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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-proximity-labeling 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.

Somatic mutations that perturb Parkin ubiquitin ligase activity and the misregulation of iron homeostasis have both been linked to Parkinson's disease. Lactotransferrin (LTF) is a member of the family of transferrin iron binding proteins that regulate iron homeostasis, and increased levels of LTF and its receptor have been observed in neurodegenerative disorders like Parkinson's disease. Here, we report that Parkin binds to LTF and ubiquitylates LTF to influence iron homeostasis. Parkin-dependent ubiquitylation of LTF occurred most often on lysines (K) 182 and 649. Substitution of K182 or K649 with alanine (K182A or K649A, respectively) led to a decrease in the level of LTF ubiquitylation, and substitution at both sites led to a major decrease in the level of LTF ubiquitylation. Importantly, Parkin-mediated ubiquitylation of LTF was critical for regulating intracellular iron levels as overexpression of LTF ubiquitylation site point mutants (K649A or K182A/K649A) led to an increase in intracellular iron levels measured by ICP-MS/MS. Consistently, RNAi-mediated depletion of Parkin led to an increase in intracellular iron levels in contrast to overexpression of Parkin that led to a decrease in intracellular iron levels. Together, these results indicate that Parkin binds to and ubiquitylates LTF to regulate intracellular iron levels. These results expand our understanding of the cellular processes that are perturbed when Parkin activity is disrupted and more broadly the mechanisms that contribute to Parkinson's disease.