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. 2016 Dec 7;6(12):4047-4058.
doi: 10.1534/g3.116.034389.

Mitochondrial Carriers Link the Catabolism of Hydroxyaromatic Compounds to the Central Metabolism in Candida parapsilosis

Affiliations

Mitochondrial Carriers Link the Catabolism of Hydroxyaromatic Compounds to the Central Metabolism in Candida parapsilosis

Igor Zeman et al. G3 (Bethesda). .

Abstract

The pathogenic yeast Candida parapsilosis metabolizes hydroxyderivatives of benzene and benzoic acid to compounds channeled into central metabolism, including the mitochondrially localized tricarboxylic acid cycle, via the 3-oxoadipate and gentisate pathways. The orchestration of both catabolic pathways with mitochondrial metabolism as well as their evolutionary origin is not fully understood. Our results show that the enzymes involved in these two pathways operate in the cytoplasm with the exception of the mitochondrially targeted 3-oxoadipate CoA-transferase (Osc1p) and 3-oxoadipyl-CoA thiolase (Oct1p) catalyzing the last two reactions of the 3-oxoadipate pathway. The cellular localization of the enzymes indicates that degradation of hydroxyaromatic compounds requires a shuttling of intermediates, cofactors, and products of the corresponding biochemical reactions between cytosol and mitochondria. Indeed, we found that yeast cells assimilating hydroxybenzoates increase the expression of genes SFC1, LEU5, YHM2, and MPC1 coding for succinate/fumarate carrier, coenzyme A carrier, oxoglutarate/citrate carrier, and the subunit of pyruvate carrier, respectively. A phylogenetic analysis uncovered distinct evolutionary trajectories for sparsely distributed gene clusters coding for enzymes of both pathways. Whereas the 3-oxoadipate pathway appears to have evolved by vertical descent combined with multiple losses, the gentisate pathway shows a striking pattern suggestive of horizontal gene transfer to the evolutionarily distant Mucorales.

Keywords: 3-oxoadipate pathway; catabolism of hydroxybenzoates; evolution of biochemical pathways; gentisate pathway; mitochondrial carrier.

