CTPS2 regulates CTP synthetase activity by interacting with CTPS1

CTP synthetases (CTPS) 1 and 2 are responsible for intracellular de novo production of the CTP nucleotide required for DNA replication and cell proliferation. CTPS1 and CTPS2 are co-expressed in most of tissues and share a high structural homology. They form homotetramers that are enzymatically active and aggregate into large intracellular storage filament-like structures termed as cytoophidia. Herein, we found that CTPS1 and CTPS2 co-localized in cytoophidia when co-expressed and CTPS2-containing filaments are dependent on CTPS1 expression. Cytoophidia were not necessary for proliferation because CTPS1^H355A and CTPS2^H355A mutants that are unable to form cytoophidia could sustain normal cell proliferation. CTPS1 and CTPS2 were found to directly interact together independent of polymerization and formation of cytoophidia. When associated with CTPS2, CTPS1 enzymatic activity was decreased and more sensitive to CTP/product negative feedback, suggesting that the presence of CTPS2 modulates CTPS activity. Therefore, our results demonstrate that CTPS1 and CTPS2 do not only function independently, but also associate and form complexes in the absence of polymerization, suggesting the possibility that they directly regulate each other through heterotetramerization.