Active oxygen species (AOS) are believed to have important roles in plants in general and in plant—pathogen interactions in particular. They are believed to be involved in signal transduction, cell wall reinforcement, hypersensitive response (HR) and phytoalexin production, and to have direct antimicrobial effects. Since current methods are inadequate for localizing AOS in intact plant tissue, most studies have been conducted using cell suspension culture/elicitors systems. 3,3‐diaminobenzidine (DAB) polymerizes instantly and locally as soon as it comes into contact with H2O2 in the presence of peroxidase, and it was found that, by allowing the leaf to take up this substrate, in‐vivo and in‐situ detection of H2O2 can be made at subcellular levels. This method was successfully used to detect H2O2 in developing papillae and surrounding haloes (cell wall appositions) and whole cells of barley leaves interacting with the powdery mildew fungus. Thus, H2O2 can be detected in the epidermal cell wall subjacent to the primary germ tube from 6 h after inoculation, and subjacent to the appressorium from 15 h. The earliest time point for observation of H2O2 in relation to epidermal cells undergoing HR is 15 h after inoculation, first appearing in the zones of attachment to the mesophyll cells underneath, and eventually in the entire epidermal cell. Furthermore, it was observed that proteins in papillae and HR cells are cross‐linked, a process believed to be fuelled by H2O2. This cross‐linking reinforces the apposition, presumably assisting the arrest of the pathogen.

In mitosis, the spindle assembly checkpoint (SAC) ensures genome stability by delaying chromosome segregation until all sister chromatids have achieved bipolar attachment to the mitotic spindle. The SAC is imposed by the mitotic checkpoint complex (MCC), whose assembly is catalysed by unattached chromosomes and which binds and inhibits the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase that initiates chromosome segregation. Here, using the crystal structure of Schizosaccharomyces pombe MCC (a complex of mitotic spindle assembly checkpoint proteins Mad2, Mad3 and APC/C co-activator protein Cdc20), we reveal the molecular basis of MCC-mediated APC/C inhibition and the regulation of MCC assembly. The MCC inhibits the APC/C by obstructing degron recognition sites on Cdc20 (the substrate recruitment subunit of the APC/C) and displacing Cdc20 to disrupt formation of a bipartite D-box receptor with the APC/C subunit Apc10. Mad2, in the closed conformation (C-Mad2), stabilizes the complex by optimally positioning the Mad3 KEN-box degron to bind Cdc20. Mad3 and p31(comet) (also known as MAD2L1-binding protein) compete for the same C-Mad2 interface, which explains how p31(comet) disrupts MCC assembly to antagonize the SAC. This study shows how APC/C inhibition is coupled to degron recognition by co-activators.

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, 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:

The ubiquitylation of cell cycle regulatory proteins by the large multimeric anaphase promoting complex (APC/C) controls sister chromatid segregation and the exit from mitosis 1,2. Selection of APC/C targets is achieved through recognition of destruction motifs, predominantly the D-box 3 and KEN-box 4. Although this process is known to involve a co-activator protein (either Cdc20 or Cdh1) together with core APC/C subunits 1,2, the structural basis for substrate recognition and ubiquitylation is not understood. Here, we investigated the APC/C using single particle electron microscopy (EM) and determined a cryo-EM map of APC/CCdh1 bound to a D-box peptide at ~10 Å resolution. We find that a combined catalytic and substrate recognition module is located within the central cavity of the APC/C assembled from Cdh1, Apc10 - a core APC/C subunit previously implicated in substrate recognition 5,6,7, and the cullin domain of Apc2. Cdh1 and Apc10, identified from difference maps, create a co-receptor for D-box following repositioning of Cdh1 towards Apc10. Using NMR spectroscopy we demonstrate specific D-box – Apc10 interactions, consistent with a role for Apc10 in directly contributing towards D-box recognition by the APC/CCdh1 complex. Our results rationalise the contribution of both co-activator and core APC/C subunits to D-box recognition 8,9 and provide a structural framework for understanding mechanisms of substrate recognition and catalysis by the APC/C.

SummaryThe anaphase-promoting complex/cyclosome (APC/C) regulates sister chromatid segregation and the exit from mitosis. Selection of most APC/C substrates is controlled by coactivator subunits (either Cdc20 or Cdh1) that interact with substrate destruction motifs—predominantly the destruction (D) box and KEN box degrons. How coactivators recognize D box degrons and how this is inhibited by APC/C regulatory proteins is not defined at the atomic level. Here, from the crystal structure of S. cerevisiae Cdh1 in complex with its specific inhibitor Acm1, which incorporates D and KEN box pseudosubstrate motifs, we describe the molecular basis for D box recognition. Additional interactions between Acm1 and Cdh1 identify a third protein-binding site on Cdh1 that is likely to confer coactivator-specific protein functions including substrate association. We provide a structural rationalization for D box and KEN box recognition by coactivators and demonstrate that many noncanonical APC/C degrons bind APC/C coactivators at the D box coreceptor.