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Spindle checkpoint protein Xmad1 recruits Xmad2 to unattached kinetochores
R H Chen1, A Shevchenko, M Mann
1Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA. rc70@cornell.edu
Abstract:
The spindle checkpoint prevents the metaphase to anaphase transition in cells containing defects in the mitotic spindle or in chromosome attachment to the spindle. When the checkpoint protein Xmad2 is depleted from Xenopus egg extracts, adding Xmad2 to its endogenous concentration fails to restore the checkpoint, suggesting that other checkpoint component(s) were depleted from the extract through their association with Xmad2. Mass spectrometry provided peptide sequences from an 85-kD protein that coimmunoprecipitates with Xmad2 from egg extracts. This information was used to clone XMAD1, which encodes a homologue of the budding yeast (Saccharomyces cerevisiae) checkpoint protein Mad1. Xmad1 is essential for establishing and maintaining the spindle checkpoint in egg extracts. Like Xmad2, Xmad1 localizes to the nuclear envelope and the nucleus during interphase, and to those kinetochores that are not bound to spindle microtubules during mitosis. Adding an anti-Xmad1 antibody to egg extracts inactivates the checkpoint and prevents Xmad2 from localizing to unbound kinetochores. In the presence of excess Xmad2, neither chromosomes nor Xmad1 are required to activate the spindle checkpoint, suggesting that the physiological role of Xmad1 is to recruit Xmad2 to kinetochores that have not bound microtubules.
Insights
The spindle assembly checkpoint ensures proper cell division. Researchers identified Xmad1, a protein crucial for recruiting Xmad2 to kinetochores, thereby maintaining the spindle checkpoint.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The spindle checkpoint is vital for preventing errors during cell division by halting the cell cycle if the mitotic spindle or chromosome attachments are defective.
- Depletion of the checkpoint protein Xmad2 from Xenopus egg extracts and subsequent addition to endogenous levels failed to restore checkpoint function, indicating the depletion of other associated proteins.
Purpose of the Study:
- To identify and characterize novel components of the spindle assembly checkpoint.
- To elucidate the role of Xmad1 in the spindle checkpoint mechanism.
Main Methods:
- Mass spectrometry was employed to identify proteins co-immunoprecipitating with Xmad2.
- Xenopus egg extracts were used to study spindle checkpoint function.
- Antibody inhibition and protein localization studies were performed.
Main Results:
- Mass spectrometry identified an 85-kD protein that co-immunoprecipitates with Xmad2. This led to the cloning of XMAD1, a homolog of yeast Mad1.
- Xmad1 was found to be essential for establishing and maintaining the spindle checkpoint in egg extracts.
- Xmad1 localizes to the nuclear envelope and kinetochores, and its depletion or antibody inhibition disrupts Xmad2 localization and checkpoint function.
Conclusions:
- Xmad1 is a critical component of the spindle assembly checkpoint in Xenopus egg extracts.
- Xmad1 functions by recruiting Xmad2 to kinetochores that lack spindle microtubule attachment, thereby ensuring proper cell cycle regulation.