Mad2

Mad2 (mitotic arrest deficient 2) is an essential spindle checkpoint protein. The spindle checkpoint system is a regulatory system that restrains progression through the metaphase-to-anaphase transition. The Mad2 gene was first identified in the yeast S. cerevisiae in a screen for genes which when mutated would confer sensitivity to microtubule poisons. The human orthologues of Mad2 (MAD2L1 and MAD2L2) were first cloned in a search for human cDNAs that would rescue the microtubule poison-sensitivity of a yeast strain in which a kinetochore binding protein was missing. The protein was shown to be present at unattached kinetochores and antibody inhibition studies demonstrated it was essential to execute a block in the metaphase-to-anaphase transition in response to the microtubule poison nocodazole. Subsequent cloning of the Xenopus laevis orthologue, facilitated by the sharing of the human sequence, allowed for the characterization of the mitotic checkpoint in egg extracts. Mad2 (mitotic arrest deficient 2) is an essential spindle checkpoint protein. The spindle checkpoint system is a regulatory system that restrains progression through the metaphase-to-anaphase transition. The Mad2 gene was first identified in the yeast S. cerevisiae in a screen for genes which when mutated would confer sensitivity to microtubule poisons. The human orthologues of Mad2 (MAD2L1 and MAD2L2) were first cloned in a search for human cDNAs that would rescue the microtubule poison-sensitivity of a yeast strain in which a kinetochore binding protein was missing. The protein was shown to be present at unattached kinetochores and antibody inhibition studies demonstrated it was essential to execute a block in the metaphase-to-anaphase transition in response to the microtubule poison nocodazole. Subsequent cloning of the Xenopus laevis orthologue, facilitated by the sharing of the human sequence, allowed for the characterization of the mitotic checkpoint in egg extracts. Progression from metaphase to anaphase is marked by sister chromatid separation. The cell cycle surveillance mechanism that prevents sister-chromatid separation and transition into anaphase is called the spindle checkpoint. As a safeguard against chromosome segregation errors, the spindle assembly checkpoint (SAC) delays anaphase until all sister chromatid pairs have become bipolarly attached. Once microtubules attach to kinetochores, chromosomes are aligned on the metaphase plate, and proper bi-orientation has been achieved, the SAC stopping mechanisms are removed. Entrance into anaphase is mediated by APCCdc20 activation. APCCdc20 is a ubiquitin-protein ligase that tags the protein, securin, for destruction. Securin destruction liberates and activates its bound protease partner, separase. Separase bound to securin remains inhibited; however, when inhibition is relieved, activated separase cleaves the cohesin complex which links the sister chromatids together. Without Cdc20, the anaphase-promoting complex (APC) cannot become activated and anaphase is not triggered. Mad2 was shown to inhibit the activity of the APC by direct physical interaction in a ternary complex with Cdc20. Kinetochores that remain unattached to microtubules catalyze the sequestration of Cdc20 by Mad2. In fact, when metaphase mammalian cells are treated with the spindle-depolymerizing agent nocodazole, Mad2 proteins become localized at the kinetochores of all sister-chromatid pairs. Mad2 is capable of forming multimers and adopts at least two structural conformations. Open Mad2 differs from closed Mad2 in the positioning of the 50 residue C-terminal segment. This “safety belt” is held tightly against the right side of the protein in the open conformation. Upon loosening, the safety belt can be re-positioned around a binding partner. In the closed conformation, the safety belt wraps around the bound ligand and interacts with a different region of Mad2. Binding partners of Mad2 include either Cdc20 or Mad1. Mad1 and Cdc20 bind Mad2 in an identical fashion. Mad2 uses the same site to bind either Mad1 or Cdc20 and, thus, can only bind one of the two proteins at a time.