Mechanisms of ubiquitin transfer by the anaphase-promoting complex

Abstract

The anaphase-promoting complex (APC) is a ubiquitin-protein ligase required for the completion of mitosis in all eukaryotes. Recent mechanistic studies reveal how this remarkable enzyme combines specificity in substrate binding with flexibility in ubiquitin transfer, thereby allowing the modification of multiple lysines on the substrate as well as specific lysines on ubiquitin itself.

Figure 1

The major biological function of the APC is to initiate chromosome segregation in mitosis. In metaphase, before APC activation, the duplicated chromosomes, or sister chromatids, are aligned at the center of the mitotic spindle. The APC promotes the ubiquitination and proteasomal destruction of securin, thereby unleashing a protease, separase, that cleaves cohesin proteins holding the sister chromatids together. The chromatids separate and are then pulled apart by the mitotic spindle in anaphase. The APC also triggers destruction of cyclins, the activating subunits of the cyclin-dependent kinases (Cdks). As a result, Cdk activity drops in anaphase. The resulting dephosphorylation of Cdk substrates is required for normal anaphase spindle function and for the completion of mitosis. The APC has numerous additional substrates, not shown here, that contribute to the control of late mitotic events and govern entry into the following G1.

Figure 2

Ubiquitination occurs by a three-enzyme cascade. (a) Ubiquitin (Ub, yellow) is first activated by an E1, or ubiquitin-activating protein (purple square), which couples ATP hydrolysis to the formation of a thioester bond between the active-site cysteine of the E1 and the carboxyl terminus of ubiquitin. The E1 then transfers the activated ubiquitin to the active-site cysteine of an E2, or ubiquitin-conjugating enzyme (blue). Finally, the E3, or ubiquitin-protein ligase (green), facilitates the transfer of the ubiquitin from the E2 to a lysine on the target protein (substrate, magenta). In the case of the APC and many other E3s, this final step is repeated several times with the same substrate, resulting in ubiquitination of multiple lysines. In addition, specific lysines on ubiquitin itself can be modified, resulting in the assembly of polyubiquitin chains. (b) The APC is a member of the RING-domain family of E3s. These proteins facilitate the final step in ubiquitination by positioning the E2-ubiquitin conjugate next to the substrate, allowing the ε-amino group of a lysine on the substrate to nucleophilically attack the E2-ubiquitin thioester bond, resulting in direct transfer of ubiquitin as shown here. Members of the other major family of E3 proteins, called the HECT-domain E3s, employ an indirect, two-step mechanism (not shown here): ubiquitin is first transferred from the E2 to an active-site cysteine in the E3, after which a lysine in the substrate attacks the E3-ubiquitin thioester bond.

Figure 3

The four major protein components in an APC reaction. Catalysis depends on cooperative interactions among the APC core, activator, substrate and E2.

Figure 4

The APC core contains multiple subcomplexes. The budding yeast APC core is an approximately 1 MDa complex of 13 subunits (Table 1), including the nine key subunits shown here. One subcomplex (dark green) contains the cullin subunit Apc2 and the RING-domain protein Apc11, which recruits E2s. Another subcomplex (light green) contains the three TPR-containing subunits, Cdc27, Cdc23 and Cdc16, as well as two subunits, Apc4 and Apc5, that help connect them to the rest of the APC via Apc1. The TPR-containing subunits provide binding sites for the activator (Cdh1 or Cdc20), which contains at least two APC-interaction motifs, the Ile-Arg (IR) motif and the C-box, as well as a large WD40 repeat sequence that is likely to form a propeller-like binding site for substrate.

Figure 5

Reaction mechanism underlying protein ubiquitination by the APC and other RING-domain ubiquitin ligases. (a) The E3 positions the substrate and E2-ubiquitin conjugate in close proximity. (b) Deprotonation of the ε-amino group of a lysine on the substrate promotes nucleophilic attack of the thioester bond between the ubiquitin carboxyl terminus and the E2 active-site cysteine. (c) The initial product of nucleophilic attack is an oxyanion intermediate. (d) Completion of the reaction results in an isopeptide bond between the ubiquitin carboxyl terminus and the substrate lysine.

Figure 6

A speculative model of protein ubiquitination by the APC. Substrate (magenta) is likely to bind multivalently to both activator and the APC using degradation sequences (red rectangles) in disordered substrate regions. The APC positions substrates so that multiple lysine side chains can effectively attack the E2-ubiquitin bond. Residues near the E2 active site promote catalysis, which is stimulated upon binding to the APC. Flexibility in both the substrate and APC may help accommodate the modification of many lysines and the formation of long polyubiquitin chains.