Compartmentalized thymidine phosphorylation by mitochondrial nucleotide kinases TK2 and CMPK2

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Biology

Background:

• Deoxynucleotides (dNTPs) are essential for DNA repair and replication in postmitotic tissues.
• Deoxynucleoside salvage pathways are crucial for maintaining dNTP pools in mitochondria.
• Previous studies indicated thymidine is the sole precursor for thymidine triphosphate (TTP) synthesis in rat heart mitochondria.

Purpose of the Study:

• To investigate the substrate utilization for TTP synthesis in mitochondria from various postmitotic tissues (heart, liver, kidney, brain).
• To elucidate the mechanism behind the observed substrate preference for TTP synthesis.
• To explore the role of enzyme interactions in mitochondrial dNTP metabolism.

Main Methods:

• Isolated mitochondria from rat tissues were used to study TTP synthesis.
• Azidothymidine was employed to inhibit mitochondrial thymidine kinase 2 (TK2).
• Proximity labeling and differential fractionation experiments were performed to identify enzyme interactions.

Main Results:

• Thymidine monophosphate (TMP) could not be directly converted to TTP in isolated mitochondria; it required prior dephosphorylation to thymidine.
• This finding was consistent across mitochondria from heart, liver, kidney, and brain.
• TK2 and cytidine/uridine monophosphate kinase 2 (CMPK2) were found to interact within the mitochondria, creating a compartment that facilitates thymidine phosphorylation but not TMP utilization.

Conclusions:

• Mitochondrial TTP synthesis is dependent on thymidine, not TMP, due to enzymatic compartmentalization.
• The interaction between TK2 and CMPK2 in the mitochondrial matrix prevents TMP from acting as a direct substrate for TTP synthesis.
• This compartmentalization ensures efficient DNA repair and replication in postmitotic cells by regulating dNTP availability.