UbcX dissociates XErp1 from the APC/C Discussion
43 a robust SAC arrest in the presence of unattached kinetochores, but at the same time a fast, switch-‐like inactivation of the SAC once it is satisfied, thus ensuring the faithful segregation of the genetic material into the two descending daughter cells.
In future, it will be interesting to test if also the Cdc20 degradation can be verified in SAC arrest conditions in Xenopus, or if the two models are mutually exclusive and only the Cdc20 activation model applies. Additionally, it could be tested whether BubR1, Mad2 and/or Bub3 are regulated by ubiquitylation similar to XErp1. If this is the case, lysine less mutants of the relevant proteins could be generated which cannot be inactivated by ubiquitylation anymore.
These mutants should be inducing a spindle checkpoint arrest when overexpressed in somatic cells or a delay in anaphase onset despite a satisfied spindle checkpoint, since the APC/C cannot inactivate inhibitory MCCs anymore.
3.2. UbcX mediated ubiquitylation of XErp1 regulates its APC/C inhibitory activity
In both, the Cdc20-‐acitvation and Cdc20-‐inactivation model, the primary target of the spindle assembly checkpoint is APC/CCdc20. Similarly, the primary target of CSF and hence XErp1 is the APC/C Cdc20, however it is unclear how XErp1 regulates the APC/C. Therefore, we tested the two contradictory models of APC/C regulation during SAC arrest in the CSF mediated metaphase arrest.
3.2.1. Cdc20 is not destabilized in CSF arrested egg extract
It has been proposed that the APC/C inhibitory MCC proteins bind to the APC/C coactivator Cdc20 and turn it into an APC/C substrate (Nilsson et al., 2008), thereby promoting its degradation and APC/C inactivation. We hypothesized that during CSF arrest, Cdc20 could be recruited via XErp1 to the APC/C, resulting in Cdc20 ubiquitylation and degradation and the APC/C is kept
UbcX dissociates XErp1 from the APC/C Discussion
inactive. Our studies however demonstrate that destruction of the APC/C activator Cdc20 does not occur and, hence, does not contribute to the CSF arrest. This important finding is based on the observation that inhibition of neither protein translation nor protein degradation affected protein levels of Cdc20 in CSF-‐extract. Given that the second APC/C activator Cdh1 is not expressed in Xenopus egg extract (Lorca et al., 1998), these experiments revealed that the amounts of Cdc20 present in translation inhibited extracts is sufficient to promote the destruction of APC/C substrates. This implicates that CSF arrest is not mediated by the destabilization of Cdc20.
3.2.2. UbcX mediated ubiquitylation of XErp1 regulates its APC/C inhibitory activity
Using UbcH10 dependent APC/C activation during SAC signaling (Reddy et al.) as a model, we tested whether the Xenopus ortholog of UbcH10, UbcX can induce APC/C activation and CSF override in Xenopus. The incubation of CSF arrested egg extract with UbcX two times the endogenous levels induced APC/C activation and the degradation of APC/C substrates. Eight times the endogenous amount of UbcX was sufficient to cause CSF override and mitotic exit, as indicated by APC/C substrate degradation, dephosphorylation of mitotic phosphoproteins like Cdc27 and Cdc20 (data not shown) and sperm nuclei decondensation. UbcX addition was accompanied by XErp1 release from the APC/C, which was analyzed by glycerol gradient centrifugation and co-‐
immunoprecipitation assays. XErp1 and UbcX bind to the APC/C, and if UbcX and XErp1 compete for the same binding site, wild type and catalytic inactive UbcX would lead to the dissociation of XErp1. Since only the catalytic active version was able to decrease XErp1 binding to the APC/C, we conclude that UbcX mediated ubiquitylation is required for this effect.
To identify the target of UbcX dependent ubiquitylation, we treated CSF arrested extracts with wild type ubiquitin or as a negative control
observe Cdc20 ubiquitylation either. Interestingly, when we concentrated for APC/C bound Cdc20 and performed an in vitro ubiquitylation assay, we could to multiubiquitylated XErp1 specifically in extracts treated with UbcX and wild type ubiquitin (see figure 2.9.). Since the purification of ubiquitylated proteins
UbcX dissociates XErp1 from the APC/C Discussion
3.2.3. Are ubiquitin hydrolases counteracting the activity of UbcX during CSF
arrest?
We could show that the APC/C can free itself from the inhibitory activity by ubiquitylating XErp1 via its E2 enzyme UbcX. The APC/C and UbcX are both present during CSF arrest, raising the question of why is XErp1 is not ubiquitylated and inactivated under this condition. In cells arrested by the SAC, Cdc20 is ubiquitylated by APC/C-‐UbcH10 and this ubiquitylation of Cdc20 needs to be removed by the activity of the deubiquitylating enzyme USP44 to maintain SAC arrest (Stegmeier et al., 2007). If the balance between ubiquitylation and deubiquitylation is disturbed, for example by increasing the activity of UbcH10 or depleting the antagonizing USP44, cells cannot maintain the SAC and exit mitosis prematurely.
During CSF arrest, shifting the balance towards ubiquitylation by increasing the amounts of UbcX caused APC/C activation. To test whether a deubiquitylating activity is required to maintain CSF arrest by counteracting APC/C-‐ UbcX dependent ubiquitylation and inactivation of XErp1, we first tested the most obvious candidate for mediating this activity-‐ the Xenopus homolog of USP44.
To test the requirement of USP44 during CSF arrest, we wanted to immunodeplete USP44 from CSF egg extracts. This approach is dependent on antibodies that can recognize the protein in vivo and that are able to specifically deplete it. To obtain such antibodies, the native protein is normally used to immunize rabbits. However, we were unable to obtain native protein due to expression and solubility problems in bacteria and insect cells.
Therefore we used two USP44 peptides to generate antibodies against USP44 in two different rabbits. Both of them recognized denatured protein by westernblotting, but were unable to deplete USP44 from egg extracts, probably because the epitopes of these antibodies are masked in the native protein.
the proteasome could efficiently degrade ubiquitylated proteins like cyclin B.
The proteasome needs for its activity ubiquitin hydrolases that remove the ubiquitin before the substrates enter the proteasome tunnel (Hershko and Ciechanover, 1998). This process is inhibited when ubiquitin-‐aldehyde is incorporated into the chains, and substrates can no longer be degraded and are thus stabilized. In our hands, extracts treated with ubiquitin-‐aldehyde efficiently degraded ubiquitylated substrates, suggesting that ubiquitin-‐
aldehyde was not efficiently incorporated in the ubiquitin chains, possibly due efficient incorporation of ubiquitin-‐aldehyde into the ubiquitin chain of XErp1.
Next, endogenous XErp1 would be replaced by ubiquitin-‐aldehyde modified
UbcX dissociates XErp1 from the APC/C Discussion
ubiquitylated and to make mutant XErp1 proteins that lack these critical lysine residues. We would expect that such a mutant of XErp1 cannot be ubiquitylated by UbcX anymore and its ability to regulate the APC/C during CSF arrest and oocyte maturation is compromised when compared to the wild type protein. Nevertheless, the discovery of apparent regulation of XErp1 by ubiquitylation is novel and adds a new type of complexity to the mechanism of CSF arrest.
3.3. Is the regulation of UbcX activity important during the meiotic cell