The C2 Domain Protein Cts1 Functions in the Calcineurin Signaling Circuit during High-Temperature Stress Responses in Cryptococcus neoformans

Abstract

Calcineurin is a conserved calcium/calmodulin-dependent serine/threonine-specific protein phosphatase that acts in cell stress responses. Calcineurin is essential for growth at 37°C and for virulence of the human fungal pathogen Cryptococcus neoformans, but its substrates remain unknown. The C2 domain-containing, phospholipid-binding protein Cts1 was previously identified as a multicopy suppressor of a calcineurin mutation in C. neoformans. Here we further characterize the function of Cts1 and the links between Cts1 and calcineurin. GFP-Cts1 localizes to cytoplasmic puncta and colocalizes with the endosomal marker FM4-64. The cts1⌬ mutant shows a distinct FM4-64 staining pattern, suggesting involvement of Cts1 in endocytic trafficking. In large budded cells, GFP-Cts1 localizes transiently at the mother bud neck, as a single ring that undergoes contraction. mCherry-Cts1 colocalizes with the GFP-tagged calcineurin catalytic subunit Cna1 at sites of mRNA processing at 37°C, suggesting that Cts1 and calcineurin function coordinately during thermal stress. GFP-Cts1 exhibits slower electrophoretic mobility for cells grown at 37°C than for cells grown at 24°C, and the shift to a higher molecular weight is more pronounced in the presence of the calcineurin inhibitor FK506. In vitro treatment with calf intestinal alkaline phosphatase (CIP) restores faster electrophoretic mobility to GFP-Cts1, suggesting that Cts1 is phosphorylated at 37°C and may be dephosphorylated in a calcineurindependent manner. mCherry-Cts1 also coimmunoprecipitates with GFP-Cna1, with greater complex formation at 37°C than at 24°C. Taken together, these findings support potential roles for Cts1 in endocytic trafficking, mRNA processing, and cytokinesis and suggest that Cts1 is a substrate of calcineurin during high-temperature stress responses.Cryptococcus neoformans is a human fungal pathogen that causes life-threatening meningitis, primarily in immunocompromised patients (5, 17). Cryptococcal meningitis is one of the most important HIV-related opportunistic infections, with ϳ1 million cases occurring each year (36). Identification of novel therapeutic targets for cryptococcosis is essential given the toxic effects of existing therapies and the rising prevalence of drug-resistant strains (38).The ability to survive, grow, and divide at human physiological temperature is an important C. neoformans virulence attribute and is orchestrated by elaborate signaling pathways that are not yet fully elucidated (3, 19). Calcineurin, a calcium/ calmodulin-dependent serine/threonine-specific protein phosphatase, is essential for C. neoformans growth at 37°C and for its virulence (1, 21, 35). Calcineurin consists of two subunits, the catalytic A (Cna1) and the regulatory B (Cnb1) subunits, both of which are necessary for enzymatic function and survival of C. neoformans at 37°C (1,12,35). Studies of both pathogenic and nonpathogenic fungi have revealed important roles for calcineurin in myriad physiological processes, including morphogenesis, cell cycle progress...

A Fungus-Specific Protein Domain Is Essential for RasA-Mediated Morphogenetic Signaling in Aspergillus fumigatus

“…The outcome of this interaction is to regulate complex morphogenetic networks. To see if RasA-Cdc42 interactions are also altered in the IRD truncation mutant, we utilized a coimmunoprecipitation assay, as previously described (27). Strains coexpressing a single copy of either GFP-RasA or GFP-RasAΔIRD along with mCherry-Cdc42 in a wild-type A. fumigatus genetic background were generated.…”

Mechanisms of cytokinesis in basidiomycetous yeasts

“…Specifically the cts1 Δ mutant cells did not form typical primary septa and instead accumulated an abnormally thick septal material, similar to S. cerevisiae inn1 Δ mutant (Fox, Cox et al 2003, Nishihama, Schreiter et al 2009). Interestingly, the constriction of Cts1 appeared to follow rather than coincide with the constriction of the AMR (Aboobakar, Wang et al 2011), in contrast to Inn1 (Nishihama, Schreiter et al 2009). Whether Inn1 depends on AMR for localization remains controversial and it would be of interest to test it for the Cts1 in C. neoformans (Sanchez-Diaz, Marchesi et al 2008, Nishihama, Schreiter et al 2009).…”

“…The U. maydis orthologue of the S. pombe myosin light chain Cdc4p localizes to the septin collar prior to mitosis and the reorganization of septins is necessary for the subsequent incorporation of Cdc4, along with the F-actin, into the AMR (Shannon and Li 2000, Bohmer, Ripp et al 2009). In C. neoformans , Myo1 localizes to the bud neck and constricts similarly to the S. cerevisiae homologue (Aboobakar, Wang et al 2011). C. neoformans genome encodes an anillin homologue, which may participate in septin organization similar to Bud4 and Mid2; the U. maydis homologue candidate seems to be relatively less conserved (Table).…”

“…C. neoformans C2 domain protein Cts1 (for calcineurin mutant temperature sensitivity suppressor 1) is homologous to the S. pombe Fic1 and S. cerevisiae Inn1 (Fox, Cox et al 2003, Roberts-Galbraith, Chen et al 2009, Aboobakar, Wang et al 2011). Cts1 localizes to the site of cytokinesis and the deletion of the CTS1 results in cell separation defect (Fox, Cox et al 2003, Aboobakar, Wang et al 2011).…”

“…Cts1 localizes to the site of cytokinesis and the deletion of the CTS1 results in cell separation defect (Fox, Cox et al 2003, Aboobakar, Wang et al 2011). Specifically the cts1 Δ mutant cells did not form typical primary septa and instead accumulated an abnormally thick septal material, similar to S. cerevisiae inn1 Δ mutant (Fox, Cox et al 2003, Nishihama, Schreiter et al 2009).…”