Explaining the oligomerization properties of the spindle assembly checkpoint protein Mad2

Abstract

Mad2 is an essential component of the spindle assembly checkpoint (SAC), a molecular device designed to coordinate anaphase onset with the completion of chromosome attachment to the spindle. Capture of chromosome by microtubules occur on protein scaffolds known as kinetochores. The SAC proteins are recruited to kinetochores in prometaphase where they generate a signal that halts anaphase until all sister chromatid pairs are bipolarly oriented. Mad2 is a subunit of the mitotic checkpoint complex, which is regarded as the effector of the spindle checkpoint. Its function is the sequestration of Cdc20, a protein required for progression into anaphase. The function of Mad2 in the checkpoint correlates with a dramatic conformational rearrangement of the Mad2 protein. Mad2 adopts a closed conformation (C-Mad2) when bound to Cdc20, and an open conformation (O-Mad2) when unbound to this ligand. Checkpoint activation promotes the conversion of O-Mad2 to Cdc20-bound C-Mad2. We show that this conversion requires a C-Mad2 template and we identify this in Mad1-bound Mad2. In our proposition, Mad1-bound C-Mad2 recruits O-Mad2 to kinetochores, stimulating Cdc20 capture, implying that O-Mad2 and C-Mad2 form dimers. We discuss Mad2 oligomerization and link our discoveries to previous observations related to Mad2 oligomerization.

Recruitment of Cdc20 to the Kinetochore Requires BubR1 but Not Mad2 in Drosophila melanogaster

“…closed arrows, and D). Thus, the mechanism of recruitment of Mad2 to the kinetochore under the SAC in Drosophila syncytial embryos has the properties predicted from earlier biochemical works in yeast and human (12,13,37). This also further proved that Mad2 kinetochore localization and modification are essential to establish proper spindle checkpoint function.…”

“…To understand the significance of the above findings, it is important to determine whether Drosophila Mad2 plays a conserved SAC role. We therefore tested whether Drosophila shares one of the crucial Mad2 kinetochore recruitment mechanisms in which Mad2 molecules dimerize in different conformations, as demonstrated in other systems (12,13,37). A GFP-tagged DmMad2⌬C construct was created by deletion of the 10 C-terminal amino acids of Mad2 (Fig.…”

Mad2 binding is not sufficient for complete Cdc20 sequestering in mitotic transition control (an in silico study)

“…Mad2 heterodimers can bind to either Mad1 or Cdc20 [12,15]. Thus, C-Mad2:Cdc20 complexes can be formed not only at Mad1 kinetochore sites but in the whole cell, amplifying complex formation.…”

“…Already this purely biochemical description gave rise to several criticisms [13,14]. Especially the question remained, why and how Mad2 first binds (as O-Mad2) to Mad1, but then dissociates off (as C-Mad2) [15]. In a subsequent sophisticated experimental investigation, [12] could precise the reaction mechanism further and developed a second model, the biochemical Template model.…”

Basic mechanism of eukaryotic chromosome segregation

“…Localization of the spindle checkpoint proteins at mitotic kinetochores (Chen et al 1996) and the inactivation of APC/C complex by Mad2 (Kim et al 1998;Hwang et al 1998) explained, at least partly, the role of spindle checkpoint proteins in mitotic progression. But further experiments, such as discussed by Musacchio (DeAntoni et al 2005), are necessary to fully understand how checkpoint proteins can arrest the mitotic progression. The other mutants are cohesion-defective scc1 and mcd1 mutants (Michaelis et al 1997;Guacci et al 1997;Ciosk et al 1998).…”