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.

为应对外部环境的突发变化，新兴酵母菌种酿酒酵母（Saccharomyces cerevisiae）协调多种生理层次的多维应答机制。众多研究已探究了内质网（ER）应激下转录组反应及其调节因子，如Ire1激酶和Hac1转录因子所扮演的角色。然而，关于其他生理层次上对ER应激的应答了解甚少。本研究中，我们采用定量磷酸化蛋白质组学和计算网络推断技术，揭示了酵母菌对还原剂二硫苏糖醇（DTT）的磷酸化蛋白质组应答及其上游调控网络。我们对比了野生型细胞和缺乏IRE1或MAPK激酶MKK1和MKK2突变的细胞在DTT处理前后的生理状态。除了揭示这些激酶的下游靶点外，我们的推断方法还预测了DTT应答中的新调节因子，包括细胞周期调节因子Cdc28和渗透压应答激酶Rck2，这些调节因子通过计算验证得到了证实。我们的研究结果还揭示了不同应激条件下应答的相似性与令人惊异的差异性，特别是蛋白激酶A靶点的应答。这些结果对产生普遍应激反应特征的信号通路程序的广泛性具有重要意义。