FIGURE 5.

NMN is the cytosolic precursor of mitochondrial NAD⁺ synthesis in 293mitoPARP cells. A, overexpression of cytoplasmic NRK1, but not mitochondrial matrix TK2, increases mitochondrial NAD⁺ content. 293mitoPARP cells were transiently transfected with vectors encoding FLAG-tagged human NRK1 or TK2. The mitochondrial localization of TK2 was verified (supplemental Fig. S3B). B and C, increase of mitochondrial NAD⁺ levels by overexpressed NRK1 depends on a source of extracellular NR (NAD and NMN are extracellularly degraded to NR, Fig. 3). D, cytosolic NMN serves as precursor for mitochondrial NAD⁺. Increased NRK activity in the cytosol enhances mitochondrial NAD⁺ content when NR, NMN, or NAD⁺ are provided as extracellular NAD⁺ precursors. The conversion of NR to NMN by mitochondrial thymidine kinase 2 (TK2) activity was ruled out (dashed line).