SPINDLE CHECKPOINT PROTEIN XMAD1 RECRUITS XMAD2 TO UNATTACHED KINETOCHORES

The spindle checkpoint prevents the metaphase to anaphase transition i n cells containing defects in the mitotic spindle or in chromosome att achment to the spindle. When the checkpoint protein Xmad2 is depleted from Xenopus egg extracts, adding Xmad2 to its endogenous concentratio n fails to restore the checkpoint, suggesting that other checkpoint co mponent(s) were depleted from the extract through their association wi th Xmad2. Mass spectrometry provided peptide sequences from an 85-kD p rotein that coimmunoprecipitates with Xmad2 from egg extracts. This in formation was used to clone XMAD1, which encodes a homologue of the bu dding yeast (Saccharomyces cerevisiae) checkpoint protein Mad1. Xmad1 is essential for establishing and maintaining the spindle checkpoint i n egg extracts. Like Xmad2, Xmad1 localizes to the nuclear envelope an d the nucleus during interphase, and to those kinetochores that are no t bound to spindle microtubules during mitosis. Adding an anti-Xmad1 a ntibody to egg extracts inactivates the checkpoint and prevents Xmad2 from localizing to unbound kinetochores. In the presence of excess Xma d2, neither chromosomes nor Xmad1 are required to activate the spindle checkpoint, suggesting that the physiological role of Xmad1 is to rec ruit Xmad2 to kinetochores that have not bound microtubules.