Mapping Proximity Associations of Core Spindle Assembly Checkpoint Proteins

The spindle assembly checkpoint (SAC) is critical for sensing defective microtubule-kinetochore attachments and tension across the kinetochore and functions to arrest cells in prometaphase to allow time to repair any errors before proceeding into anaphase. Dysregulation of the SAC leads to chromosome segregation errors that have been linked to human diseases like cancer. Although much has been learned about the composition of the SAC and the factors that regulate its activity, the proximity associations of core SAC components have not been explored in a systematic manner. Here, we have taken a BioID2-proximitylabeling proteomic approach to define the proximity protein environment for each of the five core SAC proteins BUB1, BUB3, BUBR1, MAD1L1, and MAD2L1 in mitotic-enriched populations of cells where the SAC is active. These five protein association maps were integrated to generate a SAC proximity protein network that contains multiple layers of information related to core SAC protein complexes, protein-protein interactions, and proximity associations. Our analysis validated many known SAC complexes and protein-protein interactions. Additionally, it uncovered new protein associations, including the ELYS-MAD1L1 interaction that we have validated, which lend insight into the functioning of core SAC proteins and highlight future areas of investigation to better understand the SAC.

Automated summary

**The spindle assembly checkpoint (SAC) is crucial for detecting errors in microtubule-kinetochore attachments and tension, which can lead to chromosome segregation errors linked to diseases like cancer. Despite advances in understanding SAC composition and regulatory factors, the proximity associations of core SAC components have not been systematically explored. Using a BioID2-proximity labeling proteomic approach, the proximity protein environment of five core SAC proteins has been characterized in mitotic-enriched cell populations where the SAC is active. The analysis has not only validated known SAC complexes and protein-protein interactions, but also revealed new protein associations that provide insight into SAC function and suggest future research directions for better understanding.**