The multienzymatic protein CAD leading the de novo biosynthesis of pyrimidines localizes exclusively in the cytoplasm and does not translocate to the nucleus

Abstract

CAD, the multienzymatic protein that initiates and controls the de novo biosynthesis of pyrimidines, plays a major role in nucleotide homeostasis, cell growth and proliferation. Despite its interest as a potential antitumoral target, there is a lack of understanding on CAD’s structure and functioning mechanisms. Although mainly identified as a cytosolic complex, different studies support the translocation of CAD into the nucleus, where it could have a yet undefined function. Here, we track the subcellular localization of CAD by using fluorescent chimeras, cell fractionation and immunoblotting with specific antibodies. Contradicting previous studies, we demonstrate that CAD is exclusively localized at the cytosol and discard a possible translocation to the nucleus.

Keywords:

• Carbamoyl phosphate synthetase
• aspartate transcarbamoylase
• dihydroorotase
• nucleotide metabolism
• fluorescence microscopy
• immunoblotting

Disclosure statement

The authors declare no conflict of interest.

Acknowledgments

We thank Diego Megias (CNIO Confocal Unit) for advice and help with confocal microscopy. G9C cells were kindly provided by David Patterson (University of Denver). pPEU2 and pPEU3 plasmids were kindly provided by Nick Berrow (IRB Barcelona).

Additional information

Funding

This work was supported by MINECO grants BFU2016-80570-R and RTI2018-098084-B-I00 (AEI/FEDER, UE), Secretaría de Estado de Investigación, Desarrollo e Innovación.