Updated project metadata. In order to investigate the dynamic regulation of the APC/C by intrinsically disordered regions (IDRs) upon phosphorylation, we employed cross-linking mass spectrometry (CLMS). Given that the phosphorylation of Apc1-300L (IDR in Apc1) is a key determinant in its release from the co-activator binding site, we compared the interaction profiles of unphosphorylated and hyper-phosphorylated rAPC/C_WT complexes. The CLMS analysis highlights not only known interactions consistently identified within APC/C complexes, regardless of phosphorylation status, but also dynamic IDR-mediated interactions that are lost upon phosphorylation or are phosphorylation-dependent. Additionally, the data indicate that phosphorylation significantly reduces Apc1-300L's interaction with Apc8-L (IDR in Apc8) and Apc6, suggesting Apc1-300L's dislocation from the co-activator Cdc20 binding site, thereby facilitating Cdc20 loading onto the APC/C.