List of Bait Peptides and the Identified Proteins.

★<p>Proteins in italics were previously known to bind to the bait peptides and reproduced in the study. The interactions between proteins in bold italics and the bait proteins were identified in the bioinformatics study.</p

Experimental outline of PAX methodology.

<p>(<b>A</b>) Arrays of bait peptides are synthesized onto PEG-based membrane supports. The photo-activatable amino acid cross-linker (pBpa) is incorporated into the bait peptides so that each peptide spot has pBpa at a different position in the sequence (see inset schematic). (<b>B</b>, <b>C</b>) The membrane is incubated with cell lysate and subjected to 350–365 nm light to cross-link with interacting proteins. (<b>D</b>) The indirect and non-specific interactors are removed by high stringent, denaturing washes. (<b>E</b>, <b>F</b>) Each strip of the bait peptide spots is cut off the membrane, further chopped into small pieces, and trypsinized. (<b>G</b>, <b>H</b>) The samples are filtered to remove PEG and subjected to LC-MS/MS analysis to identify the photo-trapped proteins.</p

PAX-captured proteins are MS identifiable.

<p>(<b>A</b>) PAX captures a known interacting protein following over-expression in FreeStyle 293 cells. An array of peptides consisting of a proline-rich sequence of FMNL1 and a control sequence were incubated with FreeStyle 293 cell lysate overexpressing srGAP2, photo-crosslinked and washed at high stringency. Immunoblotting with srGAP2 antibody detected phototrapping of srGAP2 by PAX. pBpa in the bait peptides is indicated in blue. (<b>B</b>) Comparative immunoblotting of srGAP2 antigen dot-blot. Different amounts of srGAP2 were spotted onto nitrocellulose membrane and simultaneously immunoblotted with srGAP2 antibody. (<b>C</b>) Identification of srGAP2 by mass spectrometry. The five spots with the highest amount of crosslinked srGAP2 from a duplicate blot of (A) were subjected to MS/MS. 3 unique peptides (underlined) covering 8% of the entire sequence of srGAP2 protein were identified. (<b>D</b>) PAX captures a known interacting protein from mouse brain lysate. Peptide array comprising mGluR5 sequences with a PPXXF motif was incubated with mouse brain lysate, photo-crosslinked and subjected to high stringency washes. Immunoblotting with Homer1 antibody indicates photo-trapping of Homer1 by PAX. pBpa in the bait peptides is indicated in blue.</p

Calcineurin Targets Involved in Stress Survival and Fungal Virulence

<div><p>Calcineurin governs stress survival, sexual differentiation, and virulence of the human fungal pathogen <i>Cryptococcus neoformans</i>. Calcineurin is activated by increased Ca²⁺ levels caused by stress, and transduces signals by dephosphorylating protein substrates. Herein, we identified and characterized calcineurin substrates in <i>C</i>. <i>neoformans</i> by employing phosphoproteomic TiO₂ enrichment and quantitative mass spectrometry. The identified targets include the transactivator Crz1 as well as novel substrates whose functions are linked to P-bodies/stress granules (PBs/SGs) and mRNA translation and decay, such as Pbp1 and Puf4. We show that Crz1 is a <i>bona fide</i> calcineurin substrate, and Crz1 localization and transcriptional activity are controlled by calcineurin. We previously demonstrated that thermal and other stresses trigger calcineurin localization to PBs/SGs. Several calcineurin targets localized to PBs/SGs, including Puf4 and Pbp1, contribute to stress resistance and virulence individually or in conjunction with Crz1. Moreover, Pbp1 is also required for sexual development. Genetic epistasis analysis revealed that Crz1 and the novel targets Lhp1, Puf4, and Pbp1 function in a branched calcineurin pathway that orchestrates stress survival and virulence. These findings support a model whereby calcineurin controls stress and virulence, at the transcriptional level via Crz1, and post-transcriptionally by localizing to PBs/SGs and acting on targets involved in mRNA metabolism. The calcineurin targets identified in this study share little overlap with known calcineurin substrates, with the exception of Crz1. In particular, the mRNA binding proteins and PBs/SGs residents comprise a cohort of novel calcineurin targets that have not been previously linked to calcineurin in mammals or in <i>Saccharomyces cerevisiae</i>. This study suggests either extensive evolutionary rewiring of the calcineurin pathway, or alternatively that these novel calcineurin targets have yet to be characterized as calcineurin targets in other organisms. These findings further highlight <i>C</i>. <i>neoformans</i> as an outstanding model to define calcineurin-responsive virulence networks as targets for antifungal therapy.</p></div

Mutations of calcineurin-dependent phosphorylation sites promotes Crz1 nuclear localization and transcriptional activity at 24°C.

