The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex, and interphase inhibitor Emi1 ensures the correct order and timing of distinct cell cycle transitions. Here, we used cryo-electron microscopy to determine atomic structures of APC/C-coactivator complexes with either Emi1 or a UbcH10-ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module, and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10-ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of experiments to further investigate APC/C functions in vivo.The activities of the diverse proteins that orchestrate the sequential biochemical and morphological changes intrinsic to the cell division cycle are controlled through the integration of protein phosphorylation, proteolysis and changes in gene expression. Two cullin-RING E3 ubiquitin ligases, the APC/C and SCF, catalyze the ubiquitination of multiple cell cycle proteins to regulate their proteasome-mediated proteolysis. By regulating the ordered degradation of substrates such as cyclins, securin, mitotic kinases, and microtubule motors and assembly factors, the APC/C controls sister chromatid segregation, cytokinesis and the initiation of chromosome duplication [1][2][3] .The APC/C is a large assembly comprising 19 subunits 4 . Its activity depends on the association of one of two coactivator subunits (either Cdc20 or Cdh1) that specify substrate recognition and stimulate the catalytic activity of APC/C -E2 complexes [4][5][6][7] . Coactivators engage the APC/C through a conserved N-terminal C box motif and a C-terminal Ile-Arg Correspondence should be addressed to D.B. (dbarford@mrc-lmb.cam.ac.uk). Author contributions. L.C. prepared grids, collected and analyzed EM data and determined the 3D reconstructions, fitted coordinates and built models, prepared figures, co-wrote the paper. Z.Z. designed and made constructs, performed biochemical analysis and purified proteins. J.Y. prepared and purified the complexes and performed biochemical analysis. S.H.McL. performed and analyzed SPR experiments. D.B. directed the project, built models and co-wrote the paper.Author information. EM maps are deposited with the EM-DB with accession codes: 2924 (APC/C Cdh1.Emi1 ), 2925 (APC/ C Cdh1.Hsl1.UbcH10-Ub) , 2926 (APC/C Cdh1.Hsl1.Apc11-UbcH10) . APC/C Cdh1.Emi1 coords have accession code 4ui9.The authors declare no competing financial interests. Europe PMC Funders GroupAuthor Manuscript Nature. Author manuscript; available in PMC 2015 December 25. Published in final edited form as:Nature. 2,3 . Cyclin-dependent kinase (CDK) phosphorylates the...

The ubiquitination of cell cycle regulatory proteins by the anaphase-promoting complex/ cyclosome (APC/C) controls sister chromatid segregation, cytokinesis and the establishment of G1. The APC/C is an unusually large multimeric cullin-RING ligase. Its activity is strictly dependent on regulatory coactivator subunits that promote APC/C -substrate interactions and stimulate its catalytic reaction. Because the structures of many APC/C subunits and their organization within the assembly are unknown, the molecular basis for these processes is poorly understood. Here, from a cryo-EM reconstruction of a human APC/C-coactivator-substrate complex at 7.4 Å resolution, we have determined the complete secondary structural architecture of the complex. With this information we identified protein folds for structurally uncharacterized subunits, and the definitive location of all 20 APC/C subunits within the 1.2 MDa assembly. Comparison with apo APC/C shows that coactivator promotes a profound allosteric transition involving displacement of the cullin-RING catalytic subunits relative to the degron recognition module of coactivator and Apc10. This transition is accompanied by increased flexibility of the cullin-RING subunits and enhanced affinity for UbcH10~ubiquitin, changes which may contribute to coactivator-mediated stimulation of APC/C E3 ligase activity.Regulation of cell division by reversible protein phosphorylation and ubiquitination involves the coordinated interplay of protein kinases and phosphatases, and ubiquitin ligases and deubiquitinases 1 . The anaphase-promoting complex/cyclosome (APC/C) is an E3 cullin-RING ligase that mediates ubiquitin-dependent proteolysis of specific regulatory proteins to control chromosome segregation in mitosis, the events of cytokinesis and mitotic exit,

In the dividing eukaryotic cell the spindle assembly checkpoint (SAC) ensures each daughter cell inherits an identical set of chromosomes. The SAC coordinates the correct attachment of sister chromatid kinetochores to the mitotic spindle with activation of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase that initiates chromosome separation. In response to unattached kinetochores, the SAC generates the mitotic checkpoint complex (MCC), a multimeric assembly that inhibits the APC/C, delaying chromosome segregation. Here, using cryo-electron microscopy we determined the near-atomic resolution structure of an APC/C-MCC complex (APC/CMCC). We reveal how degron-like sequences of the MCC subunit BubR1 block degron recognition sites on Cdc20, the APC/C coactivator subunit (Cdc20APC/C) responsible for substrate interactions. BubR1 also obstructs binding of UbcH10 (APC/C’s initiating E2) to repress APC/C ubiquitination activity. Conformational variability of the complex allows for UbcH10 association, and we show from a structure of APC/CMCC in complex with UbcH10 how the Cdc20 subunit intrinsic to the MCC (Cdc20MCC) is ubiquitinated, a process that results in APC/C reactivation when the SAC is silenced.

