Delta(7)-Sterol-C5-desaturase: molecular characterization and functional expression of wild-type and mutant alleles

An Arabidopsis thaliana recessive monogenic mutant (stel-1) presenting a de ficiency of the Delta(7)-sterol-C5(6)-desaturase step in the sterol pathway has been reported previously [12]. To further characterize stel-1, Arabido psis, Nicotiana tabacum and Homo sapiens cDNAs encoding Delta 7-sterol-C5(6 )-desaturases were isolated and identified on the basis of their ability to restore ergosterol synthesis in erg3, a yeast null mutant whose gene encod ing the Delta 7-sterol-C5(6)-desaturase was disrupted. Overexpression of th e Arabidopsis cDNA driven by a 35S promoter in transgenic stel-1 plants led to full complementation of the mutant. This result demonstrates that STE1 was the impaired component in the desaturation system. Four independent rev erse transcriptions of stel-1 RNA followed by polymerase chain reactions (R T-PCRs), yielded a single product. Alignment of the wild-type ORF with the RT-PCR derived stel-1 ORF revealed a single amino acid substitution: Thr-11 4 in the wild-type is changed to lie in stel-1. Expression in erg3 resulted in a 6-fold lowered efficiency of the stel-1 ORF in complementing the yeas t biosynthetic pathway when compared to the wild-type ORE The presence of t his mutation in the mutant stel-1 genomic sequence land no additional modif ication between stel-1 and wild-type genes) demonstrates that the change of the Thr-114 to Ile is necessary and sufficient to create the leaky allele stel-1. The occurrence of a hydroxylated amino acid (Thr or Ser) at the pos ition corresponding to Thr-114 in the five Delta(7)-sterol-C5(6) desaturase s identified so far suggests that this amino acid is important for normal e nzymatic function. Nomenclature: erg3, a Saccharomyces cerevisiae Delta(7)-sterol-C5(6)-desatu rase mutant [2]; stel-1, an Arabidopsis Delta 7-sterol-C5(6)-desaturase mut ant; ERG3, the protein encoded by ERG3, the wild-type yeast Delta(7)-sterol -C5(6)-desaturase gene; STE1, the protein encoded by STE1, the wild-type Ar abidopsis Delta(7)-sterol-C5(6)-desaturase gene. Sterol nomenclature: lanos terol, 4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol; cycloar tenol, 4,4,14 alpha-trimethyl-9 beta, 19-cyclo-5 alpha-cholest-24-en-3 beta -ol; episterol, 5 alpha-ergost-7,24(24(1))-dien-3 beta-ol; Delta(7)-avenast erol, 5 alpha-stigmast-7,Z-24(24(1))-dien-3 beta-ol; 5-dehydroepisterol, er gosta-5,7,24(24(1))-dien-3 beta-ol; 5-dehydro-Delta(7)-avenasterol, stigmas ta-5,7,2-24(24(1))-dien-3 beta-ol; ergosterol, ergosta-5,7,E-22-dien-3 beta -ol; campesterol, 24R-24-methyl-cholest-5-en-3 beta-ol; 22-dihydrobrassicas terol, 24S-24-methyl-cholest-5-en-3 beta-ol; sitosterol, 24R-24-ethyl-chole st-5-en-3 beta-ol; stigmasterol, 24S-24-ethyl-cholesta-5,E-22-dien-3 beta-o l.