How cells sense connected chromosomesCells have a "checkpoint" that pauses cell division until all chromosomes are properly arranged on the mitotic spindle to allow precise distribution of one copy ...of each chromosome to each daughter cell. Hiruma et al. and Ji et al. explain the molecular mechanism by which cells sense that they are ready to divide. The protein kinase MPS1 associates with a protein complex at the kinetochore of the chromosome. Its activity produces signals that pause the cell cycle. When the chromosome becomes properly attached to the mitotic spindle, microtubules of the spindle physically compete for binding to the same site on the kinetochore where MPS1 is bound. Thus, once the kinetochore is properly attached, MPS1 dissociates, the inhibitory signal is lost, and cell division is allowed to proceed.Science, this issue pp. 1264 and 1260 The spindle checkpoint of the cell division cycle senses kinetochores that are not attached to microtubules and prevents precocious onset of anaphase, which can lead to aneuploidy. The nuclear division cycle 80 complex (Ndc80C) is a major microtubule receptor at the kinetochore. Ndc80C also mediates the kinetochore recruitment of checkpoint proteins. We found that the checkpoint protein kinase monopolar spindle 1 (Mps1) directly bound to Ndc80C through two independent interactions. Both interactions involved the microtubule-binding surfaces of Ndc80C and were directly inhibited in the presence of microtubules. Elimination of one such interaction in human cells caused checkpoint defects expected from a failure to detect unattached kinetochores. Competition between Mps1 and microtubules for Ndc80C binding thus constitutes a direct mechanism for the detection of unattached kinetochores.
The master spindle checkpoint kinase Mps1 senses kinetochore-microtubule attachment and promotes checkpoint signaling to ensure accurate chromosome segregation. The kinetochore scaffold Knl1, when ...phosphorylated by Mps1, recruits checkpoint complexes Bub1-Bub3 and BubR1-Bub3 to unattached kinetochores. Active checkpoint signaling ultimately enhances the assembly of the mitotic checkpoint complex (MCC) consisting of BubR1-Bub3, Mad2, and Cdc20, which inhibits the anaphase-promoting complex or cyclosome bound to Cdc20 (APC/C
) to delay anaphase onset. Using in vitro reconstitution, we show that Mps1 promotes APC/C inhibition by MCC components through phosphorylating Bub1 and Mad1. Phosphorylated Bub1 binds to Mad1-Mad2. Phosphorylated Mad1 directly interacts with Cdc20. Mutations of Mps1 phosphorylation sites in Bub1 or Mad1 abrogate the spindle checkpoint in human cells. Therefore, Mps1 promotes checkpoint activation through sequentially phosphorylating Knl1, Bub1, and Mad1. This sequential multi-target phosphorylation cascade makes the checkpoint highly responsive to Mps1 and to kinetochore-microtubule attachment.
The Cdc48 adenosine triphosphatase (ATPase) (p97 or valosin-containing protein in mammals) and its cofactor Ufd1/Npl4 extract polyubiquitinated proteins from membranes or macromolecular complexes for ...subsequent degradation by the proteasome. How Cdc48 processes its diverse and often well-folded substrates is unclear. Here, we report cryo-electron microscopy structures of the Cdc48 ATPase in complex with Ufd1/Npl4 and polyubiquitinated substrate. The structures show that the Cdc48 complex initiates substrate processing by unfolding a ubiquitin molecule. The unfolded ubiquitin molecule binds to Npl4 and projects its N-terminal segment through both hexameric ATPase rings. Pore loops of the second ring form a staircase that acts as a conveyer belt to move the polypeptide through the central pore. Inducing the unfolding of ubiquitin allows the Cdc48 ATPase complex to process a broad range of substrates.
The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the ...proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C terminus through the central pore of the hexameric double ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48.
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•The Cdc48 ATPase complex binds the ubiquitin chain rather than the attached substrate•One ubiquitin molecule is unfolded by binding to Cdc48 and its Ufd1/Npl4 cofactor•Cdc48 only unfolds polypeptides C-terminal to the unfolded ubiquitin molecule•Unfolded substrate is released from the Cdc48 complex without deubiquitination
The Cdc48 ATPase (p97 or VCP in mammals) complex extracts polyubiquitinated proteins from membranes or macromolecular complexes for proteasomal degradation. Ji et al. clarify the mechanism of substrate processing by showing how the ATPase unfolds one ubiquitin molecule, how it translocates polypeptides with branchpoints, and how it releases its substrates.
The conserved ATPase p97 (Cdc48 in yeast) and adaptors mediate diverse cellular processes through unfolding polyubiquitinated proteins and extracting them from macromolecular assemblies and membranes ...for disaggregation and degradation. The tandem ATPase domains (D1 and D2) of the p97/Cdc48 hexamer form stacked rings. p97/Cdc48 can unfold substrates by threading them through the central pore. The pore loops critical for substrate unfolding are, however, not well-ordered in substrate-free p97/Cdc48 conformations. How p97/Cdc48 organizes its pore loops for substrate engagement is unclear. Here we show that p97/Cdc48 can form double hexamers (DH) connected through the D2 ring. Cryo-EM structures of p97 DH reveal an ATPase-competent conformation with ordered pore loops. The C-terminal extension (CTE) links neighboring D2s in each hexamer and expands the central pore of the D2 ring. Mutations of Cdc48 CTE abolish substrate unfolding. We propose that the p97/Cdc48 DH captures a potentiated state poised for substrate engagement.
