A Fox2-dependent fatty acid ß-oxidation pathway coexists both in peroxisomes and mitochondria of the ascomycete yeast Candida lusitaniae

Abstract

It is generally admitted that the ascomycete yeasts of the subphylum Saccharomycotina possess a single fatty acid ß-oxidation pathway located exclusively in peroxisomes, and that they lost mitochondrial ß-oxidation early during evolution. In this work, we showed that mutants of the opportunistic pathogenic yeast Candida lusitaniae which lack the multifunctional enzyme Fox2p, a key enzyme of the ß-oxidation pathway, were still able to grow on fatty acids as the sole carbon source, suggesting that C. lusitaniae harbored an alternative pathway for fatty acid catabolism. By assaying 14Cα-palmitoyl-CoA consumption, we demonstrated that fatty acid catabolism takes place in both peroxisomal and mitochondrial subcellular fractions. We then observed that a fox2Δ null mutant was unable to catabolize fatty acids in the mitochondrial fraction, thus indicating that the mitochondrial pathway was Fox2p-dependent. This finding was confirmed by the immunodetection of Fox2p in protein extracts obtained from purified peroxisomal and mitochondrial fractions. Finally, immunoelectron microscopy provided evidence that Fox2p was localized in both peroxisomes and mitochondria. This work constitutes the first demonstration of the existence of a Fox2p-dependent mitochondrial β-oxidation pathway in an ascomycetous yeast, C. lusitaniae. It also points to the existence of an alternative fatty acid catabolism pathway, probably located in peroxisomes, and functioning in a Fox2p-independent manner.

Figure 1. Fatty acid catabolism in ascomycetous yeasts.

Model adapted from , . The β-oxidation pathway is exclusively peroxisomal. The localization of the specific enzymes of the glyoxylate cycle (Icl1p and Mls1p) is susceptible to variations according to the species (i. e. Icl1p is peroxisomal in C. albicans and cytoplasmic in S. cerevisiae). Mls1p: Malate synthase. CAT: carnitine acetyl-transferase.

Figure 2. Neutral lipid composition of the wild-type, fox2Δ and pxa1Δ strains.

(A) Thin-layer chromatography (TLC) of lipid extracts obtained from the wild-type, fox2Δ and pxa1Δ strains after growth on YPD or YNB +2% C16:0. The silica gel plates were observed under UV light after spraying of primuline. PL: phospholipids, TAG: tri-acyl-glycerol. (B) Amount of FA contained in PL (light gray) and TAG (dark gray) fractions purified by TLC and quantified by gas-chromatography. C17:0 was used as standard. The sum of the PL and TAG fractions are represented with black bars. Error bars represent SE.

Figure 3. Consumption of ¹⁴C_α-palmitoyl-CoA by cellular crude extracts of the C. lusitaniae icl1Δ, fox2Δ, pxa1Δ and wild-type strains.

(A) ¹⁴C_α-palmitoyl-CoA consumption rates after 15 min at 37°C by crude extracts of the mutant and wild-type strains after analysis of the chromatograms using ImageQuant software. Error bars represent SE. (B) Corresponding chromatograms. T0: Amount of ¹⁴C_α-palmitoyl-CoA initially present in the reaction mix. WT: wild-type strain. P: ¹⁴C_α-palmitoyl-CoA. E: ¹⁴C_α-hexadecenoyl-CoA. H: ¹⁴C_α-3-hydroxyhexadecenoyl-CoA. S: start.

Figure 4. Consumption of ¹⁴C_α-palmitoyl-CoA by peroxisomal and mitochondrial fractions of the C. lusitaniae fox2Δ, pxa1Δ and wild-type strains.

(A) ¹⁴C_α-palmitoyl-CoA consumption rates after 15 min at 37°C by peroxisomal and mitochondrial fractions of the mutant and wild-type strains after analysis of the chromatograms using ImageQuant software. The activity observed in the mitochondrial fraction of fox2Δ and wild-type strains were compared using a Student's t-test (*, p = 0.016). Error bars represent SE. (B) Corresponding chromatograms. T0: Amount of ¹⁴C_α-palmitoyl-CoA initially present in the reaction mix. WT: wild-type strain. P: ¹⁴C_α-palmitoyl-CoA. E: ¹⁴C_α-hexadecenoyl-CoA. H: ¹⁴C_α-3-hydroxyhexadecenoyl-CoA. S: start.

Figure 5. Consumption of ¹⁴C_α-palmitoyl-CoA by different quantity of proteins from the peroxisomal and mitochondrial fractions of the C. lusitaniae wild-type strain.

¹⁴C_α-palmitoyl-CoA consumption rates after 2 min at 37°C by peroxisomal (diamonds) and mitochondrial (squares) fractions of the wild-type strains. Activity is expressed as nmol of ¹⁴C_α-palmitoyl-CoA consumed per min after analysis of the chromatograms using ImageQuant software. Error bars represent SE.

Figure 6. Immunodetection of Fox2p, Icl1p and cytochrome c by Western-blot in the C. lusitaniae fox2Δ and wild-type strains.

(A) Thirty µg of proteins of the crude extracts and 10 µg of proteins of the peroxisomal and mitochondrial fractions were separated by SDS-PAGE. (B) Signal integration expressed in relative intensity using Quantity One software. Relative amounts of Fox2p are represented with black bars, Icl1p with grey bars, and Cytc with white bars. Error bars represent SE. Cytc: cytochrome c, WT: wild type, CE: crude extract, P: peroxisomal fraction, M: mitochondrial fraction.

Figure 7. Characterization of the wild-type and fox2Δ strains by electron and immunoelectron microscopy.

Glucose-grown cells (A and B) or oleate-induced cells (C to F) of wild-type (A, C, and E) or fox2Δ (B, D, and F) strains were fixed and prepared for electron microscopy (A to D) or immunoelectron microscopy (E and F). In E and F, cryosections were incubated with a polyclonal hen primary antibody directed against C. lusitaniae Fox2p and antibodies were revealed with immunogold particles conjugated to anti-hen antibodies. P, peroxisome, M, mitochondrion, N, nucleus. Bars, 200 nm.