Abstract
The cytidine deaminases belong to the family of multisubunit enzymes that catalyze the hydrolytic deamination of their substrate to a corresponding uracil product. They play a major role in pyrimidine nucleoside and nucleotide salvage. The intracellular distribution of cytidine deaminase and related enzymes has previously been considered to be cytosolic. Here we show that human cytidine deaminase (HCDA) is present in the nucleus. A highly specific, affinity purified polyclonal antibody against HCDA was used to analyze the intracellular localization of native HCDA in a variety of mammalian cells by in situ immunochemistry. Native HCDA was found to be present in the nucleus as well as the cytoplasm in several cell types. Indirect immunofluorescence microscopy indicated a predominantly nuclear localization of FLAG-tagged HCDA overexpressed in these cells. We have identified an amino-terminal bipartite nuclear localization signal that is both necessary and sufficient to direct HCDA and a non-nuclear reporter protein to the nucleus. We also show HCDA binding to the nuclear import receptor, importin alpha. Similar putative bipartite nuclear localization sequences are found in other cytidine/deoxycytidylate deaminases. The results presented here suggest that the pyrimidine nucleotide salvage pathway may operate in the nucleus. This localization may have implications in the regulation of nucleoside and nucleotide metabolism and nucleic acid biosynthesis.
Highlights
The cytidine deaminases belong to the family of multisubunit enzymes that catalyze the hydrolytic deamination of their substrate to a corresponding uracil product
The enzymes are generally considered to be cytoplasmic in intracellular localization, in the sequence of human cytidine deaminase (HCDA), we identified two clusters of basic amino acids near the amino terminus with similarity to the classical bipartite nuclear localization signal
FLAG-tagged wild-type HCDA and point mutants, generated by polymerase chain reaction (PCR), were expressed in E. coli, and the expressed protein was purified on a FLAG affinity column
Summary
Angelika Somasekaram‡§, Adam Jarmuz‡, Alan How‡, James Scott¶ʈ**, and Naveenan Navaratnam‡**. The cytidine deaminases belong to the family of multisubunit enzymes that catalyze the hydrolytic deamination of their substrate to a corresponding uracil product They play a major role in pyrimidine nucleoside and nucleotide salvage. Cytidine, and deoxycytidylate deaminases belong to the family of enzymes that deaminate mononucleotide substrates and are involved in maintenance of the pyrimidine nucleotide pool in the cell [1]. The enzymes are generally considered to be cytoplasmic in intracellular localization, in the sequence of HCDA, we identified two clusters of basic amino acids near the amino terminus with similarity to the classical bipartite nuclear localization signal. Other members of this family of enzymes contain similar sequences These studies suggest that the nuclear localization of HCDA may be important in providing substrate for RNA biosynthesis, in times of high metabolic activity.
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