Yeast sterol C-8-C-7 isomerase: Identification and characterization of a high-affinity binding site for enzyme inhibitors

The yeast gene ERG2 encodes a sterol C-8-C-7 isomerase and is essential for ergosterol synthesis and cell proliferation. Its striking homology with the so-called sigma(1) receptor of guinea pig brain, a polyvalent steroid and drug binding protein, suggested that the yeast sterol C-8-C-7 isomerase (ERG2) carries a similar high affinity drug binding domain. Indeed the sigma ligands [H-3]haloperidol (K-d = 0.3 nM) and [H-3]ifenprodil (K-d = 1.4 nM) bound to a single population of sites in ERG2 wild type yeast microsomes (B-max values of 77 and 61 pmol/mg of protein, respectively), whereas binding activity was absent in strains carrying ERG2 gene mutations or disruptions. [H-3]Ifenprodil binding was inhibited by sterol isomerase inhibitors such as fenpropimorph (K-i = 0.05 nM), tridemorph (K-i = 0.09 nM), MDL28,815 (k(i) = 0.44 nM), triparanol (K-i = 1.5 nM), and AY-9944 (K-i = 5.8 nM). [H-3]Haloperidol specifically photoaffinity-labeled a protein with an apparent molecular weight of 27 400, in agreement with the molecular mass of the sterol C-8-C-7 isomerase (24 900 Da). 9E10 c-myc antibodies specifically immunoprecipitated the c-myc tagged protein after [H-3]haloperidol photolabeling, unequivocally proving that the drug binding site is localized on the ERG2 gene product. Haloperidol, trifluperidol, and ifenprodil inhibited the growth of Saccharomyces cerevisiae and reduced the ergosterol content of cells grown in their presence. Our results demonstrate that the yeast sterol C-8-C-7 isomerase has a polyvalent high-affinity drug binding site similar to mammalian sigma receptors and that in yeast sigma ligands inhibit sterol biosynthesis.