Combining Deep Sequencing, Proteomics, Phosphoproteomics, and Functional Screens To Discover Novel Regulators of Sphingolipid Homeostasis

Sphingolipids (SLs) are essential components of cell membranes and are broad-range bioactive signaling molecules. SL levels must be tightly regulated as imbalances affect cellular function and contribute to pathologies ranging from neurodegenerative and metabolic disorders to cancer and aging. Deciphering how SL homeostasis is maintained and uncovering new regulators is required for understanding lipid biology and for identifying new targets for therapeutic interventions. Here we combine omics technologies to identify the changes of the transcriptome, proteome, and phosphoproteome in the yeast Saccharomyces cerevisiae upon SL depletion induced by myriocin. Surprisingly, while SL depletion triggers important changes in the expression of regulatory proteins involved in SL homeostasis, the most dramatic regulation occurs at the level of the phosphoproteome, suggesting that maintaining SL homeostasis demands rapid responses. To discover which of the phosphoproteomic changes are required for the cell’s first-line response to SL depletion, we overlaid our omics results with systematic growth screens for genes required during growth in myriocin. By following the rate of SL biosynthesis in those candidates that are both affecting growth and are phosphorylated in response to the drug, we uncovered Atg9, Stp4, and Gvp36 as putative new regulators of SL homeostasis.鞘脂（Sphingolipids, SLs）是细胞膜的必需组成成分，同时也是广谱生物活性信号分子。鞘脂的水平必须受到严格调控，因为其失衡会影响细胞功能，并促成多种病理状态，涵盖神经退行性疾病、代谢紊乱，乃至癌症与衰老。阐明鞘脂稳态的维持机制并发掘新的调控因子，是理解脂质生物学以及确定治疗干预新靶点的必要前提。本研究结合组学技术（omics technologies），对经霉菌素（myriocin）诱导发生鞘脂耗竭的酿酒酵母（Saccharomyces cerevisiae）中的转录组（transcriptome）、蛋白质组（proteome）以及磷酸化蛋白质组（phosphoproteome）变化进行了鉴定。令人意外的是，尽管鞘脂耗竭会引发参与鞘脂稳态调控的调节蛋白表达发生显著变化，但最为剧烈的调控发生在磷酸化蛋白质组层面，这表明维持鞘脂稳态需要快速的应答反应。为了探明哪些磷酸化蛋白质组变化是细胞应对鞘脂耗竭的一线应答所必需的，我们将组学研究结果与在霉菌素培养条件下生长所必需的基因的系统性生长筛选数据进行了交叉比对。通过追踪那些既影响生长、又会响应该药物发生磷酸化的候选基因的鞘脂生物合成速率，我们发掘出Atg9、Stp4与Gvp36作为鞘脂稳态的潜在新型调控因子。