SummaryDuring interphase, the nuclear envelope (NE) serves as a selective barrier between cytosol and nucleoplasm. When vertebrate cells enter mitosis, the NE is dismantled in the process of nuclear envelope breakdown (NEBD). Disassembly of nuclear pore complexes (NPCs) is a key aspect of NEBD, required for NE permeabilization and formation of a cytoplasmic mitotic spindle. Here, we show that both CDK1 and polo-like kinase 1 (PLK1) support mitotic NPC disintegration by hyperphosphorylation of Nup98, the gatekeeper nucleoporin, and Nup53, a central nucleoporin linking the inner NPC scaffold to the pore membrane. Multisite phosphorylation of Nup53 critically contributes to its liberation from its partner nucleoporins, including the pore membrane protein NDC1. Initial steps of NPC disassembly in semi-permeabilized cells can be reconstituted by a cocktail of mitotic kinases including cyclinB-CDK1, NIMA, and PLK1, suggesting that the unzipping of nucleoporin interactions by protein phosphorylation is an important principle underlying mitotic NE permeabilization.

Highlights d Global quantification of assembly state changes in the mitotic proteome d Improved performance over thermostability measurement of proteome states d Discovery of a mitotic disassembly intermediate of the nuclear pore complex d Introduction of SECexplorer-cc, a publicly available online platform

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During mitotic entry of vertebrate cells, nuclear pore complexes (NPCs) are rapidly disintegrated. NPC disassembly is initiated by hyperphosphorylation of linker nucleoporins (Nups), which leads to the dissociation of FG repeat Nups and relaxation of the nuclear permeability barrier. However, less is known about disintegration of the huge nuclear and cytoplasmic rings, which are formed by annular assemblies of Y‐complexes that are dissociated from NPCs as intact units. Surprisingly, we observe that Y‐complex Nups display slower dissociation kinetics compared with other Nups during in vitro NPC disassembly, indicating a mechanistic difference in the disintegration of Y‐based rings. Intriguingly, biochemical experiments reveal that a fraction of Y‐complexes remains associated with mitotic ER membranes, supporting recent microscopic observations. Visualization of mitotic Y‐complexes by super‐resolution microscopy demonstrates that they form two classes of higher order assemblies: large clusters at kinetochores and small, focal ER‐associated assemblies. These, however, lack features qualifying them as persisting ring‐shaped subassemblies previously proposed to serve as structural templates for NPC reassembly during mitotic exit, which helps to refine current models of nuclear reassembly.

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