Functional characterization of the Chlamydomonas reinhardtii ERG3 ortholog, a gene involved in the biosynthesis of ergosterol

Abstract

Background: 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. 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 findings: A 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/significance: Complementation of the yeast erg3 null phenotypes strongly suggests that the gene annotated as ERG3 in C. reinhardtii functions as a sterol C-5 desaturase.

Figure 1. Schematic diagram of the putative pathway of ergosterol biosynthesis in S. cerevisiae and C. reinhardtii.

Figure 2. Schematic diagram of the reaction catalyzed by Erg3p in yeast.

Erg3p is responsible for introducing a double bond at the C-5 carbon (denoted by the star) of the B-ring of episterol to produce ergosta- 5,7,24(28)- trienol. This step is the second to last step in the biosynthetic pathway to ergosterol.

Figure 3. Amino acid sequence alignment of Sterol C-5 desaturase in different organisms.

Arabidopsis thaliana (AtERG3) NCBI Accession Number CAA62079; Zea mays (ZmERG3) ACG38774; Chlamydomonas reinhardtii (CrERG3) XP_001701457; Homo sapiens (HsERG3) BAA33729; Rattus norvegicus (RnERG3) NP_446094; Sacchromyces cerevisiae (ScERG3) NP_013157. Conserved amino acid sequences shared by all organisms are denoted by a star. The highlighted area corresponds to the putative histidine-containing metal binding domain. Dashed lines indicate gaps in the alignment. The bold, italicized font corresponds to the three putative transmembrane spanning regions of the C. reinhardtii ERG3 protein.

Figure 4. Schematic diagram of sterol C-5 desaturase in C. reinhardtii as annotated in the JGI Chlamydomonas Genome Version 4.0.

ERG3 has six exons and five introns.

Figure 5. Deletion of the ERG3 gene in yeast.

(A) Schematic diagram of homologous recombination strategy used to delete ERG3 in Sacchromyces cerevisiae. PCR primers were designed to amplify the URA3 marker gene containing the flanking regions of ERG3. (B) Ura⁺ isolates were screened by PCR to verify proper integration of URA3 and deletion of ERG3. Only correctly integrated isolates would show a 459 base pair product using primers internal to URA3 and upstream of the flanking homology. Stars denote the positive isolates selected for further analysis. PCR was also used to verify the downstream end of flanking region homology with URA3 (data not shown).

Figure 6. Phenotypic complementation of yeast erg3Δ strains.

Known phenotypes of yeast erg3 null mutants (hypersensitivity to cycloheximide and inability to grow on acetate) were complemented by plasmids expressing either S. cerevisiae ERG3 or C. reinhardtii ERG3 cDNA. A) S. cerevisiae ADH1 promoter plasmid used to express ERG3 genes. Haploid yeast carrying the erg3Δ::URA3 allele were transformed with the designated plasmids and plated on dextrose minimal media lacking leucine (YMD minus Leu) to select for transformants. Single colony isolates were then re-streaked on minimal media lacking leucine and containing cycloheximide (0.13 µg/mL) (B) minimal media lacking leucine and containing acetate as sole carbon and energy source (C), and on YMD lacking as overall growth control (D). Yeast mutant for ERG3 and containing the vector plasmid lacking an insert cannot grow on cycloheximide or acetate, while cells containing the plasmid expressing either yeast or C. reinhardtii ERG3 are able to grow. Each pair of plates represents erg3Δ yeast sporulated from two independently isolated diploid knockout isolates, 1+2 are DDY4259 and DDY4260 transformed with the vector only, 3+4 are DDY4261 and DDY4262, the same strains transformed with ADH1-yeast ERG3, and 5+6 are DDY4263 and DDY4264 with ADH1-C.reinhardtii ERG3. For the second panel, 7+8 are DDY4253 and DDY4254 transformed with the vector only, 9+10 are DDY4255 and DDY4256 transformed with ADH1-yeast ERG3, and 11+12 are DDY4257 and DDY4258 with ADH1-C.reinhardtii ERG3.

Figure 7. GC/MS data.

Total ion chromatographs were analyzed to look specifically for the 100% ion of ergosterol. Mass ion 363 gives the best indication of the presence or absence of ergosterol in the samples. (A) Ergosterol standard, (B) Lipid extract from DDY4259 vector control, (C) DDY4263 expressing C.reinhardtii ERG3, (D) DDY4261 expressing S. cerevisiae ERG3.