Aspartate is crucial for nucleotide synthesis, ammonia detoxification, and maintaining redox balance via the malate-aspartate-shuttle (MAS). To disentangle these multiple roles of aspartate metabolism, tools are required that measure aspartate concentrations in real time and in live cells. We introduce AspSnFR, a genetically encoded green fluorescent biosensor for intracellular aspartate, engineered through displaying and screening biosensor libraries on mammalian cells. In live cells, AspSnFR is able to precisely and quantitatively measure cytosolic aspartate concentrations and dissect its production from glutamine. Combining high-content imaging of AspSnFR with pharmacological perturbations exposes differences in metabolic vulnerabilities of aspartate levels based on nutrient availability. Further, AspSnFR facilitates tracking of aspartate export from mitochondria through SLC25A12, the MAS’ key transporter. We show that SLC25A12 is a rapidly responding and direct route to couple Ca²⁺ signaling with mitochondrial aspartate export. This establishes SLC25A12 as a crucial link between cellular signaling, mitochondrial respiration, and metabolism.

天冬氨酸对于核苷酸合成、氨解毒以及通过苹果酸-天冬氨酸-穿梭 (MAS) 维持氧化还原平衡至关重要。为了理清天冬氨酸代谢的多重作用，需要实时测量活细胞中天冬氨酸浓度的工具。我们推出了 AspSnFR，一种用于细胞内天冬氨酸的基因编码绿色荧光生物传感器，通过在哺乳动物细胞上展示和筛选生物传感器库进行设计。在活细胞中，AspSnFR 能够精确定量地测量胞质天冬氨酸浓度并剖析其从谷氨酰胺的产生。将 AspSnFR 的高内涵成像与药理学扰动相结合，揭示了基于营养可用性的天冬氨酸水平代谢脆弱性的差异。此外，AspSnFR 有助于通过 MAS 的关键转运蛋白 SLC25A12 跟踪线粒体的天冬氨酸输出。我们证明，SLC25A12 是一种快速响应的直接途径，可将 Ca ²⁺ 信号传导与线粒体天冬氨酸输出耦合。这使得 SLC25A12 成为细胞信号传导、线粒体呼吸和新陈代谢之间的关键纽带。