The cytochrome b5 dependent C-5(6) sterol desaturase DES5A from the endoplasmic reticulum of Tetrahymena thermophila complements ergosterol biosynthesis mutants in Saccharomyces cerevisiae

Abstract

Tetrahymena thermophila is a free-living ciliate with no exogenous sterol requirement. However, it can perform several modifications on externally added sterols including desaturation at C5(6), C7(8), and C22(23). Sterol desaturases in Tetrahymena are microsomal enzymes that require Cyt b(5), Cyt b(5) reductase, oxygen, and reduced NAD(P)H for their activity, and some of the genes encoding these functions have recently been identified. The DES5A gene encodes a C-5(6) sterol desaturase, as shown by gene knockout in Tetrahymena. To confirm and extend that result, and to develop new approaches to gene characterization in Tetrahymena, we have now, expressed DES5A in Saccharomyces cerevisiae. The DES5A gene was codon optimized and expressed in a yeast mutant, erg3Δ, which is disrupted for the gene encoding the S. cerevisiae C-5(6) sterol desaturase ERG3. The complemented strain was able to accumulate 74% of the wild type level of ergosterol, and also lost the hypersensitivity to cycloheximide associated with the lack of ERG3 function. C-5(6) sterol desaturases are expected to function at the endoplasmic reticulum. Consistent with this, a GFP-tagged copy of Des5Ap was localized to the endoplasmic reticulum in both Tetrahymena and yeast. This work shows for the first time that both function and localization are conserved for a microsomal enzyme between ciliates and fungi, notwithstanding the enormous evolutionary distance between these lineages. The results suggest that heterologous expression of ciliate genes in S. cerevisiae provides a useful tool for the characterization of genes in Tetrahymena, including genes encoding membrane protein complexes.

Fig. 1

Phylogenetic analysis of cytochrome b5 proteins. The phylogenetic tree of 43 amino acid sequences (UniProtKB) was generated using a Maximum likelihood method with 500 bootstrap replicates with MEGA5 software, as described in Materials and Methods. The black rhombus indicate Tetrahymena thermophila putative cytb5 enzymes. The black box indicates a Mus musculus Acyl-CoA desaturase protein, containing a Cytb₅ domain, which is used as an outgroup. The bar indicates percentages of substitution.

Fig. 2

Hypersensitivity to cycloheximide in the erg3 null strain is resuced by expression of the DES5A gene. Serial dilutions of the wild type, erg3 null and erg3+pC-5T complemented strains were spotted onto plates containing minimum medium plus 0.01 μg/ml of cycloheximide and grown for 4 days at 28 °C.

Fig. 3

GC-MS analysis of sterols. A: S. cerevisiae WT 303. B: erg3 null. C: erg3+p C-5T strain. D and E: mass spectra of 5α-ergosta-7,22-dien-3β-ol and ergosterol trimethylsilyl derivatives respectively. The compounds were identified with the NIST library. Peaks: 1 - zymosterol, 2 - 5α-ergosta-8,22-dien-3β-ol, 3 - ergosterol, 4 - 5α-ergosta-7,22-dien-3β-ol, 5 - fecosterol, 6 - 5α-ergosta-8-en-3β-ol, 7 - 5α-ergosta-7,24-dien-3β-ol, 8 - 5α-ergosta-7-en-3β-ol, 9 - lanosterol.

Fig. 4

Localization of GFP tagged Des5Ap in Tetrahymena thermophila (A) Schematic representation of gene replacement in the WT locus of the DES5A gene by targeting the DES5A-GFP construct by homologous recombination, using a cassette in which neo4 confers paramomycin resistance. (B) Immunofluorescence localization of GFP-tagged DES5A. Wild-type (WT) and DES5A-GFP strains were grown to Log phase and fixed for indirect immunofluorescence staining. The GFP was localized by anti-GFP primary antibody followed by labeling with anti-rabbit–Alexa Fluor 488 secondary antibody. DIC: Differential interference contrast microscopy. White arrows indicate the nucleus of the ciliate.

Fig. 5

Localization of GFP-tagged Des5Ap in Saccharomyces cerevisiae. The erg3 strain expressing GFP by itself (top) and GFP-tagged DES5A (bottom) were grown in minimum medium and analyzed at early log phase by direct fluorescence microscopy. DNA was stained with Hoechst.