The role of ABC proteins Aus1p and Pdr11p in the uptake of external sterols in yeast: dehydroergosterol fluorescence study.

Uptake of external sterols in the yeast Saccharomyces cerevisiae is a multistep process limited to anaerobiosis or heme deficiency. It includes crossing the cell wall, insertion of sterol molecules into plasma membrane and their internalization and integration into intracellular membranes. We applied the fluorescent ergosterol analog dehydroergosterol (DHE) to monitor the initial steps of sterol uptake by three independent approaches: fluorescence spectroscopy, fluorescence microscopy and sterol quantification by HPLC. Using specific fluorescence characteristics of DHE we showed that the entry of sterol molecules into plasma membrane is not spontaneous but requires assistance of two ABC (ATP-binding cassette) pumps--Aus1p or Pdr11p. DHE taken up by uptake-competent hem1ΔAUS1PDR11 cells could be directly visualized by UV-sensitive wide field fluorescence microscopy. HPLC analysis of sterols revealed significant amounts of exogenous ergosterol and DHE (but not cholesterol) associated with uptake-deficient hem1Δaus1Δpdr11Δ cells. Fluorescent sterol associated with these cells did not show the characteristic emission spectrum of membrane-integrated DHE. The amount of cell-associated DHE was significantly reduced after enzymatic removal of the cell wall. Our results demonstrate that the yeast cell wall is actively involved in binding and uptake of ergosterol-like sterols.

Characterization of squalene epoxidase of Saccharomyces cerevisiae by applying terbinafine-sensitive variants.

Squalene epoxidase (SE) is the target of terbinafine, which specifically inhibits the fungal enzyme in a noncompetitive manner. On the basis of functional homologies to p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, the Erg1 protein contains two flavin adenine dinucleotide (FAD) domains and one nucleotide binding (NB) site. By in vitro mutagenesis of the ERG1 gene, which codes for the Saccharomyces cerevisiae SE, we isolated erg1 alleles that conferred increased terbinafine sensitivity or that showed a lethal phenotype when they were expressed in erg1-knockout strain KLN1. All but one of the amino acid substitutions affected conserved FAD/nucleotide binding sites. The G(25)S, D(335)X (W, F, P), and G(210)A substitutions in the FADI, FADII, and NB sites, respectively, rendered the SE variants nonfunctional. The G(30)S and L(37)P variants exhibited decreased enzymatic activity, accompanied by a sevenfold increase in erg1 mRNA levels and an altered sterol composition, and rendered KLN1 more sensitive not only to allylamines (10 to 25 times) but also to other ergosterol biosynthesis inhibitors. The R(269)G variant exhibited moderately reduced SE activity and a 5- to 10-fold increase in allylamine sensitivity but no cross-sensitivity to the other ergosterol biosynthesis inhibitors. To further elucidate the roles of specific amino acids in SE function and inhibitor interaction, a homology model of Erg1p was built on the basis of the crystal structure of PHBH. All experimental data obtained with the sensitive Erg1 variants support this model. In addition, the amino acids responsible for terbinafine resistance, although they are distributed along the sequence of Erg1p, cluster on the surface of the Erg1p model, giving rise to a putative binding site for allylamines.

Terbinafine resistance in a pleiotropic yeast mutant is caused by a single point mutation in the ERG1 gene.

A terbinafine-resistant mutant of the yeast Saccharomyces cerevisiae with a complex pleiotropic phenotype (resistance to terbinafine and itraconazole, sensitivity to several antifungal compounds, respiration deficiency, and temperature sensitivity) has been isolated after chemical mutagenesis. Detailed analysis revealed that some of its traits (thermosensitive growth, sensitivity to the polyene antimycotic nystatin and to calcofluor white) are linked to alterations in the cell wall. A single C1288G base change in the ERG1 gene resulting in the substitution of proline by alanine at position 430 in the enzyme squalene epoxidase (Erg1p) was identified as the sole cause of terbinafine resistance. This novel mutation in the ERG1 gene confers only partial resistance of Erg1p to terbinafine, however, even the low level of resistance enables terbinafine-treated mutant cells to maintain adequate ergosterol levels over longer cultivation periods. Lack of interference of squalene accumulation with growth of terbinafine-treated mutant cells indicates that the antimycotic effect of terbinafine in S. cerevisiae may be linked primarily to ergosterol depletion.