Isolation and characterization of an Arabidopsis thaliana C-8,7 sterol isomerase: functional and structural similarities to mammalian C-8,7 sterol isomerase/emopamil-binding protein.
The yeast C-8,7 sterol isomerase contains a polyvalent high-affinity drug binding site similar to mammalian sigma receptors. Exogenously supplied sigma ligands inhibit sterol biosynthesis in yeast, demonstrating a pharmacological relationship between sigma ligand-binding and C-8,7 sterol isomerase activity. We report the isolation of an Arabidopsis thaliana C-8,7 sterol isomerase by functional complementation of the corresponding sterol mutant in yeast and its characterization by exposure to sigma ligands. The yeast erg2 mutant, which lacks the C-8,7 sterol isomerase gene and activity, was transformed with an Arabidopsis cDNA yeast expression library. Transformed colonies were selected for restoration of C-8,7 sterol isomerase activity (i.e. wild-type ergosterol production) by enhanced resistance to the antibiotic cycloheximide. Sterols produced in complemented lines were characterized by gas chromatography-mass spectroscopy (GC-MS). The full-length A. thaliana cDNA (pA.t.SI1) that complemented the erg2 mutation contains an open reading frame encoding a 21 kDa protein that shares 68% similarity and 35% amino acid identity to the recently isolated mouse C-8,7 sterol isomerase. The sigma ligands, haloperidol, ifenprodil and verapamil inhibited the production of ergosterol in wild-type Saccharomyces cerevisiae and in the erg2 mutant complemented with pA.t.SI1. Structural and biochemical similarities between the A. thaliana C-8,7 sterol isomerase and the mammalian emopamil-binding protein (EBP) are discussed.
Grebenok
R J
RJ
Department of Biology, Canisius College, Buffalo, NY 14208, USA.
Ohnmeiss
T E
TE
Yamamoto
A
A
Huntley
E D
ED
Galbraith
D W
DW
Della Penna
D
D
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D029681:Arabidopsis Proteins; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018744:DNA, Plant / Q000032:analysis / Q000235:genetics; D005816:Genetic Complementation Test; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010944:Plants / Q000201:enzymology / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013251:Steroid Isomerases / Q000235:genetics* / Q000378:metabolism
D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; D018744:DNA, Plant; C490379:C-8,7 sterol isomerase, Arabidopsis; C490380:Ebp protein, mouse; D013251:Steroid Isomerases; C022669:delta(8)-delta(7)-sterol isomerase