<p><b>(A)</b> Schematic diagram of the Crz1 protein drawn to scale. The yellow pins indicate the locations of 7 of the 12 predicted phosphorylation sites acted upon by calcineurin and mutated in the Crz1^7S-A strain; the green and red boxes show the PolyQ domain and the zinc finger domains, respectively. <b>(B)</b> The Crz1^WT (ECt3) and Crz1^7S-A (ECt335) strains were grown at 24°C and then shifted to 37°C for 15 minutes prior to fixation with 4% formaldehyde and visualized by direct fluorescence microscopy. GFP-Nop1 served as a nucleolar marker. <b>(C)</b> Quantification of cells with Crz1 nuclear localization at 24°C and after thermal stress (37°C) for 15 minutes. Error bars represent standard deviation; quantifications were conducted using three biological replicates. <b>(D)</b> To assay Crz1 gel migration mobility, the Crz1^WT (ECt3), Crz1^4S-A (ECt223), Crz1^6S-A (ECt231) and Crz1^7S-A (ECt335) strains were grown to log phase at 24°C and then shifted to 37°C for 1 hr in the presence or absence of 1 μg/ml FK506. Protein was extracted and analysed by western blot with mCherry antibody. <b>(E)</b> Expression levels of the <i>CHS6</i> gene in WT (H99), <i>crz1</i>Δ (AFA3-3), Crz1^WT, Crz1^6S-A, and Crz1^7S-A strains employed in panel 3D were compared. Strains were grown at 24°C in YPD medium with or without 1 μg/ml FK506. RNA was isolated and the expression of the <i>CHS6</i> gene was assessed by real-time PCR. Results shown are representative of three biological replicates. <b>(F)</b> The wild-type (H99), <i>cna1</i>Δ (KK1), <i>crz1</i>Δ (AFA3-3), Crz1^7S-A (ECt335 and ECt362), and Crz1^WT (ECt3 and ECt4) strains were grown in YPD media. Five 10-fold serial dilutions of each strain were spotted onto YPD containing the indicated additives and incubated at 30°C for 48 hours. Cultures with no additives were incubated at 30°C or 37°C for 48 hours or 39°C for 72 hours. CR: Congo Red; CFW: calcofluor white.</p

Identification of calcineurin-dependent targets.

<p><b>(A)</b> Venn diagram illustrating the number of potential calcineurin targets identified by the phosphopeptide screens and target overlap between the 44 phosphorylated proteins enriched by at least 2-fold in the two calcineurin-deficient samples (FK506 exposure or <i>cna1</i> mutation). Phosphopeptide profiles from WT (H99) cells grown at 25°C and shifted to 37°C for 1 hr and exposed or not to FK506 were aligned and compared (FK506). A similar comparison was performed for phosphopeptides profiles from the WT (strain H99) culture shifted from 25°C to 37°C and a <i>cna1</i>Δ (strain KK1) culture subjected to the same temperature shift (<i>cna1Δ</i>). Biological cultures were performed in duplicate for each condition tested and following trypsin digestion each sample was divided into three to access TiO2 enrichment and analytical reproducibility. <b>(B)</b> Functional category of the forty-four putative calcineurin targets. <b>(C)</b> Summary of potential calcineurin targets identified at 37°C. Note that this table only lists a subset of the potential calcineurin targets at 37°C with at least a 2-fold or greater signal difference (T-test p-value <0.05) in the two different screens and for which a characterized <i>S</i>. <i>cerevisiae</i> ortholog is known (based on best hit blast searches against the <i>S</i>. <i>cerevisiae</i> genome database) and does not include the identified but uncharacterized ORFs. The differential fold change signal for each individual phosphopeptide (see <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005873#ppat.1005873.s007" target="_blank">S2</a></b> and <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005873#ppat.1005873.s008" target="_blank">S3</a> Tables</b> for detailed and complete individual phosphopeptide data) within the same protein observed under the FK506 or <i>cna1</i>Δ condition is indicated. * denotes proteins characterized as components of PB/SGs in <i>C</i>. <i>neoformans</i>.</p

Phenotypes of the calcineurin target mutants exposed to various stresses.

<p>Spot dilution assays with WT (H99), <i>cna1</i>Δ (HP243), <i>crz1</i>Δ (HP235), <i>lhp1</i>Δ (HP22), <i>pbp1</i>Δ (HP6), <i>puf4</i>Δ (HP17), <i>vts1</i>Δ (HP24), <i>anb1</i>Δ (HP36), <i>tif3</i>Δ (HP9), <i>gcd2</i>Δ (HP28), and <i>gwo1</i>Δ (HP1) mutants were performed under several stress conditions as indicated. Strain cultures were incubated overnight, serially diluted 10-fold, and plated on YPD medium without or with FK506, CaCl₂, Congo red (CR), dithiothreitol (DTT), sodium dodecyl sulfate (SDS), and tunicamycin (TM) at the indicated concentrations. Cells were incubated for 2 to 3 days at 30°C, 37°C, 38°C, or 39°C as indicated and all cultures with compound additions were incubated at 30°C. Results shown are representative of two independent experimental replicates.</p

Calcineurin regulates the phosphorylation of Crz1.

