title: 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 cts1Δ 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 calcineurin-dependent 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.
authors: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
authors: Eanas F
authors: EF
authors: Aboobakar
authors: Xuying
authors: X
authors: Wang
authors: Joseph
authors: J
authors: Heitman
authors: Lukasz
authors: L
authors: Kozubowski
mesh_terms: D019703:Calcineurin / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D003455:Cryptococcus neoformans / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*; D048749:Cytokinesis; D003594:Cytoplasmic Granules / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006358:Hot Temperature; D025301:Hyphae / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*; D010641:Phenotype; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012323:RNA Processing, Post-Transcriptional; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013312:Stress, Physiological; D016559:Tacrolimus / Q000494:pharmacology
chemical_list: D065095:Calcineurin Inhibitors; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019703:Calcineurin; D016559:Tacrolimus