Abstract #10

Functional Analysis of Mad3 in the Spindle Assembly Checkpoint in Budding Yeast. Janet Burton, Mark Solomon. Molecular Biophys. & Biochem., Yale University, New Haven, CT.
   The spindle assembly checkpoint monitors the attachment/tension between chromosome kinetochores and microtubules. Defects in the attachment/tension activate the checkpoint, which delays anaphase onset to prevent chromosome missegregation. The checkpoint delays anaphase by inhibiting the ubiquitin ligase activity of APC^Cdc20, which targets the anaphase inhibitor securin/Pds1p for ubiquitin-mediated proteolysis. The molecular players involved in the spindle checkpoint include Mad1, Mad2, Mad3, Bub1, Bub3 and Mps1. Both Mad2 and Mad3 are known to bind directly to Cdc20, but how this translates into inhibition of APC^Cdc20 activity has remained elusive. We and others have observed that Mad3 from many species contains a KEN box motif, a degradation signal often found in APC^Cdh1 substrates. We were therefore interested in investigating what role this motif may have in Mad3 function. We found that although the Mad3p KEN box plays no obvious role in Mad3p stability, it is nevertheless required for Mad3p checkpoint function. Unlike cells with wild-type Mad3p, cells containing Mad3p with a mutant KEN box (mkb) rapidly lose viability after brief exposure to microtubule disrupting agents and are able to grow in the presence of overexpressed Mps1p, indicating that they lack a functional spindle checkpoint. In agreement with this conclusion, Mad3-mkb cells (but not wild-type cells) continue to degrade Pds1p when challenged with spindle disrupting agents. Unlike wild-type Mad3p, Mad3-mkb is unable to bind Cdc20p in vivo, though it retains its ability to bind Bub3p, and displays reduced binding to recombinant Cdc20p in vitro. Interestingly, the APC-substrate Hsl1p can compete with Mad3p for Cdc20p binding in vitro in a D-box/KEN box dependent manner, suggesting that Cdc20p recognizes Mad3p using the same binding surface it uses for interaction with APC substrates. Taken together, these findings suggest that Cdc20p, like Cdh1p, recognizes the KEN box motif in vivo and raise the possibility that the spindle checkpoint may function at least in part by inhibiting Cdc20p-substrate interactions.

Return to YGM 2006 Home at SGD