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Quantitative DIA/SWATH proteomic profiling of SAGA complex mutants in Cryptococcus neoformans

ABSTRACT: This project investigates the proteomic consequences of deleting key subunits of the SAGA transcriptional coactivator complex (Hfi1, Spt7 and Spt20) in Cryptococcus neoformans. Quantitative SWATH/DIA LC-MS/MS was performed on wild-type and mutant strains grown at 30°C and 37°C to identify pathway-level alterations associated with impaired SAGA function. The dataset provides peptide- and protein-level quantification to support differential abundance analysis and functional interpretation of SAGA-dependent regulatory processes.

INSTRUMENT(S):

ORGANISM(S): Fungi Cryptococcus Neoformans Var. Grubii Serotype A (strain H99 / Atcc 208821 / Cbs 10515 / Fgsc 9487) (filobasidiella Neoformans Var. Grubii)

TISSUE(S): Cell Culture

SUBMITTER: Chendi Yu

LAB HEAD: James Fraser

PROVIDER: PXD071194 | Pride | 2026-01-03

REPOSITORIES: Pride

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Project description:Cryptococcus neoformans is an opportunistic, human fungal pathogen which undergoes fascinating switches in cell cycle control and ploidy when it encounters stressful environments such as the human lung. Here we carry out a mechanistic analysis of the spindle checkpoint which regulates the metaphase to anaphase transition, focusing on Mps1 kinase and the downstream checkpoint components Mad1 and Mad2. We demonstrate that Cryptococcus mad1D or mad2D strains are unable to respond to microtubule perturbations, continuing to re-bud and divide, and die as a consequence. Fluorescent tagging of Chromosome 3, using a lacO array and mNeonGreen-lacI fusion protein, demonstrates that mad mutants are unable to maintain sister-chromatid cohesion in the absence of microtubule polymers. Thus, the classic checkpoint functions of the SAC are conserved in Cryptococcus. In interphase, GFP-Mad1 is enriched at the nuclear periphery, and it is recruited to unattached kinetochores in mitosis. Purification of GFP-Mad1 followed by mass spectrometric analysis of associated proteins show that it forms a complex with Mad2 and that it interacts with other checkpoint signalling components (Bub1) and effectors (Cdc20 and APC/C sub-units) in mitosis. We also demonstrate that overexpression of Mps1 kinase is sufficient to arrest Cryptococcus cells in mitosis, and show that this arrest is dependent on both Mad1 and Mad2. We find that a C-terminal fragment of Mad1 is an effective in vitro substrate for Mps1 kinase and map several Mad1 phosphorylation sites. Some sites are highly conserved within the C-terminal Mad1 structure and we demonstrate that mutation of threonine 667 (T667A) leads to loss of checkpoint signalling and abrogation of the GAL-MPS1 arrest. Thus Mps1-dependent phosphorylation of C-terminal Mad1 residues is a critical step in Cryptococcus spindle checkpoint signalling. We conclude that CnMps1 protein kinase, Mad1 and Mad2 proteins have all conserved their important, spindle checkpoint signalling roles helping ensure high fidelity chromosome segregation.

Project description:Carbon dioxide (CO2) sensing, transport, and metabolism play a pivotal role in survival and proliferation of pathogenic microbes infecting human host from natural environments due to the drastic difference of CO2 levels. Carbonic anhydrases (CAs) are not only key CO2-metabolic enzymes catalyzing reversible interconversion between CO2 and bicarbonate (HCO3-), but also important CO2-signaling modulators. Cryptococcus neoformans that causes fatal fungal meningitis contains two functional CAs, Can1 and Can2, among which Can2 plays essential roles for growth and sexual differentiation of the pathogen. However, downstream genes and signaling network regulated by CAs have not been studied thus far. In this study, we constructed a C. neoformans strain where CAN2 expression is controlled by the CTR4 (copper transporter) promoter and elucidated its transcriptome patterns by using DNA microarray analysis to elucidate downstream target genes of Can2. Expectedly, the CAN2 promoter replacement strain showed growth defects in a CO2-dependent manner when CAN2 is repressed. The CA-transcriptome analysis discovered a number of Can2-dependent genes, including those involved in fatty acid biosynthesis (FAS1), sexual differentiation (GPB1), capsule structure organization (CAS3), iron-metabolism (CFO2), and a number of stress-regulated genes, including an oxidative stress-responsive Atf1 transcription factor, although a majority of them do not have any orthologs in other fungi. The present study revealed other roles of Atf1 in capsule and melanin production and diverse stress responses, including thermotolerance and antifungal drug resistance, of C. neoformans. This study provides further insights into the signaling network of CA/CO2-sensing pathway in pathogenic fungi. 17 slides are used in this analysis, 3 or 2 biological replicate experiments are performed, total RNAs are extracted from 2 strains (H99 Wild type strain, CTR4::CAN2) at 2 time points (0hr time, 12hr time) in 2 conditions (BCS, CuSO4). We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy5 as Sample dye and Cy3 as a control dye. Several experiment samples are dye swaped.

			Action	DRS
	20240326_SWATH_output_Lib.tsv	Tabular
	WT_30C_rep1.wiff	Wiff
	WT_30C_rep1.wiff.scan	Wiff
	WT_30C_rep1.wiff2	Wiff
	WT_30C_rep2.wiff	Wiff

Dataset Information

Quantitative DIA/SWATH proteomic profiling of SAGA complex mutants in Cryptococcus neoformans

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