Inducible gene deletion reveals essentiality of protein kinases and a septation initiation network in Candida albicans

Protein kinases are key components of many signaling pathways that regulate cellular activities, and some of them are indispensable for the viability of cells. We used inducible gene deletion to assess the importance of a set of putative essential protein kinases for growth and viability of the pathogenic yeast Candida albicans and to get clues about the functions of uncharacterized essential kinases. We found that bud32Δ, ctk1Δ, rio1Δ, and rio2Δ mutants were viable but grew very slowly, explaining previous failures to generate homozygous deletion mutants. PTK2 was essential, but under certain conditions ptk2Δ mutants remained viable and over time could acquire suppressor mutations in the Ptk2-dependent plasma membrane ATPase Pma1 that restored growth. Deletion of the uncharacterized orf19.5376 was lethal and the null mutants formed pseudohyphae that lacked normal septa and eventually lysed, a phenotype that was phenocopied by auxin-induced protein depletion. The mutants were defective in septin organization, indicating that the orf19.5376-encoded kinase is functionally similar to the nonessential kinase Elm1 of Saccharomyces cerevisiae, but is indispensable for viability in C. albicans. Mutants lacking orf19.3456, which does not have a homolog in S. cerevisiae, were also nonviable and grew as aseptate, sometimes multinucleate hyphae before cell death. Co-immunoprecipitation followed by liquid chromatography-mass spectrometry identified a protein, encoded by the uncharacterized orf19.193, as a candidate regulatory subunit of the orf19.3456-encoded kinase, as mutants lacking this protein exhibited the same terminal phenotype as orf19.3456 mutants. These results provide strong evidence that instead of using a mitotic exit network (MEN) with only two kinases (Cdc15 and Dbf2), as was previously thought, C. albicans regulates septum formation and cytokinesis via a septation initiation network (SIN), known from fission yeast and filamentous fungi, which contains a protein kinase cascade consisting of the upstream kinase Cdc15, the orf19.3456-encoded kinase, and the downstream kinase Dbf2.