Identification And Characterization Of Candida Albicans CaPTC2, CaPPH3 And CaPTC5

Candida albicans is the most important opportunitive fungal pathogen. Type 2C protein phosphatases are one group of serine/threonine protein phosphatases involved in the reversible protein phosphorylation. To identify potential drug targets in C. albicans, we have characterized the functions of CaPTC2, CaPPH3 and CaPTC5 genes in this study.From the Candida genome database, we identified CaPTC2, CaPPH3 and CaPTC5 gene sequences. CaPTC2 has an open reading frame (ORF) of 1749 bp in length, and encodes a protein of 583 amino acids sharing 31.9% identity with ScPtc2p at amino acid level. We cloned and expressed the catalytic domain of CaPtc2p, and found that the purified catalytic domain protein exhibited dephosphorylation activity, which was dependent on the presence of Mg²⁺ or Mn²⁺ ion and inhibited by the protein Ser/Thr phosphatase inhibitor NaF. These results indicate that CaPTC2 encodes a PP2C phosphatase. We disrupted the two alleles of CaPTC2, and the homozygous mutant JR4 for CaPTC2 was sensitive to SDS, azole, and genotoxic agents such as HU and MMS. To our surprise, CaPtc2p is predominantly present in the mitochondria and also in the cytoplasm of C. albicans cells.CaPPH3 has an ORF of 783 bp, which encodes a protein of 261 amino acids sharing 68.2% identity with ScPph3p at the amino acid level. We constructed the homozygous mutant of CaPPH3. Its homozygous mutant shows sensitivity to genotoxic agents HU and MMS. Deletion of CaPPH3 promotes the filamentous growth of C. albicans in liquid SLAD and FBS media. Furthermore, we have constructed the double mutant for CaPTC2 and CaPPH3, which shows a severer sensitivity to genotoxic agents such as HU and MMS than their single gene deletion mutants. Therefore, both CaPtc2p and CaPph3p play important roles in checkpoint deactivation pathway.CaPTC5 has an ORF of 1740 bp, which encodes a protein of 580 amino acids sharing 39.7% identity with ScPtc5p at the amino acid level. We deleted two alleles of CaPTC5 but didn't found any phenotype for this mutant under various growth conditions. CaPtc2p is present in the mitochondria of C. albicans cells.