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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 early mitotic inhibitor 1 (Emi1) ensures the correct order and timing of distinct cell-cycle transitions. Here we use 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 future experiments to investigate APC/C functions in vivo . A cryo-electron microscopy determination of the atomic structures of anaphase-promoting complex (APC/C)–coactivator complexes with either Emi1 or a UbcH10–ubiquitin conjugate. APC/C ubiquitination mechanism This paper describes the atomic structures of the anaphase-promoting complex (APC/C) bound to the coactivator Cdh1 and the early mitotic inhibitor Emi1, or the E2 enzyme UbcH10 conjugated to ubiquitin. The (APC/C) is a multisubunit E3 ubiquitin ligase enzyme that controls chromosome segregation and the subsequent exit from mitotic cell division. The resolutions the authors achieved (3.6 Å and 4.1 Å) allow them to define the architecture of all APC/C subunits and inter-subunit interactions within the assembly and the position of the catalytic module. The structures explain how Emi1 mediates inhibition of not just UbcH10 but also another E2 enzyme, Ube2S. Among other insights gained is how the association of APC/C with the coactivator Cdh1 is antagonized by Cdh1 phosphorylation.

The interplay between E2 and E3 enzymes regulates the polyubiquitination of substrates in eukaryotes. Among the several RING-domain E3 ligases in humans, many utilize two distinct E2s for polyubiquitination. For example, the cell cycle regulatory E3, human anaphase-promoting complex/cyclosome (APC/C), relies on UBE2C to prime substrates with ubiquitin (Ub) and on UBE2S to extend polyubiquitin chains. However, the potential coordination between these steps in ubiquitin chain formation remains undefined. While numerous studies have unveiled how RING E3s stimulate individual E2s for Ub transfer, here we change perspective to describe a case where the chain-elongating E2 UBE2S feeds back and directly stimulates the E3 APC/C to promote substrate priming and subsequent multiubiquitination by UBE2C. Our work reveals an unexpected model for the mechanisms of RING E3–dependent ubiquitination and for the diverse and complex interrelationship between components of the ubiquitination cascade.The cell cycle regulatory E3 ligase APC/C cooperates with UBE2C to prime substrates with ubiquitin and UBE2S to extend the ubiquitin chains. Careful analysis reveals that binding of the UBE2S to APC/C accelerates the rate-limiting step of APC/C–UBE2C.

Phosphorylation of the anaphase-promoting complex (APC/C) allows for its control by the co-activator Cdc20; a mechanism that has relevance to understanding the control of other large multimeric complexes by phosphorylation. Coactivator Cdc20 control of APC/C The anaphase-promoting complex/cyclosome (APC/C) is a large E3 ubiquitin ligase that coordinates sister chromatid segregation, cytokinesis and the initiation of chromosome duplication. It is regulated by elaborate mechanisms including by its coactivator subunits (Cdc20 and Cdh1), reversible phosphorylation, and the spindle assembly checkpoint. Here using cryo-electron microscopy and biochemical analysis, David Barford and colleagues define how mitotic phosphorylation of APC/C allows for its control by Cdc20. Of almost 150 phospho-sites in mitotic APC/C, only a few directly regulate Cdc20 binding through displacement of a newly identified auto-inhibitory segment. This study has relevance to understanding the control of other large multimeric complexes by multi-site phosphorylation. In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase 1 , 2 . The catalytic activity of the APC/C and its 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 degrons 3 , and enhance the affinity of the APC/C for its cognate E2 (refs 4 , 5 , 6 ). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C 7 , 8 , 9 , 10 , 11 , 12 , whereas phosphorylation of Cdh1 prevents its association with the APC/C 9 , 13 , 14 . Since both coactivators associate with the APC/C through their common C-box 15 and Ile-Arg tail motifs 16 , 17 , the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory 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 auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk–cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl- l -arginine methyl ester, preferentially suppresses APC/C Cdc20 rather than APC/C Cdh1 , and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. 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.

