Parallel proteomics and phosphoproteomics defines starvation signal specific processes in cell quiescence

Cells arrest growth and enter a quiescent state upon nutrient deprivation. However, the molecular processes by which cells respond to different starvation signals to regulate exit from the cell division cycle and initiation of quiescence remains poorly understood. To study the role of protein expression and signaling in quiescence we combined temporal profiling of the proteome and phosphoproteome using stable isotope labeling with amino acids in cell culture (SILAC) in Saccharomyces cerevisiae (budding yeast). We find that carbon and phosphorus starvation signals activate quiescence through largely distinct proteome and phosphoproteome remodeling. However, increased expression of mitochondrial proteins is essential for quiescence establishment in response to both starvation signals. Whereas the quiescent proteome is established within 6 hours of starvation the quiescent phosphoproteome undergoes continuous changes for at least 30 hours following initial starvation. Deletion of the putative quiescence regulator RIM15, which encodes a serine-threonine kinase, results in reduced survival of cells starved for phosphorus and nitrogen, but not carbon. However, we identified common protein phosphorylation roles for RIM15 in quiescence that are enriched for RNA metabolism and translation. We also find evidence for RIM15-mediated phosphorylation of some targets, including IGO1, prior to starvation consistent with a functional role for RIM15 beyond quiescence regulation. Finally, we find evidence for widespread catabolism of amino acids in response to nitrogen starvation, indicating widespread amino acid recycling via salvage pathways in conditions lacking environmental nitrogen. Our study defines an expanded quiescent proteome and phosphoproteome in yeast, and highlights the multiple coordinated molecular processes at the level of protein expression that are required for quiescence.

细胞在营养剥夺时会停止生长并进入静息状态。然而，细胞响应不同饥饿信号以调控细胞分裂周期退出、启动静息状态的分子过程，目前仍知之甚少。为探究蛋白质表达与信号转导在静息状态中的作用，我们以酿酒酵母（Saccharomyces cerevisiae，又称出芽酵母）为研究模型，通过细胞培养氨基酸稳定同位素标记（SILAC）技术，对蛋白质组与磷酸化蛋白质组开展时序分析。我们发现，碳饥饿与磷饥饿信号主要通过截然不同的蛋白质组与磷酸化蛋白质组重塑，诱导细胞进入静息状态。不过，线粒体蛋白表达的上调，对于细胞响应两种饥饿信号以建立静息状态均不可或缺。相较而言，静息状态的蛋白质组可在饥饿开始后6小时内完成构建，而静息状态的磷酸化蛋白质组则在初始饥饿后的至少30小时内持续发生动态变化。敲除编码丝氨酸-苏氨酸激酶的推定静息状态调控因子RIM15，会降低细胞在磷饥饿与氮饥饿条件下的存活率，但对碳饥饿条件下的细胞存活率无显著影响。不过，我们发现RIM15在静息状态中存在共通的蛋白质磷酸化调控功能，这些功能显著富集于RNA代谢与蛋白质翻译过程。我们还发现，RIM15可在饥饿发生前即对部分靶标（包括IGO1）进行磷酸化，这一证据表明RIM15的功能并不局限于静息状态的调控。最后，我们发现细胞在氮饥饿条件下会发生广泛的氨基酸分解代谢，这表明在环境氮缺乏的条件下，细胞可通过补救途径实现大规模的氨基酸循环再利用。本研究明确了酿酒酵母中扩展版的静息状态蛋白质组与磷酸化蛋白质组，并揭示了静息状态建立所必需的、蛋白质表达层面的多种协同调控分子过程。