Dissection of the Spindle Assembly Checkpoint by Proximity Proteomics

Abstract

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 prior to proceeding into anaphase. The SAC has a central role in ensuring the fidelity of chromosome segregation and its dysregulation has been linked to the development of human diseases like cancer. The establishment and maintenance of the SAC relies on multiple protein complexes that are intricately regulated in a spatial and temporal manner through posttranslational modifications like phosphorylation. Over the past few decades the SAC has been highly investigated and much has been learned about its protein constituents and the pathways and factors that regulate its activity. However, the spatio-temporal proximity associations of the core SAC components have not been explored in a systematic manner. Here, we have taken a BioID2 proximity-labeling proteomic approach to define the proximity protein environment for each of the five core SAC proteins BUB1, BUB3, BUBR1, MAD1L1, and MAD2L1 under conditions where the SAC is active in prometaphase. These five protein association maps were integrated to generate the 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 of the known SAC complexes and protein-protein interactions. Additionally, it uncovered new protein associations that lend insight into the functioning of the SAC and highlighted future areas that should be investigated to generate a comprehensive understanding of the SAC.3

Quantitative Measurement of Secretory Protein Mistargeting by Proximity Labeling and Parallel Reaction Monitoring

“…Immunodetection of cyt APEX in avidin-enriched samples is difficult, since common epitopes including the FLAG tag are disrupted by proximity labeling, presumably due to direct modification. We considered endogenously biotin-binding proteins in mitochondrial matrix 52,[60][61][62] as potential global standards for data normalization. These include PC, PCCA, MCCC1 and ACACB.…”

“…This factor should control for injection efficiency, loss of material during sample preparation, differences in the recovery of cells or protein, errors in protein quantification prior to avidin purification, and to the extent that the signal is dominated by cyt APEX-labeled proteins, the labeling activity of cyt APEX in a given experiment. Mitochondrial carboxylases such as PC, which are endogenously biotinylated, have been used for normalization in proximity labeling experiments when the peroxidases is localized elsewhere than the mitochondria 52,[60][61][62] , controlling for the total amount protein loaded onto (strept)avidin beads. These proteins as normalization factors are valid if the assumptions of consistent expression level and consistent proximity labeling activity are maintained across conditions.…”