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Figures

Figure 1
Figure 1
Relative mRNA expression of C. parapsilosis genes involved in the 3-OAP (MNX1, MNX3, HDX1, OSC1, and OCT1) and GP (MNX2, GDX1, and FPH1). RNA samples were prepared from CLIB214 cells grown in synthetic medium supplemented with 2% glucose (SD, bars no. 1), 10 mM 3-hydroxybenzoate (S3OH, bars no. 2), 10 mM 4-hydroxybenzoate (S4OH, bars no. 3), or 3% glycerol (SGly, bars no. 4). Quantification of mRNA was performed as described in Materials and Methods. Relative expression was normalized to EFB1 gene expression. The assays were performed in at least three independent experiments with two parallel replicates in each case (error bars, mean ± SEM) and the significance of differences between the samples (S3OH, S4OH, and SGly) and the control (SD) was evaluated by Student’s t-test (* P < 0.05).
Figure 2
Figure 2
Intracellular localization of Hdx1p, Osc1p, Oct1p involved in the 3-OAP and Fph1p involved in the GP. C. parapsilosis strain CDU1 was transformed with pBP8-derived plasmid constructs expressing indicated proteins fused with yEGFP3. Transformants with the vector pBP8 were used as a control. Cells were grown at 28° in synthetic media containing 3-hydroxybenzoate (GP), or 4-hydroxybenzoate (3-OAP), as a sole carbon source, and examined by fluorescence microscopy. Mitochondria were stained with MitoTracker Red CMXRos as described in Materials and Methods. Bar, 25 μm.
Figure 3
Figure 3
Relative mRNA expression of the C. parapsilosis MC genes (CRC1, DIC1, LEU5, MPC1, MPC3, OAC1, ODC1, SFC1, YHM2, YMC1, and YMC2). RNA samples were prepared from CLIB214 cells grown in synthetic medium supplemented with 2% glucose (SD), 10 mM 3-hydroxybenzoate (S3OH), 10 mM 4-hydroxybenzoate (S4OH), or 3% glycerol (SGly). Quantification of mRNA was performed as described in Materials and Methods. Relative expression was normalized to EFB1 gene expression. The assays were performed in at least three independent experiments, with two parallel replicates in each case (error bars, mean ± SEM), and the significance of differences between the samples (S3OH, S4OH, and SGly) and the control (SD) was evaluated by Student’s t-test (* P < 0.05, ** P < 0.01, *** P < 0.001).
Figure 4
Figure 4
Functional complementation of S. cerevisiae Δsfc1 by the SFC1 gene from C. parapsilosis. Strains BY4742 and BY4742 Δsfc1 transformed with pYES2/CT (control) and pYES2/CT-SFC1 were spotted as serial dilutions onto SGlyGal0.1 plates. Growth was evaluated either after 2 d incubation at 28° (left panel) or after 7 d at 37° (middle panel) and 20° (right panel).
Figure 5
Figure 5
Intracellular localization of C. parapsilosis Sfc1p. S. cerevisiae strains BY4742 and BY4742 Δsfc1 were transformed with plasmid pUG36-SFC1. C. parapsilosis strain SR23 was transformed with plasmid pPK6-SFC1. Cells transformed with the vector pUG36 (S. cerevisiae) and pPK6 (C. parapsilosis) were used as controls. Transformants were grown at 28° in SGlyGal2.0 medium and examined by fluorescence microscopy. DNA in cells was stained with DAPI as described in Materials and Methods. Bar, 25 μm.
Figure 6
Figure 6
Proposed integration of the 3-OAP and GP with mitochondrial metabolism through MC proteins. The enzymes of the 3-OAP and GP are highlighted with bold letters. Enzyme abbreviations: Mnx1, 4-hydroxybenzoate 1-hydroxylase; Mnx3, hydroquinone hydroxylase; Hdx1, hydroxyquinol 1,2-dioxygenase; Osc1, 3-oxoadipate CoA-transferase; Oct1, 3-oxoadipyl-CoA thiolase; Mnx2, 3-hydroxybenzoate 6-hydroxylase; Gdx1, gentisate 1,2-dioxygenase; Fph1, fumarylpyruvate hydrolase. Carrier acronyms: Odc1, oxodicarboxylate carrier; Sfc1 succinate/fumarate carrier; Leu5, CoA carrier; Dic1, dicarboxylate carrier; Yhm2, oxoglutarate/citrate carrier; Oac1, oxaloacetate/isopropylmalate carrier; Mpc1 and 3, subunits of pyruvate carrier; Crc1, carnitine carrier; Aac1, ADP/ATP carrier; Mir1, phosphate carrier; GSH, glutathione.
Figure 7
Figure 7
Molecular phylogenies of proteins encoded by HDX1 and GDX1 genes. The sequences of key enzymes of the 3-OAP and GP, i.e., Hdx1 (upper panel) and Gdx1 (lower panel), respectively, were used for constructing the phylogenies. The whole tree structure is shown schematically, with lineages colored according to their taxonomic classification: Pezizomycotina in red, Basidiomycotina in blue, Mucoromycotina in green, Saccharomycotina in yellow, Taphrinomycotina in pink, and Bacteria in cyan. Saccharomycotina-containing subtrees are shown in more detail in the corresponding insets, to show the name of the species.
Figure 8
Figure 8
Phylogenetic profile showing the presence and absence of genes of the 3-OAP (left panel) and GP (right panel) across several sequenced Saccharomycotina species. The species phylogeny has been adapted from Gerecova et al. (2015). Presence and absence profiles are based on results from a Blast search (E-value < 10−25), which are indicated with the following colors: black (no homolog), violet (exactly one homolog), and red (two or more homologs).

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