Saccharomyces cerevisiae Is Dependent on Vesicular Traffic between the Golgi Apparatus and the Vacuole When Inositolphosphorylceramide Synthase Aur1 Is Inactivated
Saccharomyces cerevisiae Is Dependent on Vesicular Traffic between the Golgi Apparatus and the Vacuole When Inositolphosphorylceramide Synthase Aur1 Is Inactivated
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DOI：
10.1128/ec.00117-15
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发表时间：
2015-10
期刊：
影响因子：
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通讯作者：
N. S. Voynova;C. Roubaty;Hector M. Vazquez;Shamroop Mallela;Christer S. Ejsing;A. Conzelmann
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文献类型：
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作者：
N. S. Voynova;C. Roubaty;Hector M. Vazquez;Shamroop Mallela;Christer S. Ejsing;A. Conzelmann
摘要肌醇磷酸神经酰胺（IPC）及其甘露糖化衍生物是酵母中唯一的复合鞘脂。它们的合成可被aureobasidin A（阿坝）减少，该物质特异性抑制IPC合酶Aur 1。据报道，通过降低IPC水平，阿坝引起内质网（ER）应激、胞质钙增加、活性氧产生和线粒体损伤，导致细胞凋亡。我们发现，当Aur 1被转录下调逐渐耗尽时，神经酰胺的积累成为细胞存活的主要障碍。碱性神经酰胺酶YPC 1的过表达在这种条件下拯救细胞。我们建立了羟基化C26脂肪酸作为神经酰胺水解的可靠标志。这种水解仅在YPC 1过表达时发生。相反，当Aur 1被阿坝A急性抑制时，YPC 1的过表达没有有益的作用。高通量的遗传筛选显示，囊泡介导的高尔基体，内体和液泡之间的运输成为生存的关键时，Aur 1被压抑，不论模式的镇压。此外，当细胞被阿坝A急性应激时，空泡酸化变得至关重要，并且奎纳克林摄取到空泡中表明阿坝A激活空泡酸化。抗氧化剂N-乙酰半胱氨酸不改善阿坝上的细胞生长，表明由阿坝诱导的活性氧自由基在其毒性中起次要作用。阿坝强烈诱导细胞壁完整性途径，但渗透支持并不能提高野生型细胞在阿坝上的生存力。总之，这些数据支持并完善了目前AbA介导的细胞死亡模型，并将液泡蛋白运输和酸化作为抗逆的新关键要素。
ABSTRACT Inositolphosphorylceramide (IPC) and its mannosylated derivatives are the only complex sphingolipids of yeast. Their synthesis can be reduced by aureobasidin A (AbA), which specifically inhibits the IPC synthase Aur1. AbA reportedly, by diminishing IPC levels, causes endoplasmic reticulum (ER) stress, an increase in cytosolic calcium, reactive oxygen production, and mitochondrial damage leading to apoptosis. We found that when Aur1 is gradually depleted by transcriptional downregulation, the accumulation of ceramides becomes a major hindrance to cell survival. Overexpression of the alkaline ceramidase YPC1 rescues cells under this condition. We established hydroxylated C26 fatty acids as a reliable hallmark of ceramide hydrolysis. Such hydrolysis occurs only when YPC1 is overexpressed. In contrast, overexpression of YPC1 has no beneficial effect when Aur1 is acutely repressed by AbA. A high-throughput genetic screen revealed that vesicle-mediated transport between Golgi apparatus, endosomes, and vacuole becomes crucial for survival when Aur1 is repressed, irrespective of the mode of repression. In addition, vacuolar acidification becomes essential when cells are acutely stressed by AbA, and quinacrine uptake into vacuoles shows that AbA activates vacuolar acidification. The antioxidant N-acetylcysteine does not improve cell growth on AbA, indicating that reactive oxygen radicals induced by AbA play a minor role in its toxicity. AbA strongly induces the cell wall integrity pathway, but osmotic support does not improve the viability of wild-type cells on AbA. Altogether, the data support and refine current models of AbA-mediated cell death and add vacuolar protein transport and acidification as novel critical elements of stress resistance.