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 b5, Cyt b5 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.

Lathosterol oxidase (sterol C5-desaturase) deletion confers resistance to amphotericin B and sensitivity to acidic stress inLeishmania major

“…1B-F), suggesting that LSO is primarily located at the ER. This result is similar to the localizations of C14DM and SMT in Leishmania (18, 19), as well as LSO in T. thermophila (28).…”

Introducing the concept of biocatalysis in the classroom: The conversion of cholesterol to provitamin D3

“…5B, the Des7p:eGFP strain showed a strong signal around the nucleus, in a pattern that is consistent with nuclear envelope localization, and a more diffuse one in the cytoplasm. This pattern is consistent with localization of the enzyme in the endoplasmic reticulum (ER) of these cells [28].…”

“…The tagged gene was inserted in its endogenous locus to avoid potential miss-expression artifacts (Fig. This pattern is consistent with localization of the enzyme in the endoplasmic reticulum (ER) of these cells [28]. Because of high autofluorescence background in wild type cells, we increased the selectivity of the signal using an anti-GFP antibody and indirect immunofluorescence microscopy for detection.…”

Characterization of Tt ALV2, an Essential Charged Repeat Motif Protein of the Tetrahymena thermophila Membrane Skeleton

“…S2D). It is also evident from the literature that tagging of proteins in Tetrahymena does not predispose those constructs to associate with cytoskeletal structures and ciliary basal bodies (22)(23)(24). Furthermore, expression of GFP alone from the same plasmid under the control of same promoter results in accumulation of GFP in the cytosol and not the cytoskeleton (see Fig.…”