In eukaryotes, the anaphase-promoting complex/cyclosome (APC/C) regulates the ubiquitin-dependent proteolysis of specific cell cycle proteins to coordinate chromosome segregation in mitosis and entry into G1 (refs 1,2). The APC/C’s catalytic activity and ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits (Cdc20 and Cdh1). Coactivators recognize substrate degrons3, and enhance the APC/C’s affinity for its cognate E2 (refs 4–6). During mitosis, cyclin-dependent kinase and polo kinase control Cdc20 and Cdh1-mediated activation of the APC/C. Hyper-phosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C7–12, whereas phosphorylation of Cdh1 prevents its association with the APC/C9,13,14. Since both coactivators associate with the APC/C through their common C box15 and IR (Ile-Arg) tail motifs16,17, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy (cryo-EM) and biochemical analysis, we define the molecular basis of how APC/C phosphorylation allows for its control by Cdc20. An auto-inhibitory (AI) segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the AI segment displaces it from the C-box binding site. Efficient phosphorylation of the AI segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin-Cks to a hyper-phosphorylated loop of Apc3. We also find that the small molecule inhibitor, tosyl-L-arginine methyl ester (TAME), preferentially suppresses APC/CCdc20 rather than APC/CCdh1, and interacts with both the C-box and IR-tail binding sites. Our results reveal the mechanism for the regulation of mitotic APC/C by phosphorylation and provide a rationale for the development of selective inhibitors of this state.

In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes, that by assembling onto specialized Cenp-A nucleosomes 1,2 , function to connect centromeric chromatin to microtubules of the mitotic spindle 3,4 . Whereas the centromeres of vertebrate chromosomes comprise Mb of DNA and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules 5,6 . All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-A Nuc ), each of which is perfectly centred on its cognate centromere [7][8][9] . The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here, we describe the cryo-EM structure of the S. cerevisiae inner kinetochore module -the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN-Cenp-A Nuc ). The structure explains the inter-dependency of CCAN's constituent sub-complexes and shows how the 'Y'-shaped opening of CCAN accommodates Cenp-A Nuc to allow specific Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Phycobilisomes (PBSs) are light-harvesting antennae that transfer energy to photosynthetic reaction centers in cyanobacteria and red algae. PBSs are supermolecular complexes composed of phycobiliproteins (PBPs) that bear chromophores for energy absorption and linker proteins. Although the structures of some individual components have been determined using crystallography, the three-dimensional structure of an entire PBS complex, which is critical for understanding the energy transfer mechanism, remains unknown. Here, we report the structures of an intact PBS and a PBS in complex with photosystem II (PSII) from Anabaena sp. strain PCC 7120 using single-particle electron microscopy in combination with biochemical and molecular analyses. In the PBS structure, all PBP trimers and the conserved linker protein domains were unambiguously located, and the global distribution of all chromophores was determined. We provide evidence that ApcE and ApcF are critical for the formation of a protrusion at the bottom of PBS, which plays an important role in mediating PBS interaction with PSII. Our results provide insights into the molecular architecture of an intact PBS at different assembly levels and provide the basis for understanding how the light energy absorbed by PBS is transferred to PSII.

The maintenance of genome stability during mitosis is coordinated by the spindle assembly checkpoint (SAC) through its effector the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex (APC/C, also known as the cyclosome). Unattached kinetochores control MCC assembly by catalysing a change in the topology of the β-sheet of MAD2 (an MCC subunit), thereby generating the active closed MAD2 (C-MAD2) conformer. Disassembly of free MCC, which is required for SAC inactivation and chromosome segregation, is an ATP-dependent process driven by the AAA+ ATPase TRIP13. In combination with p31, an SAC antagonist, TRIP13 remodels C-MAD2 into inactive open MAD2 (O-MAD2). Here, we present a mechanism that explains how TRIP13-p31 disassembles the MCC. Cryo-electron microscopy structures of the TRIP13-p31-C-MAD2-CDC20 complex reveal that p31 recruits C-MAD2 to a defined site on the TRIP13 hexameric ring, positioning the N terminus of C-MAD2 (MAD2) to insert into the axial pore of TRIP13 and distorting the TRIP13 ring to initiate remodelling. Molecular modelling suggests that by gripping MAD2 within its axial pore, TRIP13 couples sequential ATP-driven translocation of its hexameric ring along MAD2 to push upwards on, and simultaneously rotate, the globular domains of the p31-C-MAD2 complex. This unwinds a region of the αA helix of C-MAD2 that is required to stabilize the C-MAD2 β-sheet, thus destabilizing C-MAD2 in favour of O-MAD2 and dissociating MAD2 from p31. Our study provides insights into how specific substrates are recruited to AAA+ ATPases through adaptor proteins and suggests a model of how translocation through the axial pore of AAA+ ATPases is coupled to protein remodelling.

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