PXD052327 is an
original dataset announced via ProteomeXchange.
Dataset Summary
Title | Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint |
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. |
HostingRepository | PRIDE |
AnnounceDate | 2024-10-22 |
AnnouncementXML | Submission_2024-10-22_06:42:55.811.xml |
DigitalObjectIdentifier | |
ReviewLevel | Peer-reviewed dataset |
DatasetOrigin | Original dataset |
RepositorySupport | Unsupported dataset by repository |
PrimarySubmitter | Kevin Hardwick |
SpeciesList | scientific name: Cryptococcus neoformans; NCBI TaxID: 5207; |
ModificationList | phosphorylated residue |
Instrument | Orbitrap Exploris 480 |
Dataset History
Revision | Datetime | Status | ChangeLog Entry |
0 | 2024-05-16 17:06:02 | ID requested | |
1 | 2024-05-27 03:31:37 | announced | |
⏵ 2 | 2024-10-22 06:42:56 | announced | 2024-10-22: Updated project metadata. |
Publication List
Keyword List
submitter keyword: Cryptococcus neoformans, Mad2, Mad1, spindle checkpoint, Mps |
Contact List
Professor Kevin Hardwick |
contact affiliation | The University of Edinburgh |
contact email | kevin.hardwick@ed.ac.uk |
lab head | |
Kevin Hardwick |
contact affiliation | University of Edinburgh |
contact email | kevin.hardwick@ed.ac.uk |
dataset submitter | |
Full Dataset Link List
Dataset FTP location
NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2024/05/PXD052327 |
PRIDE project URI |
Repository Record List
[ + ]
[ - ]
- PRIDE
- PXD052327
- Label: PRIDE project
- Name: Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint