Figure 1.
Fatty acid catabolism in ascomycetous yeasts.
Model adapted from [3], [4]. 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.
Table 1.
Growth characteristics of mutant, reintegrant, and wild-type strains on different carbon sources at 30°C.
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 14Cα-palmitoyl-CoA by cellular crude extracts of the C. lusitaniae icl1Δ, fox2Δ, pxa1Δ and wild-type strains.
(A) 14Cα-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 14Cα-palmitoyl-CoA initially present in the reaction mix. WT: wild-type strain. P: 14Cα-palmitoyl-CoA. E: 14Cα-hexadecenoyl-CoA. H: 14Cα-3-hydroxyhexadecenoyl-CoA. S: start.
Figure 4.
Consumption of 14Cα-palmitoyl-CoA by peroxisomal and mitochondrial fractions of the C. lusitaniae fox2Δ, pxa1Δ and wild-type strains.
(A) 14Cα-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 14Cα-palmitoyl-CoA initially present in the reaction mix. WT: wild-type strain. P: 14Cα-palmitoyl-CoA. E: 14Cα-hexadecenoyl-CoA. H: 14Cα-3-hydroxyhexadecenoyl-CoA. S: start.
Table 2.
Purification of peroxisomes and mitochondria from cells grown in induction medium.
Figure 5.
Consumption of 14Cα-palmitoyl-CoA by different quantity of proteins from the peroxisomal and mitochondrial fractions of the C. lusitaniae wild-type strain.
14Cα-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 14Cα-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.
Table 3.
Number and size of peroxisomes and number of mitochondria sections observed in Candida lusitaniae cells by electron microscopy.