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

• Published in: Eukaryotic Cell 2011-12, Vol.10 (12), p.1714-1723
• Main Authors: Aboobakar, Eanas F, Wang, Xuying, Heitman, Joseph, Kozubowski, Lukasz
• Format: Article
• Language: English
• Subjects: alkaline phosphatase; Calcineurin - metabolism; Calcineurin Inhibitors; calves; Cryptococcus neoformans; Cryptococcus neoformans - growth & development; Cryptococcus neoformans - metabolism; Cryptococcus neoformans - physiology; Cytokinesis; Cytoplasmic Granules - metabolism; electrophoresis; Endocytosis; Endosomes - metabolism; Fungal Proteins - genetics; Fungal Proteins - metabolism; fungi; Green Fluorescent Proteins - metabolism; Hot Temperature; humans; Hyphae - growth & development; Hyphae - metabolism; Hyphae - physiology; messenger RNA; molecular weight; mutants; mutation; pathogens; Phenotype; phosphoprotein phosphatase; Phosphorylation; Protein Binding; Protein Structure, Tertiary; protein subunits; Protein Transport; Recombinant Fusion Proteins - metabolism; RNA Processing, Post-Transcriptional; Signal Transduction; stress response; Stress, Physiological; Tacrolimus - pharmacology; thermal stress; virulence
• ISSN: 1535-9778; 1535-9786
• DOI: 10.1128/EC.05148-11

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.