8-Dehydrosterols induce membrane traffic and autophagy defects through V-ATPase dysfunction in Saccharomyces cerevisae

Abstract

8-Dehydrosterols are present in a wide range of biologically relevant situations, from human rare diseases to amine fungicide-treated fungi and crops. However, the molecular bases of their toxicity are still obscure. We show here that 8-dehydrosterols, but not other sterols, affect yeast vacuole acidification through V-ATPases. Moreover, erg2Δ cells display reductions in proton pumping rates consistent with ion-transport uncoupling in vitro. Concomitantly, subunit Vph1p shows conformational changes in the presence of 8-dehydrosterols. Expression of a plant vacuolar H(+)-pumping pyrophosphatase as an alternative H(+)-pump relieves Vma(-)-like phenotypes in erg2Δ-derived mutant cells. As a consequence of these acidification defects, endo- and exo-cytic traffic deficiencies that can be alleviated with a H(+)-pumping pyrophosphatase are also observed. Despite their effect on membrane traffic, 8-dehydrosterols do not induce endoplasmic reticulum stress or assembly defects on the V-ATPase. Autophagy is a V-ATPase dependent process and erg2Δ mutants accumulate autophagic bodies under nitrogen starvation similar to Vma(-) mutants. In contrast to classical Atg(-) mutants, this defect is not accompanied by impairment of traffic through the CVT pathway, processing of Pho8Δ60p, GFP-Atg8p localisation or difficulties to survive under nitrogen starvation conditions, but it is concomitant to reduced vacuolar protease activity. All in all, erg2Δ cells are autophagy mutants albeit some of their phenotypic features differ from classical Atg(-) defective cells. These results may pave the way to understand the aetiology of sterol-related diseases, the cytotoxic effect of amine fungicides, and may explain the tolerance to these compounds observed in plants.

Vacuolar H+-Pyrophosphatase AVP1 is Involved in Amine Fungicide Tolerance in Arabidopsis thaliana and Provides Tridemorph Resistance in Yeast

“…Vacuole vesicles extracted from cells treated with 3 μM tridemorph for 5 h were not capable to form proton gradients ( Figure 2A ). This impairment was similar using erg2Δ -derived vesicles ( Hernandez et al, 2015 ). Also in agreement with previous results, hydrolytic activity assays showed that V-ATPase activity was inhibited to a minor extent (435 ± 97 vs. 341 ± 8 μmol Pi min -1 mg -1 protein ± SEM for vesicles obtained from control and tridemorph-treated yeast cells respectively).…”

“…In a previous report ( Hernandez et al, 2015 ), we described that the presence of 8-dehydrosterols in yeast membranes induced a change in subunit a of the V-ATPase that made it more susceptible to proteolytic attack. We hypothesized that limiting the proteolytic turnover of vacuolar proteins might help yeast to withstand tridemorph treatment.…”

“…Tridemorph is a fungicide that phenocopies a defect on ERG2 , which causes V-ATPase dysfunction ( Hernandez et al, 2015 ). Bearing in mind the differences found in terms of cell death induction between tridemorph treatment and an erg2Δ mutation, we were interested in ascertaining if inhibition of V-ATPase proton pumping was reproduced by tridemorph ( Figure 2 ).…”

“…It has been shown that fungicides inducing the accumulation of Δ 14 -sterols and 14α-methylated sterols, through Erg24p and Erg11p inhibition respectively, exert part of their effects via inhibition of the vacuolar H + -ATPase in Saccharomyces cerevisiae and Candida albicans ( Zhang and Rao, 2010 ; Zhang et al, 2010 ). Similarly, yeast mutants bearing mutations in the ERG2 gene has been shown to display faulty V-ATPase-dependent proton pumping and intraorganellar acidification leading to a plethora of cellular defects ( Hernandez et al, 2015 ). The S. cerevisiae ERG2 gene encodes the yeast sterol-Δ 8 ,Δ 7 -isomerase, an ortholog of Arabidopsis thaliana HYD1 ( Souter et al, 2002 ) and it is a target for amine fungicides like tridemorph.…”

Transcriptome analysis of fungicide-responsive gene expression profiles in two Penicillium italicum strains with different response to the sterol demethylation inhibitor (DMI) fungicide prochloraz

“…Fungal resistance to DMIs can also be ascribed to overexpression of CYP51s, especially by some enhancer elements [9,[27][28][29][30][31][32][33]. In addition to CYP51s, recently, other genes encoding fungal ergosterol biosynthesis-related enzymes have been proposed to be potential targets, including ERG2 (encoding C − 8 sterol isomerase) [34][35][36] and ERG6 (encoding C − 24 sterol methyltransferase) [37][38][39][40]. The importance of both ERG2 and ERG6 to cycloheximide resistance for S. cerevisiae has also been genetically emphasized [41].…”