is the precursor for lipoic acid, an important mitochondrial cofactor. Following ACP-dependent octanoate synthesis, Lip2, Lip3, Lip5, and Gcv3 support the synthesis of lipoic acid and ligation to its target proteins including the E3 component of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. Ppt2 – 4’phophopantetheine transferase; MCT – malonyl-coA transferase; KAS – ketoacyl synthetase; KAR – ketoacyl reductase; HTD2 – hydroxyacyl-thioester reductase type 2; ETR – enoyl-thioester reductase; Lip2, Lip3, Lip5, Gcv3 – lipoic acid biosynthesis and ligation. (B) acp1Δ heterozygous diploids in the W303 strain background were dissected with and without a plasmid expressing Acp1-V5 and spores were grown on YPAD medium for 2 days. (C) ppt2Δ, lip5Δ, lip2Δ, lip3Δ, and gcv3Δ heterozygous diploids were dissected and spores were grown on YPAD medium for 2 days.