The APC Subunit Doc1 Promotes Recognition of the Substrate Destruction Box

Departments of Physiology and Biochemistry and Biophysics, University of California-San Francisco, San Francisco, CA 94143-2200, USA.
Current Biology (Impact Factor: 9.57). 02/2005; 15(1):11-8. DOI: 10.1016/j.cub.2004.12.066
Source: PubMed


Accurate chromosome segregation during mitosis requires the coordinated destruction of the mitotic regulators securin and cyclins. The anaphase-promoting complex (APC) is a multisubunit ubiquitin-protein ligase that catalyzes the polyubiquitination of these and other proteins and thereby promotes their destruction. How the APC recognizes its substrates is not well understood. In mitosis, the APC activator Cdc20 binds to the APC and is thought to recruit substrates by interacting with a conserved target protein motif called the destruction box. A related protein, called Cdh1, performs a similar function during G1. Recent evidence, however, suggests that the core APC subunit Doc1 also contributes to substrate recognition.
To better understand the mechanism by which Doc1 promotes substrate binding to the APC, we generated a series of point mutations in Doc1 and analyzed their effects on the processivity of substrate ubiquitination. Mutations that reduce Doc1 function fall into two classes that define spatially and functionally distinct regions of the protein. One region, which includes the carboxy terminus, anchors Doc1 to the APC but does not influence substrate recognition. The other region, located on the opposite face of Doc1, is required for Doc1 to enhance substrate binding to the APC. Importantly, stimulation of binding by Doc1 also requires that the substrate contain an intact destruction box. Cells carrying DOC1 mutations that eliminate substrate recognition delay in mitosis with high levels of APC substrates.
Doc1 contributes to recognition of the substrate destruction box by the APC. This function of Doc1 is necessary for efficient substrate proteolysis in vivo.

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Available from: Christopher Walton Carroll, Aug 28, 2015
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    • "Substrate recognition is conferred by short destruction motifs (D box, KEN box and ABBA motifs [16] [17] [18] [19]) that interact with coactivator subunits [7] [8] [9] [10] [11] [12]. The core APC/C subunit Apc10 contributes to D box recognition through a D box coreceptor formed with coactivator [13] [20] [21]. Coactivators increase APC/C activity by promoting an increase in affinity for E2 and by enhancing E2 catalytic efficiency [13] [14] [15]. "
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    ABSTRACT: Many essential biological processes are mediated by complex molecular machines comprising multiple subunits. Knowledge of the architecture of individual subunits and their positions within the overall multimeric complex is key to understanding the molecular mechanisms of macromolecular assemblies. The anaphase-promoting complex/cyclosome (APC/C) is a large multi-subunit complex that regulates cell cycle progression by ubiquitinating cell cycle proteins for proteolysis by the proteasome. The holo-complex is composed of 15 different proteins that assemble to generate a complex of 20 subunits. Here, we describe the crystal structures of Apc4 and the N-terminal domain of Apc5 (Apc5N). Apc4 comprises a WD40 domain split by a long α-helical domain, whereas Apc5N has an α-helical fold. In a separate study, we had fitted these atomic models to a 3.6 Å-resolution cryo-EM map of the APC/C. We describe how in the context of the APC/C, regions of Apc4 disordered in the crystal assume order through contacts to Apc5, whereas Apc5N shows small conformational changes relative to its crystal structure. We discuss the complementary approaches of high-resolution electron microscopy and protein crystallography to the structure determination of subunits of multimeric complexes.
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    • "Under these conditions, we were able to efficiently pull down the APC/C complex together with cyclin B1. Hyper-phosphorylated APC3 mediates cyclin B1 recruitment to the APC/C while the spindle checkpoint is still active, which becomes effective after checkpoint release, when APC10 and Cdc20 direct cyclin B1 to the catalytic site of the APC/ C (Carroll et al., 2005; Chang et al., 2014). Our results reveal that the stability of geminin and securin, two other known APC/C Cdc20 substrates (Clijsters et al., 2013; Hagting et al., 2002), is less dependent on the phosphorylation state of the APC/C. "
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    Full-text · Article · Mar 2015 · Biology Open
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    • "Other APC/C subunits assemble onto this minimal module in order to confer specificity to the E3 ligase. The substrate recognition and poly-ubiquitination requires the additional function of APC10/DOC1 (Carroll et al., 2005). This selective target recognition is activated during the cell cycle by co-activators such as CDC20 (cell division cycle protein 20) and Cdh1 (cadherin 1) (Vodermaier et al., 2003). "
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