Abstract
BackgroundThe predominant sterol in the membranes of the alga Chlamydomonas reinhardtii is ergosterol, which is commonly found in the membranes of fungi, but is rarely found in higher plants. Higher plants and fungi synthesize sterols by different pathways, with plants producing cycloartenol as a precursor to end-product sterols, while non-photosynthesizing organisms like yeast and humans produce lanosterol as a precursor. Analysis of the C. reinhardtii genome sequence reveals that this algae is also likely to synthesize sterols using a pathway resembling the higher plant pathway, indicating that its sterols are synthesized somewhat differently than in fungi. The work presented here seeks to establish experimental evidence to support the annotated molecular function of one of the sterol biosynthetic genes in the Chlamydomonas genome.Methodology/Principal FindingsA gene with homology to the yeast sterol C-5 desaturase, ERG3, is present in the Chlamydomonas genome. To test whether the ERG3 ortholog of C. reinhardtii encodes a sterol C-5 desaturase, Saccharomyces cerevisiae ERG3 knockout strains were created and complemented with a plasmid expressing the Chlamydomonas ERG3. Expression of C. reinhardtii ERG3 cDNA in erg3 null yeast was able to restore ergosterol biosynthesis and reverse phenotypes associated with lack of ERG3 function.Conclusions/SignificanceComplementation of the yeast erg3 null phenotypes strongly suggests that the gene annotated as ERG3 in C. reinhardtii functions as a sterol C-5 desaturase.
Highlights
Sterols are isoprenoid-derived molecules found in the membranes of eukaryotic organisms and have been shown to play an important role in membrane fluidity and permeability [1,2,3,4]
In the proposed pathway for ergosterol biosynthesis in C. reinhardtii (Fig. 1), ERG3 would catalyze the conversion of episterol to ergosta- 5,7,24 (28)-trienol
ERG3 in C. reinhardtii is located on chromosome 16 of the genome [12], and the predicted amino acid sequence of ERG3 aligns well with other C-5 sterol desaturases (Table 1 and Fig. 3)
Summary
Sterols are isoprenoid-derived molecules found in the membranes of eukaryotic organisms and have been shown to play an important role in membrane fluidity and permeability [1,2,3,4]. Fungi and vertebrates synthesize sterols with lanosterol as an intermediate (Fig. 1), while plants synthesize sterols using cycloartenol as an intermediate [5]. In these pathways the biosynthetic steps from isopentanyl PP to squalene epoxide are the same. The predominant sterol in the membranes of the alga Chlamydomonas reinhardtii is ergosterol, which is commonly found in the membranes of fungi, but is rarely found in higher plants. Analysis of the C. reinhardtii genome sequence reveals that this algae is likely to synthesize sterols using a pathway resembling the higher plant pathway, indicating that its sterols are synthesized somewhat differently than in fungi. The work presented here seeks to establish experimental evidence to support the annotated molecular function of one of the sterol biosynthetic genes in the Chlamydomonas genome
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