Evidence for physical association of mitochondrial fatty acid oxidation and oxidative phosphorylation complexes

Abstract

Fatty acid β-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are key pathways involved in cellular energetics. Reducing equivalents from FAO enter OXPHOS at the level of complexes I and III. Genetic disorders of FAO and OXPHOS are among the most frequent inborn errors of metabolism. Patients with deficiencies of either FAO or OXPHOS often show clinical and/or biochemical findings indicative of a disorder of the other pathway. In this study, the physical and functional interactions between these pathways were examined. Extracts of isolated rat liver mitochondria were subjected to blue native polyacrylamide gel electrophoresis (BNGE) to separate OXPHOS complexes and supercomplexes followed by Western blotting using antisera to various FAO enzymes. Extracts were also subjected to sucrose density centrifugation and fractions analyzed by BNGE or enzymatic assays. Several FAO enzymes co-migrated with OXPHOS supercomplexes in different patterns in the gels. When palmitoyl-CoA was added to the sucrose gradient fractions containing OXPHOS supercomplexes in the presence of potassium cyanide, cytochrome c was reduced. Cytochrome c reduction was completely blocked by myxothiazol (a complex III inhibitor) and 3-mercaptopropionate (an inhibitor of the first step of FAO), but was only partially inhibited by rotenone (a complex I inhibitor). Although palmitoyl-CoA and octanoyl-CoA provided reducing equivalents to OXPHOS-containing supercomplex fractions, no accumulation of their intermediates was detected. In contrast, short branched acyl-CoA substrates were not metabolized by OXPHOS-containing supercomplex fractions. These data provide evidence of a multifunctional FAO complex within mitochondria that is physically associated with OXPHOS supercomplexes and promotes metabolic channeling.

FIGURE 1.

Identification of ETC supercomplexes by BNGE. A, rat liver mitochondria were permeabilized with digitonin and resolved by BNGE followed by Coomassie staining to visualize ETC bands. Molecular mass and ETC activity stains identify the top two bands as ETC supercomplexes (SC). The migration of the individual ETC complexes is also indicated. B, permeabilized rat liver mitochondria were subjected to sucrose density gradient centrifugation, and the resulting 10 fractions were separated by BNGE and visualized with Coomassie Blue. The supercomplexes and complex I appeared in the first three fractions. These fractions were pooled and designated as supercomplex (SC) containing fraction in subsequent experiments.

FIGURE 2.

Supercomplexes react with antibodies against FAO proteins. A, relative migration of SC bands and complex I on a blue native gel. Complex I activity stain reacts with the individual complex I band as well as the SC bands. B, the complexes were transferred from the blue native gel to a nylon membrane and Western blotted with antibodies against FAO enzymes including the acyl-CoA dehydrogenases (very long chain acyl-CoA dehydrogenase (VLCAD), long chain acyl-CoA dehydrogenase (LCAD), medium chain acyl-CoA dehydrogenase (MCAD)), their redox partner ETF, and the mitochondrial trifunctional protein (TFP). The supercomplex (SC) and complex I bands reacted variably with these antibodies. In contrast, an antibody against isovaleryl-CoA dehydrogenase, an acyl-CoA dehydrogenase active in amino acid degradation rather than FAO, did not react with either the SC or complex I bands. C and D, the gel strip from A was treated with Laemmli sample buffer for 30 min, placed at the top of a 12.5% SDS-PAGE gel, subjected to electrophoresis, and either treated with silver staining (C) or Western blotted with purified VLCAD antibody (D). The scanned images were superimposed to identify the location of VLCAD among ETC complexes and supercomplexes (arrows). I, V, III, and IV: complexes I, V, III, and IV, respectively.

FIGURE 3.

Verification of the presence of FAO enzymes in ETC supercomplexes by SDS-PAGE and Western blotting. Permeabilized rat liver mitochondria were fractionated by sucrose density centrifugation. The supercomplex (SC)-containing fractions were pooled and resolved by SDS-PAGE followed by Western blotting for very long chain acyl-CoA dehydrogenase (VLCAD), long chain acyl-CoA dehydrogenase (LCAD), medium chain acyl-CoA dehydrogenase (MCAD), short chain acyl-CoA dehydrogenase (SCAD), ETF, and isovaleryl-CoA dehydrogenase (IVD). Purified recombinant proteins (P) were used as positive control for each blot. All of the FAO proteins were present in the supercomplex fractions whereas the closely related enzyme isovaleryl-CoA dehydrogenase was not. (ETF is a heterodimer, thus there are two bands representing the α and β subunits.)

