Calcineurin is a calcium-calmodulin-activated serine/threonine-specific phosphatase that operates during cellular responses to stress and plays a prominent role in transcriptional control, whereas regulatory events beyond transcription are less well characterized. This study reveals a novel transcription-independent role of calcineurin during the temperature stress response in the human fungal pathogen Cryptococcus neoformans. The diffusely cytoplasmic calcineurin catalytic subunit Cna1 relocates to endoplasmic reticulum (ER)-associated puncta and the mother-bud neck when cells are subjected to 37°C. More than 50% of Cna1 puncta contain the P-body constituent decapping enzyme Dcp1 and the stress granule constituent poly(A)-binding protein Pub1. These results support a model in which calcineurin orchestrates thermal stress responses by associating with sites of mRNA processing.

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...