Mitochondrial fatty acid synthesis coordinates mitochondrial oxidative metabolism

Abstract

19Cells harbor two systems for fatty acid synthesis, one in the cytoplasm (catalyzed by fatty 20 acid synthase, FASN) and one in the mitochondria (mtFAS). In contrast to FASN, mtFAS is 21 poorly characterized, especially in higher eukaryotes, with the major product(s), metabolic roles, 22 and cellular function(s) being essentially unknown. Here we show that hypomorphic mtFAS 23 mutants display a severe loss of electron transport chain (ETC) complexes and exhibit 24 compensatory metabolic activities including reductive carboxylation. This effect on ETC 25 complexes appears to be independent of protein lipoylation, the best characterized function of 26 2 mtFAS, as mutants lacking lipoylation have an intact ETC. Finally, mtFAS impairment blocks the 27 differentiation of skeletal myoblasts in vitro. Together, these data suggest that ETC activity in 28 mammals is profoundly controlled by mtFAS function, thereby connecting anabolic fatty acid 29 synthesis with the oxidation of carbon fuels. 30 31 Keywords: 32 Mitochondria, fatty acid synthesis, electron transport chain, reductive carboxylation, lipoylation, 33 TCA cycle, metabolism, respiration, OXPHOS, muscle 34 35 Abbreviations: 36 ACP -acyl carrier protein, ETC -electron transport chain, FAS -fatty acid synthesis, FASN -37 fatty acid synthase, LYRM -leucine tyrosine arginine motif, mtFAS -mitochondrial fatty acid 38 synthesis, OGDH -oxoglutarate dehydrogenase, OXPHOS -oxidative phosphorylation, PAGE 39 -polyacrylamide gel electrophoresis, PDH -pyruvate dehydrogenase, TCA -tricarboxylic acid 40 41 Introduction 42 43Fatty acids play diverse cellular roles, including providing the hydrophobic tails of membrane 44 phospholipids, energy storage in the form of triglycerides, and as cell signaling molecules. Aside 45 from the import of exogenous fatty acids, mammalian cells use the well-known and well-studied 46 cytoplasmic enzyme fatty acid synthase (FASN) to make palmitate, which is further modified to 47 form the diverse array of cellular fatty acids (Smith, 1994). However, it is much less appreciated 48 that mitochondria also harbor a spatially and genetically distinct fatty acid synthesis pathway 49 (mtFAS) (reviewed in (Nowinski et al., 2018)). In contrast to FASN, which is a very large protein 50 that contains several domains and encompasses all of the enzymatic activities necessary for 51 FAS condensed in a single polypeptide chain, the mtFAS pathway is comprised of at least six 52 enzymes all encoded by separate genes. These enzymes catalyze sequential steps to achieve 53 one cycle of two-carbon addition to a growing acyl chain ( Figure 1A). The nascent fatty acids 54 are covalently attached to the mitochondrial acyl carrier protein (ACP), which acts as a soluble 55 scaffold upon which the acyl chains are built. In each cycle of the pathway, malonyl-CoA is 56 converted to malonyl-ACP, which then undergoes a condensation reaction with the growing fatty 57 acyl chain on ACP, extending the chain by two carbons and releasing a CO2 molecule. 58Thereafter, the remaining...

Recessive pathogenic variants inMCATcause combined oxidative phosphorylation deficiency

“…We therefore assessed how reexpression of wild type and p.T271I human MCAT affects mitochondrial respiration. In comparison to controls, Mcat hypomorphic mutant C2C12 exhibited significantly decreased basal and maximal respiration rates, as previously described (Figure 4B,C) [5]. Expression of a mitochondrially targeted DSRed construct (mtDSRed) minimally affected respiration in the Mcat mutant cells, while expression of wild type human MCAT fully rescued both basal and maximal respiration rates.…”

“…Mcat clones have a 30-40% decreased basal respiration rate compared to controls, which is explained by markedly decreased levels of fully assembled complexes I, II, and IV. Metabolomics and carbon-labeling studies are all consistent with decreased function of the TCA cycle and oxidative phosphorylation in the hypomorphic Mcat mutant cells [5]. This work demonstrates that the role of mtFAS extends beyond protein lipoylation and includes a crucial role in the maintenance of ETC activity.…”

“…These cells display a roughly 90% reduction in MCAT expression, and similar to the proband fibroblasts have defects in assembly and stability of ETC complexes I, II, and IV. However, the hypomorphic Mcat C2C12 lines also have undetectable protein lipoylation by western blot [5]. Re-expression of wild type and p.T271I human MCAT rescued protein lipoylation (Figure 4A).…”

“…Although rescue of fully assembled electron transport chain complexes in patient fibroblasts by re-expression of MCAT was incomplete, there are several difficulties associated with this approach, including a lack of isogenic control cell lines and high variability among control cell lines, along with low infection efficiencies in the proband fibroblasts. We therefore decided to test whether human MCAT with the p.T271I mutation could efficiently restore protein lipoylation, ETC complex assembly, and cellular respiration in our previously generated hypomorphic Mcat mutant C2C12 cell lines [5]. These cells display a roughly 90% reduction in MCAT expression, and similar to the proband fibroblasts have defects in assembly and stability of ETC complexes I, II, and IV.…”

“…Cells were harvested with 0.25% trypsin-EDTA (Gibco, 25200-072), pelleted, washed with sterile PBS (Gibco, 10010-023), pelleted again and frozen in liquid nitrogen. Mitochondria were isolated as previously described [5]. Briefly, cells were suspended in 1 mL CP-1 buffer (100mM KCl, 50 mM Tris-HCl, 2 mM EGTA, pH 7.4), lysed by passing through a 27-gauge needle, and centrifuged at 700 x g to pellet debris.…”