Significance The anaphase-promoting complex/cyclosome (APC) is a multisubunit RING E3 ubiquitin (Ub) ligase that regulates mitosis, meiosis, and numerous facets of neurobiology by targeting key regulatory proteins for Ub-mediated degradation. Despite great importance, it remains unclear how APC, or most of the other 600 RING E3s in humans, targets Ub to lysines in disordered substrates. Here, we report the structural and molecular basis for substrate ubiquitination by APC and its partner E2, UBCH10. UBCH10 is recruited to APC, activated for ubiquitination, and positioned for substrate targeting through multisite interactions with the APC cullin–RING core. We propose that many RING E3–E2 assemblies work similarly, with multisite interactions establishing specificity, harnessing ubiquitination machineries to accelerate searching for target lysines, and facilitating regulation. For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2∼Ub intermediate to a lysine on a remotely bound disordered substrate. One such E3 is the gigantic, multisubunit 1.2-MDa anaphase-promoting complex/cyclosome (APC), which controls cell division by ubiquitinating cell cycle regulators to drive their timely degradation. Intrinsically disordered substrates are typically recruited via their KEN-box, D-box, and/or other motifs binding to APC and a coactivator such as CDH1. On the opposite side of the APC, the dynamic catalytic core contains the cullin-like subunit APC2 and its RING partner APC11, which collaborates with the E2 UBCH10 (UBE2C) to ubiquitinate substrates. However, how dynamic RING–E2∼Ub catalytic modules such as APC11–UBCH10∼Ub collide with distally tethered disordered substrates remains poorly understood. We report structural mechanisms of UBCH10 recruitment to APC Cᴰᴴ¹ and substrate ubiquitination. Unexpectedly, in addition to binding APC11’s RING, UBCH10 is corecruited via interactions with APC2, which we visualized in a trapped complex representing an APC Cᴰᴴ¹–UBCH10∼Ub–substrate intermediate by cryo-electron microscopy, and in isolation by X-ray crystallography. To our knowledge, this is the first structural view of APC, or any cullin–RING E3, with E2 and substrate juxtaposed, and it reveals how tripartite cullin–RING–E2 interactions establish APC’s specificity for UBCH10 and harness a flexible catalytic module to drive ubiquitination of lysines within an accessible zone. We propose that multisite interactions reduce the degrees of freedom available to dynamic RING E3–E2∼Ub catalytic modules, condense the search radius for target lysines, increase the chance of active-site collision with conformationally fluctuating substrates, and enable regulation.

The transmission of a complete set of chromosomes to daughter cells during cell division is vital for development and tissue homeostasis. The spindle assembly checkpoint (SAC) ensures correct segregation by informing the cell cycle machinery of potential errors in the interactions of chromosomes with spindle microtubules prior to anaphase. To do so, the SAC monitors microtubule engagement by specialized structures known as kinetochores and integrates local mechanical and chemical cues such that it can signal in a sensitive, responsive and robust manner. In this Review, we discuss how SAC proteins interact to allow production of the mitotic checkpoint complex (MCC) that halts anaphase progression by inhibiting the anaphase-promoting complex/cyclosome (APC/C). We highlight recent advances aimed at understanding the dynamic signalling properties of the SAC and how it interprets various naturally occurring intermediate attachment states. Further, we discuss SAC signalling in the context of the mammalian multisite kinetochore and address the impact of the fibrous corona. We also identify current challenges in understanding how the SAC ensures high-fidelity chromosome segregation.The spindle assembly checkpoint (SAC) ensures correct chromosome segregation during mitosis by inhibiting anaphase until all kinetochores are attached to microtubules. Recent studies highlight the dynamic properties of SAC signalling and begin to explain signal integration at mammalian kinetochores, which feature multiple attachment points.

The ubiquitination of cell cycle regulatory proteins by the anaphase-promoting complex/cyclosome (APC/C) controls sister chromatid segregation, cytokinesis and the establishment of the G1 phase of the cell cycle. 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-electron microscopy 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 the 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. The anaphase-promoting complex/cyclosome (APC/C) is a large E3 ligase that mediates ubiquitin-dependent proteolysis of cell cycle regulatory proteins; here the complete secondary structure architecture of human APC/C complexed with its coactivator CDH1 and substrate HSL1 is determined at 7.4 Å resolution, revealing allosteric changes induced by the coactivator that enhance affinity for UBCH10–ubiqutin. Human anaphase-promoting complex structure The anaphase-promoting complex/cyclosome (APC/C) is a large E3 ligase that mediates ubiquitin-dependent proteolysis of cell cycle regulatory proteins to control various events during replication and cell division. Here, using cryo-electron microscopy, David Barford and colleagues determine the complete secondary structural architecture of human APC/C in complex with its coactivator CDH1 and substrate HSL1 at 7.4 Å resolution. The structural information allows the position and architecture of all 20 APC/C subunits to be defined and provides insights into how CDH1 stimulates APC/C ubiquitination activity.

