Coenzyme A (CoA) is one of the central cofactors of metabolism, yet a method for measuring its concentration in living cells is missing. Here we introduce the first biosensor for measuring CoA levels in different organelles of mammalian cells. The semisynthetic biosensor is generated through the specific labeling of an engineered GFP–HaloTag fusion protein with a fluorescent ligand. Its readout is based on CoA-dependent changes in Förster resonance energy transfer efficiency between GFP and the fluorescent ligand. Using this biosensor, we probe the role of numerous proteins involved in CoA biosynthesis and transport in mammalian cells. On the basis of these studies, we propose a cellular map of CoA biosynthesis that suggests how pools of cytosolic and mitochondrial CoA are maintained.

辅酶 A (CoA) 是新陈代谢的核心辅助因子之一，但缺乏测量其在活细胞中浓度的方法。在这里，我们介绍了第一个用于测量哺乳动物细胞不同细胞器中 CoA 水平的生物传感器。该半合成生物传感器是通过用荧光配体对工程化 GFP-HaloTag 融合蛋白进行特异性标记而产生的。其读数基于 GFP 和荧光配体之间 Förster 共振能量转移效率的 CoA 依赖性变化。使用这种生物传感器，我们探究了哺乳动物细胞中参与 CoA 生物合成和运输的众多蛋白质的作用。在这些研究的基础上，我们提出了 CoA 生物合成的细胞图谱，表明细胞质和线粒体 CoA 库是如何维持的。