FIGURE 4.

ETC supercomplexes demonstrate enzymatic activity for ACADs involved in FAO but not for ACADs involved in amino acid degradation. Permeabilized rat liver mitochondria were fractionated by sucrose density centrifugation. The supercomplex-containing fractions were pooled and used for ACAD activity assays with straight chain acyl-CoA substrates for very long chain ACAD and long chain ACAD (C20–C14), medium chain acyl-CoA dehydrogenase (C12–C8), and short chain ACAD (C6–C4). Considerable activity was detected with these substrates. In contrast, only trace activity was detected with the branched chain acyl-CoA substrates for isovaleryl-CoA dehydrogenase (iC5), isobutyryl-CoA dehydrogenase (iC4), short branched chain ACAD (2-methylbutyryl-CoA (2MeC4)), or glutaryl-CoA dehydrogenase (Glutaryl). Columns represent means ± S.D (error bars) of triplicate assays with the corresponding acyl-CoA substrate.

FIGURE 5.

Supercomplex-containing fractions show FAO substrate channeling. The pooled supercomplex fraction was incubated with palmitoyl-CoA, and the reaction products were analyzed by HPLC. A, curve a, palmitoyl-CoA at 50 μm is shown. Retention time is 38.5 min. Curve b, palmitoyl-CoA was incubated with 2 of μg purified very long chain ACAD in the presence of ETF, NAD⁺, CoASH, and ATP. The retention time for the generated C16-enoyl-CoA is 40.5 min. These were used as standards for subsequent reactions with the supercomplex fractions. B, supercomplex fraction (2.0 μg of protein) was incubated with 0.5 nm palmitoyl-CoA in the presence of ETF, NAD⁺, CoASH, and ATP. Curve a, zero time shows the initial amount of palmitoyl-CoA. Curve b, after 20 min the amount of palmitoyl-CoA was dramatically reduced, but there was no accumulation of enoyl-CoA or other intermediate products, indicating efficient channeling of the FAO substrates by the supercomplex fraction. Curve c, supercomplex fraction was preincubated for 10 min with the long chain ACAD/very long chain ACAD inhibitor MPA prior to reaction with palmitoyl-CoA in the presence of ETF, NAD⁺, CoASH, and ATP. MPA effectively inhibited the metabolism of palmitoyl-CoA by the supercomplex-containing fraction.

SCHEME 1.

Relationship between FAO and the ETC. FAO provides reducing equivalents directly to the ETC through two mechanisms. In the first mechanism, reducing equivalents from ACADs are transferred via ETF to ETF:ubiquinone oxidoreductase (also known as ETF dehydrogenase), resulting in reduction of CoQ, the substrate for complex III. In the second mechanism, NAD is reduced by 3-hydroxyacyl-CoA dehydrogenase to form NADH, the substrate for complex I. 3-Mercaptopropionic acid (MPA) inhibits the long chain ACADs. Myxothiazol and rotenone are inhibitors of complex III and complex I, respectively. KCN inhibits the electron transfer from cytochrome c to complex IV.

FIGURE 6.

ETC supercomplexes bridge the activities of FAO and the ETC. A, supercomplexes isolated by sucrose density gradient centrifugation were incubated with palmitoyl-CoA and cytochrome c. The addition of exogenous ETF was required to drive the flow of electrons from palmitoyl-CoA through the ETC to cytochrome c, as measured by an increase in absorbance at 550 nm. Myxothiazol, a complex III inhibitor, blocks the reaction. B, complex I inhibitor rotenone reduces bridging activity by about half by blocking the contribution of electrons from the 3-hydroxyacyl-CoA dehydrogenase step of fatty acid oxidation (see Scheme 1). C, MPA, an inhibitor of long chain ACAD and very long chain ACAD, completely inhibited the bridging activity. The ETC was still functional and responsive to stimulation by exogenous NADH. Arrowheads indicate the time at which each reagent was added to the reaction.

FIGURE 7.

ACAD enzymatic activity and FAO-ETC bridging co-separate in sucrose gradient fractions. A, ACAD activity was measured in sucrose gradient fractions using palmitoyl-CoA as substrate. The highest activity was in fractions 1–4, which contain ETC supercomplexes. B, bridging activity was also the highest in the supercomplex-containing fractions. Bridging was measured as palmitoyl-CoA reduction of cytochrome c reduction (absorbance at 550 nm). Columns represent means ± S.D. (error bars) of triplicate assays.