<p><b>(A)</b> Crz1 electrophoretic mobility is altered by conditions that inhibit or activate calcineurin. Cultures of the <i>crz1</i>Δ <i>CRZ1-</i>4xFLAG complemented strain (SEC435) were grown to log phase at 24°C or shifted from 24°C to 37°C for 1 hour in the presence or absence of 1 μg/ml FK506. <b>(B)</b> Crz1 is a phosphoprotein. The Crz1-4xFLAG protein, immunoprecipitated from logarithmically growing cultures of strain SEC435 (see panel A) exposed or not to 1 μg/ml FK506 for 1 hr, was treated with λ phosphatase in the presence or absence of phosphatase inhibitors. <b>(C)</b> The Crz1-FLAG protein was immunoprecipitated from whole cell extracts of the <i>crz1</i>Δ + <i>CRZ1-</i>4xFLAG complemented strain (SEC435) or the <i>crz1</i>Δ + <i>CRZ1-</i>4xFLAG <i>cna1</i>Δ (HP289) strain. Immunoprecipitated Crz1-FLAG protein was treated with λ phosphatase, with and without phosphatase inhibitors. <b>(D)</b> Human calcineurin dephosphorylates Crz1 <i>in vitro</i>. Immunopurified Crz1-4xFLAG protein from the <i>crz1</i>Δ + <i>CRZ1-</i>4xFLAG complemented strain (SEC435) or the <i>crz1</i>Δ + <i>CRZ1-</i>4xFLAG <i>cna1</i>Δ (HP289) strain was treated with human calcineurin or λ phosphatase <i>in vitro</i>. In <b>Fig 2 A-D</b> following the different treatments, samples were resolved by SDS-PAGE and Crz1-4xFLAG mobility was assessed by western blot analysis as indicated in material methods. <b>(E)</b> Localization of Crz1-mCherry in WT (XW252) and the <i>cna1</i>Δ (HP282) mutant at 24°C and 37°C (upper panel). Each strain culture was grown to log phase at 24°C or shifted from 24°C to 37°C for 1 hr and visualized with a DeltaVision Elite Deconvolution microscope. The graph represents quantification of cells in which Crz1-mCherry and GFP-Nop1 were co-localized (bottom panel). <b>(F)</b> Expression of chitin synthase genes including <i>CHS5</i>, <i>CHS6</i>, and <i>CHS7</i> in WT (H99), <i>cna1</i>Δ (KK1), and <i>crz1</i>Δ (LK343) strains. WT, <i>cna1</i>Δ, and <i>crz1</i>Δ strains were grown to log phase at 24°C or shifted from 24°C to 37°C for 1 hr. RNA was isolated from the indicated strains and the expression of the chitin synthase genes was assessed by real-time PCR. Error bars depict the standard deviations from the mean of three independent experiments. Results shown in <b>Fig 2 A-D</b> and <b>2F</b> are representative of two independent experimental replicates.</p

The calcineurin pathway controls growth at 37°C, virulence, and sexual reproduction via multiple targets.

<p>The calcineurin signaling network is branched to control distinct targets including Crz1, Lhp1, Puf4, and Pbp1 in different intracellular localizations (nucleus and PB/SGs) to evoke transcriptional and post-transcriptional programs necessary for <i>Cryptococcus</i> thermotolerance, virulence, and sexual reproduction (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005873#sec008" target="_blank">Discussion</a>).</p

Sexual reproduction phenotypes of calcineurin target mutants.

<p><b>(A)</b> Sexual reproduction assays for calcineurin target mutants were conducted. The WT (H99, KN99), and the <i>cna1</i>Δ (HP242, HP243), <i>crz1</i>Δ (HP235, HP239), <i>lhp1</i>Δ (HP22, HP258), <i>pbp1</i>Δ (HP6, HP246), <i>puf4</i>Δ (HP17, HP254), <i>vts1</i>Δ (HP24, HP261), <i>anb1</i>Δ (HP36, HP263), <i>tif3</i>Δ (HP9, HP250), <i>gcd2</i>Δ (HP28, HP264), and <i>gwo1</i>Δ (HP1, HP266) mutant strains were co-cultured with the opposite mating type strain on MS media and incubated at room temperature in the dark for 7 days. Several calcineurin targets including <i>LHP1</i>, <i>PUF4</i>, and <i>TIF3</i> are required for proper sexual reproduction. Results shown are representative of two independent experimental replicates. <b>(B)</b> Pheromone gene (<i>MF</i>α<i>1</i>) expression was analyzed in WT, <i>cna1</i>Δ, and the indicated calcineurin target mutants mating crosses following incubation in V8 media at room temperature for 24 hrs as described in the legend to <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005873#ppat.1005873.g003" target="_blank">Fig 3C</a></b>. The results shown are the mean of three independent determinations with standard deviation as error bars.</p