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

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• 作者：Agustí ; n Herná ; ndez ; ^{agustin.hernandez@ibvf.csic.es" class="auth_mail" title="E-mail the corresponding author} ; Gloria Serrano-Bueno ; José ; Romá ; n Perez-Castiñ ; eira ; Aurelio Serrano ; ^{aurelio@ibvf.csic.es" class="auth_mail" title="E-mail the corresponding author}
• 关键词：GFP ; Green fluorescent protein ; LY ; Lucifer yellow ; PPi ; Inorganic pyrophosphate ; H+-PPase ; H+-pumping pyrophosphatase ; PPase ; Inorganic pyrophosphatase ; UPR ; Unfolded protein response ; PI ; Propidium iodide ; IP ; Immunoprecipitation
• 刊名：Biochimica et Biophysica Acta (BBA)-Molecular Cell Research
• 出版年：2015
• 出版时间：November 2015
• 年：2015
• 卷：1853
• 期：11
• 页码：2945-2956
• 全文大小：1424 K

文摘

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, erg2螖 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 erg2螖-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 erg2螖 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 Pho8螖60p, GFP-Atg8p localisation or difficulties to survive under nitrogen starvation conditions, but it is concomitant to reduced vacuolar protease activity. All in all, erg2螖 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.