Spindle Checkpoint Protein Xmad1 Recruits Xmad2 to Unattached Kinetochores

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.

[1]  A. Shevchenko,et al.  Rapid 'de novo' peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer. , 1997, Rapid communications in mass spectrometry : RCM.

[2]  Andrew W. Murray,et al.  Association of Spindle Assembly Checkpoint Component XMAD2 with Unattached Kinetochores , 1996, Science.

[3]  A. Murray,et al.  Activation of the Budding Yeast Spindle Assembly Checkpoint Without Mitotic Spindle Disruption , 1996, Science.

[4]  A Khodjakov,et al.  The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores , 1995, The Journal of cell biology.

[5]  A. Murray,et al.  Mad1p, a phosphoprotein component of the spindle assembly checkpoint in budding yeast , 1995, The Journal of cell biology.

[6]  A. Murray,et al.  Budding yeast Cdc20: a target of the spindle checkpoint. , 1998, Science.

[7]  A. Murray,et al.  Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase , 1992, The Journal of cell biology.

[8]  M. Kirschner,et al.  A 20s complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B , 1995, Cell.

[9]  M. Hoyt,et al.  Bub1p Kinase Activates the Saccharomyces cerevisiae Spindle Assembly Checkpoint , 1998, Molecular and Cellular Biology.

[10]  J. R. Daum,et al.  Mammalian p55CDC Mediates Association of the Spindle Checkpoint Protein Mad2 with the Cyclosome/Anaphase-promoting Complex, and is Involved in Regulating Anaphase Onset and Late Mitotic Events , 1998, The Journal of cell biology.

[11]  Stephen S. Taylor,et al.  The Human Homologue of Bub3 Is Required for Kinetochore Localization of Bub1 and a Mad3/Bub1-related Protein Kinase , 1998, The Journal of cell biology.

[12]  Kim Nasmyth,et al.  Closing the cell cycle circle in yeast: G2 cyclin proteolysis initiated at mitosis persists until the activation of G1 cyclins in the next cycle , 1994, Cell.

[13]  A. Murray,et al.  Cyclin is degraded by the ubiquitin pathway , 1991, Nature.

[14]  R. Benezra,et al.  Identification of a Human Mitotic Checkpoint Gene: hsMAD2 , 1996, Science.

[15]  M. Kirschner,et al.  Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. , 1996, Genes & development.

[16]  V. Simanis,et al.  The fission yeast dma1 gene is a component of the spindle assembly checkpoint, required to prevent septum formation and premature exit from mitosis if spindle function is compromised. , 1996, The EMBO journal.

[17]  A. Hershko,et al.  Methylated ubiquitin inhibits cyclin degradation in clam embryo extracts. , 1991, The Journal of biological chemistry.

[18]  G. Woude,et al.  The c-mos proto-oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs , 1989, Nature.

[19]  A. Shevchenko,et al.  Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. , 1996, Analytical chemistry.

[20]  A. Murray,et al.  Localization of Mad2 to Kinetochores Depends on Microtubule Attachment, Not Tension , 1998, The Journal of cell biology.

[21]  K Nasmyth,et al.  Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast. , 1993, The EMBO journal.

[22]  Andrew W. Murray,et al.  Anaphase is initiated by proteolysis rather than by the inactivation of maturation-promoting factor , 1993, Cell.

[23]  Tim Hunt,et al.  Cut2 proteolysis required for sister-chromatid separation in fission yeast , 1996, Nature.

[24]  E. Nishida,et al.  MAP Kinase Is Required for the Spindle Assembly Checkpoint but Is Dispensable for the Normal M Phase Entry and Exit in Xenopus Egg Cell Cycle Extracts , 1997, The Journal of cell biology.

[25]  Michael Schwab,et al.  Yeast Hct1 Is a Regulator of Clb2 Cyclin Proteolysis , 1997, Cell.

