Cloning by metabolic interference in yeast and enzymatic characterization of Arabidopsis thaliana sterol Delta 7-reductase

Reduction of the Delta 7 double bond of sterols, a key biosynthetic step in higher eukaryotes, is lacking in lower eukaryotes like the yeast Saccharomyces cerevisiae, leading to terminal sterols with a Delta 5,7-conjugated diene structure, Genes encoding two sterol reductases involved, respectively, in the reduction of sterol Delta 14 and Delta 24(28) double bonds have been cloned to date, but no sequence information was available on the enzyme responsible for Delta 7-bond reduction, This study presents the cloning of the NADPH-sterol Delta 7-reductase (Delta 7-red) from Arabidopsis thaliana, based on a metabolic interference approach in yeast, The principle is the functional expression of a plant cDNA library in the yeast strain FY1679-28C tolerant to sterol modifications and the selection of clones resistant to the polyene fungicide nystatin, The toxicity of this compound is dependent on the presence of Delta 5,7-unsaturated sterols in the yeast plasma membrane, One clone out of 10(5) transformants exhibits a cDNA-dependent alteration of cell sterol composition, The 1290-base pair cDNA open reading frame was isolated and sequenced, The corresponding protein presents a significant sequence similarity with yeast Delta 14- and Delta 24(28)-reductases and with human lamin B receptor. The coding sequence was extracted by polymerase chain reaction and inserted into a galactose-inducible yeast expression vector to optimize expression, Analysis using transformed wild type yeast or sterol altered mutants, indicated that Delta 5,7-ergosta- and cholesta-sterols are efficiently reduced in vivo, regardless of the structural variations on the side chain, No reductase activity was observed toward the Delta 14 or the Delta 5 positions of sterols, In vivo extensive Delta 7-reduction of the free and esterified pools of sterols was observed upon induction of the enzyme, Ergosterol present before induction was reduced into ergosta-5,22-dieneol, whereas ergosta-5-eneol is the new end product of sterol neosynthesis, indicating that the yeast Delta 22 desaturase may be no longer active on C-7-saturated sterols, In vitro tests indicated that Delta 7-reductase activity is preferentially associated with the endoplasmic reticulum membrane and confirmed the previous finding that NADPH is the reducing agent.