An architectural map of the anaphase-promoting complex

Abstract

Ubiquitin ligases (E3s) serve as critical regulators of metabolism, the cell cycle, DNA damage response, stress response, and receptor signaling. These enzymes catalyze the transfer of ubiquitin from a ubiquitin conjugase (E2) to a substrate, often resulting in substrate degradation via the proteasome. Ubiquitin ligases fall into three categories: the HECT domain ligases, the U-box ligases, and RING-finger ligases (Pickart and Eddins 2004). The RING-finger ligases can be further divided into those that act as single subunits and those that act as part of a multisubunit complex, typified by an associated cullin subunit (cullin-RING ligases). Two of the most heavily studied cullin-RING ligases are the anaphase-promoting complex (APC) and SCF, both of which are composed of multiple subunits and serve as important regulators of the cell cycle (Vodermaier 2004). The better characterized is SCF, which is composed of a modular specificity factor and three core subunits: a RING-finger subunit, a cullin subunit, and a scaffolding subunit (Willems et al. 2004; Petroski and Deshaies 2005). Crystal structures of the complex show that SCF holds an associated E2 and substrate in close proximity (Zheng et al. 2002). Although the precise mechanism of SCF catalysis has yet to be determined, the structure has provided great insights into how the enzyme functions.

Structural studies of the APC have proven more of a challenge. The APC is a cullin-RING ligase that ubiquitinates key regulators of mitosis, such as the mitotic and S-phase cyclins and the anaphase inhibitor securin (Peters 2002; Passmore 2004). Studies of the APC have lagged behind SCF because of the size of the complex and the inability to reconstitute it from purified components. The APC is composed of 13 core subunits (Yoon et al. 2002) and is activated by either of two weakly associating subunits Cdc20 or Cdh1 (Schwab et al. 1997; Visintin et al. 1997). Eight of the 13 core subunits are essential in budding yeast (Sikorski et al. 1991; Yu et al. 1998; Zachariae et al. 1998b; Peters 2002), making their study more challenging. Insights into the functions of nonessential subunits have been gained by studying strains in which they have been deleted. Deletions of SWM1, APC9, or CDC26 result in partial loss of Cdc27 and Cdc16 from purified APC (Zachariae et al. 1998b; Schwickart et al. 2004). Deletion of DOC1, on the other hand, reduces substrate binding and therefore enzyme processivity, but otherwise leaves the enzyme intact (Carroll and Morgan 2002; Passmore et al. 2003).

Like SCF, the APC contains a conserved RING-finger subunit (Apc11) and cullin-domain subunit (Apc2) that are essential and have been shown to be sufficient for limited catalytic activity in vitro (Gmachl et al. 2000; Leverson et al. 2000; Tang et al. 2001). The budding yeast APC contains three essential subunits with tetratricopeptide repeats (TPRs): Cdc27, Cdc23, and Cdc16, one of which (Cdc27) has been implicated in binding the activating subunit Cdh1 (Vodermaier et al. 2003; Kraft et al. 2005). The functions of the remaining subunits are unknown.

APC activity is primarily regulated by the binding of the activating subunits, Cdc20 and Cdh1 (Zachariae et al. 1998a; Jaspersen et al. 1999; Kramer et al. 2000; Rudner and Murray 2000). Two domains are thought to play roles in the binding of these subunits to the APC: a short internal motif called the C-box (Schwab et al. 2001) and a C-terminal IR dipeptide (Vodermaier et al. 2003). Cdc20 and Cdh1 also contain WD40 repeats that bind directly to APC targets, and thus may serve as a bridge between enzyme and substrate (Ohtoshi et al. 2000; Burton and Solomon 2001; Hilioti et al. 2001; Pfleger et al. 2001; Schwab et al. 2001; Burton et al. 2005; Kraft et al. 2005). The TPR protein Cdc27 has been implicated as an important binding site for the IR dipeptide motif of these subunits. Peters and colleagues have shown that Cdc27 is capable of binding an IR-containing peptide derived from the C terminus of Cdh1 (Vodermaier et al. 2003) and that Cdh1 cross-links directly to Cdc27 in vitro in an IR-dependent fashion (Kraft et al. 2005).

Previously, we showed that the only obligatory targets of the APC for cell cycle progression are securin and the B-type cyclins (Thornton and Toczyski 2003). By deleting the genes encoding securin (Pds1) and the S-phase cyclin Clb5, while overexpressing the CDK inhibitor Sic1, we constructed a yeast strain that is capable of proliferating in the absence of the APC. In the present work, we used this strain to study the impact of deleting each of the essential APC subunits on enzyme structure. We find that the largest subunit, Apc1, binds independently to two subcomplexes. One contains the three TPR subunits as well as Cdc26, while the other contains Apc2, Apc11, and Doc1. The subunits of the TPR subcomplex bind sequentially to Apc1, with Cdc23 being the most directly associated and Cdc27 the most peripheral. Apc4, Apc5, Apc1, and Cdc23 bind interdependently, such that loss of any one subunit disrupts association between the remaining three. Using APC purified from strains lacking individual subunits, we also find that both Cdc27 and Apc2 are required for full binding of Cdh1 to the APC. Finally, we present biochemical and genetic evidence for the importance of an additional domain or domains besides the IR dipeptide in activator function.

Acknowledgments

Notes

Supplemental material is available at http://genesdev.org.

Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1396906.