Although nicotinamide adenine dinucleotide phosphate (NADPH) is produced and consumed in both the cytosol and mitochondria, the relationship between NADPH fluxes in each compartment has been difficult to assess due to technological limitations. Here we introduce an approach to resolve cytosolic and mitochondrial NADPH fluxes that relies on tracing deuterium from glucose to metabolites of proline biosynthesis localized to either the cytosol or mitochondria. We introduced NADPH challenges in either the cytosol or mitochondria of cells by using isocitrate dehydrogenase mutations, administering chemotherapeutics or with genetically encoded NADPH oxidase. We found that cytosolic challenges influenced NADPH fluxes in the cytosol but not NADPH fluxes in mitochondria, and vice versa. This work highlights the value of using proline labeling as a reporter system to study compartmentalized metabolism and reveals that NADPH homeostasis in the cytosolic and mitochondrial locations of a cell are independently regulated, with no evidence for NADPH shuttle activity.

尽管烟酰胺腺嘌呤二核苷酸磷酸 (NADPH) 在细胞质和线粒体中产生和消耗，但由于技术限制，每个区室中 NADPH 通量之间的关系很难评估。在这里，我们介绍了一种解决细胞溶质和线粒体 NADPH 通量的方法，该方法依赖于追踪从葡萄糖到定位于细胞溶质或线粒体的脯氨酸生物合成代谢物的氘。我们通过使用异柠檬酸脱氢酶突变、给予化疗药物或使用基因编码的 NADPH 氧化酶，在细胞的细胞质或线粒体中引入 NADPH 挑战。我们发现细胞质挑战影响细胞质中的 NADPH 通量，但不影响线粒体中的 NADPH 通量，反之亦然。这项工作强调了使用脯氨酸标记作为报告系统来研究区室化代谢的价值，并揭示了细胞胞质和线粒体位置的 NADPH 稳态是独立调节的，没有证据表明 NADPH 穿梭活性。