Compartmentalized thymidine phosphorylation by mitochondrial nucleotide kinases TK2 and CMPK2.

Deoxynucleotides (dNTPs) in post-mitotic tissues rely on deoxynucleoside salvage pathways in order to repair and replicate nuclear and mitochondrial DNA (mtDNA). Previous work from our laboratory showed in perfused rat heart and isolated mitochondria that the only substrate for TTP synthesis is thymidine. When thymidylate (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 post-mitotic tissues, including rat liver, kidney, and brain. Using AZT to block mitochondrial thymidine kinase 2, we demonstrate that TMP cannot serve as a precursor for TTP synthesis in isolated mitochondria from any of these tissues unless it is de-phosphorylated 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 CMPK2 in the mitochondria. Differential fraction experiments provide additional evidence that association with TK2 allows CMPK2 to display TMPK2 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.