Compartmentalized thymidine phosphorylation by mitochondrial nucleotide kinases TK2 and CMPK2

Deoxynucleotides (dNTPs) in postmitotic tissues rely on deoxynucleoside salvage pathways in order to repair and replicate nuclear and mitochondrial DNA. Previous work from our laboratory showed in perfused rat hearts and isolated mitochondria that the only substrate for thymidine triphosphate synthesis is thymidine. When thymidylate (thymidine monophosphate [TMP]) is provided to bypass thymidine kinase 2 (TK2), the substrate is readily dephosphorylated to thymidine before salvage occurs, suggesting compartmentalization within the heart mitochondrial matrix. The goal of this work extends these findings in the heart to mitochondria from other postmitotic tissues, including rat liver, kidney, and brain. Using azidothymidine to block mitochondrial TK2, we demonstrate that TMP cannot serve as a precursor for thymidine triphosphate synthesis in isolated mitochondria from any of these tissues unless it is dephosphorylated to thymidine first. Broken mitochondria incubated with labeled TMP showed similar results as intact mitochondria, suggesting the findings are not related to TMP transport across the inner mitochondrial membrane. Further, using proximity labeling with immunofluorescence microscopy, we provide evidence supporting the hypothesis that TMP compartmentation is accounted for by the interaction of TK2 and cytidine/uridine monophosphate kinase 2 (CMPK2) in the mitochondria. Differential fraction experiments provide additional evidence that association with TK2 allows CMPK2 to display cytosolic thymidylate kinase 2 activity. Together, the results indicate that a two-step phosphorylation of thymidine to TDP occurs because the proximity of TK2 and CMPK2 in the mitochondria prevents TMP from diffusing from the two enzymes.