Phosphoproteome Response to Dithiothreitol Reveals Unique Versus Shared Features of Saccharomyces cerevisiae Stress Responses

To cope with sudden changes in the external environment, the budding yeast Saccharomyces cerevisiae orchestrates a multifaceted response that spans many levels of physiology. Several studies have interrogated the transcriptome response to endoplasmic reticulum (ER) stress and the role of regulators such as the Ire1 kinase and Hac1 transcription factors. However, less is known about responses to ER stress at other levels of physiology. Here, we used quantitative phosphoproteomics and computational network inference to uncover the yeast phosphoproteome response to the reducing agent dithiothreitol (DTT) and the upstream signaling network that controls it. We profiled wild-type cells and mutants lacking IRE1 or MAPK kinases MKK1 and MKK2, before and at various times after DTT treatment. In addition to revealing downstream targets of these kinases, our inference approach predicted new regulators in the DTT response, including cell-cycle regulator Cdc28 and osmotic-response kinase Rck2, which we validated computationally. Our results also revealed similarities and surprising differences in responses to different stress conditions, especially in the response of protein kinase A targets. These results have implications for the breadth of signaling programs that can give rise to common stress response signatures.为应对外界环境的突发变化，酿酒酵母（Saccharomyces cerevisiae）会启动一套覆盖多个生理层面的多维度响应程序。已有多项研究解析了细胞对内质网（endoplasmic reticulum，ER）应激的转录组响应，以及Ire1激酶（Ire1 kinase）、Hac1转录因子（Hac1 transcription factor）等调控因子的作用。然而，目前对于内质网应激在其他生理层面的响应机制仍所知甚少。本研究利用定量磷酸化蛋白质组学（quantitative phosphoproteomics）与计算网络推断（computational network inference）技术，解析了酿酒酵母在还原剂二硫苏糖醇（dithiothreitol，DTT）处理下的磷酸化蛋白质组响应，以及调控该响应的上游信号网络。我们对DTT处理前及处理后不同时间点的野生型细胞，以及缺失IRE1基因或MAPK激酶（MAPK kinases）MKK1、MKK2的突变体细胞进行了组学表征。本研究不仅揭示了上述激酶的下游靶标，还通过推断方法预测了DTT响应中的新型调控因子，包括细胞周期调控因子Cdc28与渗透压响应激酶Rck2，并通过计算手段验证了这些预测结果。本研究结果还揭示了酵母在不同应激条件下的响应存在相似性与令人意外的差异，尤其是在蛋白激酶A（protein kinase A）靶标的响应模式上。上述研究结果对于解析可产生共同应激响应特征的信号通路程序的广度具有重要启示意义。