Anaphase Promoting Complex (APC/C)

Regulated cell cycle progression is dependent upon controlled ubiquitin dependent proteolysis, mediated by the SCF and the anaphase promoting complex or cyclosome (APC/C), coupled to reversible protein phosphorylation.

The APC/C is an unusually large multi-subunit complex that functions as an E3 ubiquitin ligase to regulate progression through the cell cycle via controlled degradation of cell cycle regulatory proteins via the ubiquitin proteasome system. The budding yeast APC contains 13 different proteins including the tetratricopeptide repeat (TPR) proteins Cdc16, Cdc23, and Cdc27, which form a complex with a molecular mass in excess of 1.2 MDa.

Substrate specificity is dictated by the association of the APC/C with two WD40-repeat containing regulatory subunits, Cdc20 and Cdh1. In yeast, Cdc20 is responsible for destruction of securin at the metaphase to anaphase transition, allowing sister chromatid separation, while Cdh1 is responsible for destruction of mitotic cyclins, facilitating mitotic exit. Inactivation of the APC/C in response to the spindle checkpoint occurs by inhibition of Cdc20 function via its sequestration with Mad2.

Our aim is to determine a high-resolution structure of the APC/C to address questions of its assembly and molecular basis for substrate specificity, regulation and catalysis. We are pursuing two complementary approaches.

First, we have used single particle electron microscopy to determine structures of yeast APC/C isolated from budding yeast. By forming complexes with the co-activator Cdh1, and synthetic peptides comprising the destruction signals (D-box and KEN-box), we have generated 3D maps of ternary complexes of APC/CCdh1 with substrates.

This work identified the D-box receptor on the APC/C to be composed of Cdh1 and a core APC/C subunit Apc10. Our current cryo-EM maps are approaching 10 Å resolution. With modifications of the cryo-grids we are planning to extend this resolution to 6 Å in the near-future.

To understand the structure of the APC/C derived from the cryo-EM maps, we have established methods for accurate mapping of APC/C subunits. In this approach using recombinant methods we can selectively reconstruct the entire APC/C and APC/C sub-complexes and analyse these complexes by mass-spectrometry and electron microscopy. We have assigned all 13 core APC/C proteins to the electron density maps.

The subunit segmentation of the APC/C, coupled to the high resolution cryo-EM maps at 10 Å resolution, provide the means to accurately fit crystal structures of individual APC/C subunits and sub-complexes and homology models into the APC/C molecular envelop.

Recently we have determined the structure of the N-terminal dimerisation domain of Cdc27 and the full-length heterotetramer of Cdc16-Cdc26. This hybrid approach of X-ray crystallography and electron microscopy allowed us to generate a pseudo-atomic model of the APC/C.

Crystallography and mass spectrometry of APC/C subunits, sub-complexes and intact APC/C.
The quantities of endogenous APC/C purified from budding yeast are very small (2-5 g/L of yeast culture), hampering efforts to crystallise the APC/C. To improve the quantities of APC/C for crystallisation purposes, and to provide control over subunit composition, and allow the possibility of recombinant modification of subunits to facilitate crystallisation, we have invested considerable effort in developing methods for the over-expression of the intact APC/C using the baculovirus/insect cell system.

Using the multi-bac system we can now generate sub-complexes of the APC/C (such as the catalytic subcomplex and the tetratricopeptide subcomplex of 8 and 6 subunits respectively) and intact APC/C that appears biochemically and structurally indistinguishable from endogenous APC/C. Generation of these sub-complexes also provides insights into the APC/C assembly process and we have generated binary, ternary and quaternary sub-complexes of APC/C subunits.

In collaboration with Carol Robinson’s group (PhD student F. Stengel), we have determined the accurate mass of the larger APC/C sub-complexes, and this provides a precise definition of the subunit stoichiometry, revealing that many subunits are present as dimers, indicating that there are 20 protein subunits of the core APC/C (from 13 gene products) with a total mol. mass of 1.2 MDa.

By expression screening of individual APC/C subunits and sub-complexes from budding and fission yeast, human and E. cuniculi, we have managed to purify and crystallise a number of APC/C subunits. Most success has been achieved with the TPR subunits.

We have crystallised the N-terminus of Cdc27 and the full length of Cdc16 in complex with Cdc26. Both Cdc27 and Cdc16 are TPR subunits, and homo-dimerise via their N-termini by means of a similar (and as yet previously unreported) mechanism. The dimerisation of Cdc27 and Cdc16 is consistent with the stoichiometry of these subunits defined by mass spectrometry.

Subunit organization and pseudo-atomic model of APC/C. Atomic coordinates of Cdc16–Cdc26, Cdc23, Cdc27, Apc2, Apc10 and Cdh1 were docked in the 11A˚ cryo-EM map of the APC/C represented in grey mesh. From Schreiber et al., (2011).