[26]  S. Artavanis-Tsakonas,et al.  The Drosophila cell cycle gene fizzy is required for normal degradation of cyclins A and B during mitosis and has homology to the CDC20 gene of Saccharomyces cerevisiae , 1995, The Journal of cell biology.

[27]  A. Hershko.  Roles of ubiquitin-mediated proteolysis in cell cycle control. , 1997, Current opinion in cell biology.

[28]  Kuan-Teh Jeang,et al.  Human T Cell Leukemia Virus Type 1 Oncoprotein Tax Targets the Human Mitotic Checkpoint Protein MAD1 , 1998, Cell.

[29]  M. Kirschner,et al.  The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation. , 1998, Genes & development.

[30]  J. Blenis,et al.  Nuclear localization and regulation of erk- and rsk-encoded protein kinases , 1992, Molecular and cellular biology.

[31]  Kim Nasmyth,et al.  Genes involved in sister chromatid separation are needed for b-type cyclin proteolysis in budding yeast , 1995, Cell.

[32]  A. Murray,et al.  Characterization of spindle assembly checkpoint in Xenopus egg extracts. , 1997, Methods in enzymology.

[33]  M. Wilm,et al.  Analytical properties of the nanoelectrospray ion source. , 1996, Analytical chemistry.

[34]  Eric L. Weiss,et al.  The Saccharomyces cerevisiae spindle pole body duplication gene MPS1 is part of a mitotic checkpoint , 1996, The Journal of cell biology.

[35]  A. Murray,et al.  A MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts , 1994, Cell.

[36]  A. Murray,et al.  Cell cycle extracts. , 1991, Methods in cell biology.

[37]  B. Roberts,et al.  S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function , 1991, Cell.

[38]  M. Ashburner.  A Laboratory manual , 1989 .

[39]  Stephen S. Taylor,et al.  Kinetochore Localization of Murine Bub1 Is Required for Normal Mitotic Timing and Checkpoint Response to Spindle Damage , 1997, Cell.

[40]  Bert Vogelstein,et al.  Mutations of mitotic checkpoint genes in human cancers , 1998, Nature.

[41]  B. Roberts,et al.  The Saccharomyces cerevisiae checkpoint gene BUB1 encodes a novel protein kinase. , 1994, Molecular and cellular biology.

[42]  A. Hershko,et al.  The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis. , 1995, Molecular biology of the cell.

[43]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[44]  Andrew W. Murray,et al.  Feedback control of mitosis in budding yeast , 1991, Cell.

[45]  D. Koshland,et al.  The metaphase-to-anaphase transition: avoiding a mid-life crisis. , 1997, Current opinion in cell biology.

[46]  Tomohiro Matsumoto,et al.  Fission yeast Slp1: an effector of the Mad2-dependent spindle checkpoint. , 1998, Science.

[47]  S. Sazer,et al.  The Schizosaccharomyces pombe spindle checkpoint protein mad2p blocks anaphase and genetically interacts with the anaphase-promoting complex. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[48]  R. Benezra,et al.  MAD2 associates with the cyclosome/anaphase-promoting complex and inhibits its activity. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[49]  A. Bruno,et al.  Hologram-based refractive index detector for capillary electrophoresis: separation of metal ions , 1994 .

[50]  C. A. Thomas,et al.  Molecular cloning. , 1977, Advances in pathobiology.

[51]  R. Nicklas,et al.  Mitotic forces control a cell-cycle checkpoint , 1995, Nature.

[52]  S. Prinz,et al.  CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis. , 1997, Science.

[53]  M. Jones,et al.  Mph1, a member of the Mps1-like family of dual specificity protein kinases, is required for the spindle checkpoint in S. pombe. , 1998, Journal of cell science.

[54]  A. Murray,et al.  Microinjection of Antibody to Mad2 Protein into Mammalian Cells in Mitosis Induces Premature Anaphase , 1998, The Journal of cell biology.