Lactate is abundant in rapidly dividing cells owing to the requirement for elevated glucose catabolism to support proliferation 1 – 6 . However, it is not known whether accumulated lactate affects the proliferative state. Here we use a systematic approach to determine lactate-dependent regulation of proteins across the human proteome. From these data, we identify a mechanism of cell cycle regulation whereby accumulated lactate remodels the anaphase promoting complex (APC/C). Remodelling of APC/C in this way is caused by direct inhibition of the SUMO protease SENP1 by lactate. We find that accumulated lactate binds and inhibits SENP1 by forming a complex with zinc in the SENP1 active site. SENP1 inhibition by lactate stabilizes SUMOylation of two residues on APC4, which drives UBE2C binding to APC/C. This direct regulation of APC/C by lactate stimulates timed degradation of cell cycle proteins, and efficient mitotic exit in proliferative human cells. This mechanism is initiated upon mitotic entry when lactate abundance reaches its apex. In this way, accumulation of lactate communicates the consequences of a nutrient-replete growth phase to stimulate timed opening of APC/C, cell division and proliferation. Conversely, persistent accumulation of lactate drives aberrant APC/C remodelling and can overcome anti-mitotic pharmacology via mitotic slippage. In sum, we define a biochemical mechanism through which lactate directly regulates protein function to control the cell cycle and proliferation. Discovery of a biochemical mechanism through which lactate binds and inhibits the SUMO protease SENP1, stimulating timed degradation of cell cycle proteins, and resulting in mitotic exit.

Error-free genome duplication and segregation are ensured through the timely activation of ubiquitylation enzymes. The anaphase-promoting complex or cyclosome (APC/C), a multisubunit E3 ubiquitin ligase, is regulated by phosphorylation. However, the mechanism remains elusive. Using systematic reconstitution and analysis of vertebrate APC/Cs under physiological conditions, we show how cyclin-dependent kinase 1 (CDK1) activates the APC/C through coordinated phosphorylation between Apc3 and Apc1. Phosphorylation of the loop domains by CDK1 in complex with p9/Cks2 (a CDK regulatory subunit) controlled loading of coactivator Cdc20 onto APC/C. A phosphomimetic mutation introduced into Apc1 allowed Cdc20 to increase APC/C activity in interphase. These results define a previously unrecognized subunit-subunit communication over a distance and the functional consequences of CDK phosphorylation. Cdc20 is a potential therapeutic target, and our findings may facilitate the development of specific inhibitors.

Ubiquitin (Ub)-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of Ub regulation are conjugation through E1-E2-E3 enzymatic cascades and recognition by Ub-binding domains. An emerging theme in the Ub field is that these 2 properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to Ub’s C terminus for Ub transfer reactions, conjugation enzymes often bind noncovalently and weakly to Ub at “exosites.” However, identification of such sites is typically empirical and particularly challenging in large molecular machines. Here, studying the 1.2-MDa E3 ligase anaphase-promoting complex/cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected Ub-binding sites. Using a panel of Ub variants (UbVs), we identify a protein-based inhibitor that blocks Ub ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo-electron microscopy (cryo-EM) structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a Ub-binding exosite with preference for K48-linked chains. The results provide a tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic Ub-binding sites within large multiprotein complexes.

Faithful chromosome segregation is required for both mitotic and meiotic cell divisions and is regulated by multiple mechanisms including the anaphase-promoting complex/cyclosome (APC/C), which is the largest known E3 ubiquitin-ligase complex and has been implicated in regulating chromosome segregation in both mitosis and meiosis in animals. However, the role of the APC/C during plant meiosis remains largely unknown. Here, we show that Arabidopsis APC8 is required for male meiosis. We used a combination of genetic analyses, cytology and immunolocalisation to define the function of AtAPC8 in male meiosis. Meiocytes from apc8-1 plants exhibit several meiotic defects including improper alignment of bivalents at metaphase I, unequal chromosome segregation during anaphase II, and subsequent formation of polyads. Immunolocalisation using an antitubulin antibody showed that APC8 is required for normal spindle morphology. We also observed mitotic defects in apc8-1, including abnormal sister chromatid segregation and microtubule morphology. Our results demonstrate that Arabidopsis APC/C is required for meiotic chromosome segregation and that APC/C-mediated regulation of meiotic chromosome segregation is a conserved mechanism among eukaryotes.