Budding yeast uses the TORC1-Sch9p and cAMP-PKA signalling pathways to regulate adaptations to changing nutrient environments. Dynamic and single-cell measurements of the activity of these cascades will improve our understanding of cellular adaptation of yeast. Here, we employed the AKAR3-EV biosensor developed for mammalian cells to measure the cellular phosphorylation status determined by Sch9p and PKA activity in budding yeast. Using various mutant strains and inhibitors, we show that AKAR3-EV measures the Sch9p- and PKA-dependent phosphorylation status in intact yeast cells. At the single-cell level, we found that the phosphorylation responses are homogenous for glucose, sucrose and fructose, but heterogeneous for mannose. Cells that start to grow after a transition to mannose correspond to higher normalized FRET levels, in line with the involvement of Sch9p and PKA pathways to stimulate growth-related processes. The Sch9p and PKA pathways have a relatively high affinity for glucose (K0.5 of 0.24 mM) under glucose derepressed conditions. Lastly, steady-state FRET levels of AKAR3-EV seem to be independent of growth rates, suggesting that Sch9p- and PKA-dependent phosphorylation activities are transient responses to nutrient transitions. We believe that the AKAR3-EV sensor is an excellent addition to the biosensor arsenal for illuminating cellular adaptation in single yeast cells.

中文翻译：

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使用 AKAR3-EV 生物传感器评估芽殖酵母中的 Sch9p 和 PKA 信号传导

芽殖酵母使用 TORC1-Sch9p 和 cAMP-PKA 信号通路来调节对不断变化的营养环境的适应。对这些级联活性的动态和单细胞测量将提高我们对酵母细胞适应的理解。在这里，我们采用为哺乳动物细胞开发的 AKAR3-EV 生物传感器来测量出芽酵母中由 Sch9p 和 PKA 活性确定的细胞磷酸化状态。使用各种突变菌株和抑制剂，我们证明 AKAR3-EV 可测量完整酵母细胞中 Sch9p 和 PKA 依赖性磷酸化状态。在单细胞水平上，我们发现葡萄糖、蔗糖和果糖的磷酸化反应是同质的，但甘露糖的磷酸化反应是异质的。过渡到甘露糖后开始生长的细胞对应于更高的标准化 FRET 水平，与 Sch9p 和 PKA 途径的参与一致，可刺激生长相关过程。在葡萄糖去抑制条件下，Sch9p 和 PKA 途径对葡萄糖具有相对较高的亲和力（K0.5 为 0.24 mM）。最后，AKAR3-EV 的稳态 FRET 水平似乎与生长速率无关，表明 Sch9p 和 PKA 依赖性磷酸化活性是对营养转变的瞬时反应。我们相信 AKAR3-EV 传感器是生物传感器库的绝佳补充，可用于阐明单个酵母细胞的细胞适应。表明 Sch9p 和 PKA 依赖性磷酸化活性是对营养转变的瞬时反应。我们相信 AKAR3-EV 传感器是生物传感器库的绝佳补充，可用于阐明单个酵母细胞的细胞适应。表明 Sch9p 和 PKA 依赖性磷酸化活性是对营养转变的瞬时反应。我们相信 AKAR3-EV 传感器是生物传感器库的绝佳补充，可用于阐明单个酵母细胞的细胞适应。