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

Robert J. Grebenok, Thomas E. Ohnmeiss, Alvin Yamamoto, Edward D. Huntley, David W. Galbraith, Dean Della Penna

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)807-815
Number of pages9
JournalPlant Molecular Biology
Volume38
Issue number5
DOIs
StatePublished - Nov 1 1998

Keywords

  • Arabidopsis thaliana
  • Cloning
  • Isomerase
  • Phytosterol
  • Sigma receptor
  • Yeast complementation

ASJC Scopus subject areas

  • Agronomy and Crop Science
  • Genetics
  • Plant Science

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