In this issue of Developmental Cell, two complementary studies by Jiang et al. (2014) and Toledo et al. (2014) identify BuGZ as an interacting protein of the kinetochore adaptor Bub3 and show that it ...promotes the stabilization and kinetochore loading of Bub3, chromosome alignment, and mitotic progression.
Many polyubiquitinated proteins are extracted from membranes or complexes by the conserved ATPase Cdc48 (in yeast; p97 or VCP in mammals) before proteasomal degradation. Each Cdc48 hexamer contains ...two stacked ATPase rings (D1 and D2) and six N-terminal (N) domains. Cdc48 binds various cofactors, including the Ufd1-Npl4 heterodimer. Here, we report structures of the Cdc48-Ufd1-Npl4 complex from Chaetomium thermophilum. Npl4 interacts through its UBX-like domain with a Cdc48 N domain, and it uses two Zn
-finger domains to anchor the enzymatically inactive Mpr1-Pad1 N-terminal (MPN) domain, homologous to domains found in several isopeptidases, to the top of the D1 ATPase ring. The MPN domain of Npl4 is located above Cdc48's central pore, a position similar to the MPN domain from deubiquitinase Rpn11 in the proteasome. Our results indicate that Npl4 is unique among Cdc48 cofactors and suggest a mechanism for binding and translocation of polyubiquitinated substrates into the ATPase.
Most eukaryotic proteins are degraded by the 26S proteasome after modification with a polyubiquitin chain. Substrates lacking unstructured segments cannot be degraded directly and require prior ...unfolding by the Cdc48 ATPase (p97 or VCP in mammals) in complex with its ubiquitin-binding partner Ufd1-Npl4 (UN). Here, we use purified yeast components to reconstitute Cdc48-dependent degradation of well-folded model substrates by the proteasome. We show that a minimal system consists of the 26S proteasome, the Cdc48-UN ATPase complex, the proteasome cofactor Rad23, and the Cdc48 cofactors Ubx5 and Shp1. Rad23 and Ubx5 stimulate polyubiquitin binding to the 26S proteasome and the Cdc48-UN complex, respectively, allowing these machines to compete for substrates before and after their unfolding. Shp1 stimulates protein unfolding by the Cdc48-UN complex rather than substrate recruitment. Experiments in yeast cells confirm that many proteins undergo bidirectional substrate shuttling between the 26S proteasome and Cdc48 ATPase before being degraded.
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•Cdc48 ATPase-dependent proteasomal protein degradation reconstituted in vitro•Substrate shuttling between the proteasome and Cdc48 is mediated by Rad23 and Ubx5•Shp1 stimulates protein unfolding by Cdc48, rather than substrate recruitment•Bidirectional substrate shuttling between the molecular machines occurs in yeast
Folded proteins cannot be degraded directly by the 26S proteasome and, therefore, require prior unfolding by the Cdc48 ATPase. Li et al. use purified yeast components to reconstitute Cdc48-dependent protein degradation by the proteasome. They show that substrates are shuttled between the two molecular machines before being degraded.
Telomeres and telomere-binding proteins form complex secondary nucleoprotein structures that are critical for genome integrity but can present serious challenges during telomere DNA replication. It ...remains unclear how telomere replication stress is resolved during S phase. Here, we show that the BUB3-BUB1 complex, a component in spindle assembly checkpoint, binds to telomeres during S phase and promotes telomere DNA replication. Loss of the BUB3-BUB1 complex results in telomere replication defects, including fragile and shortened telomeres. We also demonstrate that the telomere-binding ability of BUB3 and kinase activity of BUB1 are indispensable to BUB3-BUB1 function at telomeres. TRF2 targets BUB1-BUB3 to telomeres, and BUB1 can directly phosphorylate TRF1 and promote TRF1 recruitment of BLM helicase to overcome replication stress. Our findings have uncovered previously unknown roles for the BUB3-BUB1 complex in S phase and shed light on how proteins from diverse pathways function coordinately to ensure proper telomere replication and maintenance.
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•BUB3-BUB1 complex binds to telomeres and promotes their replication during S phase•BUB3-BUB1 complex prevents telomere shortening and fragility•TRF2 binds BUB3 and targets BUB1-BUB3 to telomeres during S phase•BUB1 phosphorylates TRF1 to recruit BLM helicase to overcome replication stress
Telomeres and telomere-binding proteins form nucleoprotein structures that can present serious challenges during telomere DNA replication. Li et al. show that TRF2 targets the BUB1-BUB3 complex to telomeres during S phase and promotes telomere DNA replication. BUB1 phosphorylates TRF1 to promote recruitment of BLM helicase to overcome replication stress.
The spindle checkpoint of the cell division cycle senses kinetochores that are not attached to microtubules and prevents precocious onset of anaphase, which can lead to aneuploidy. The nuclear ...division cycle 80 complex (Ndc80C) is a major microtubule receptor at the kinetochore. Ndc80C also mediates the kinetochore recruitment of checkpoint proteins. We found that the checkpoint protein kinase monopolar spindle 1 (Mps1) directly bound to Ndc80C through two independent interactions. Both interactions involved the microtubule-binding surfaces of Ndc80C and were directly inhibited in the presence of microtubules. Elimination of one such interaction in human cells caused checkpoint defects expected from a failure to detect unattached kinetochores. Competition between Mps1 and microtubules for Ndc80C binding thus constitutes a direct mechanism for the detection of unattached kinetochores.
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