Recombinant expression, reconstitution and structure of human anaphase-promoting complex (APC/C)

Abstract

Mechanistic and structural studies of large multi-subunit assemblies are greatly facilitated by their reconstitution in heterologous recombinant systems. In the present paper, we describe the generation of recombinant human APC/C (anaphase-promoting complex/cyclosome), an E3 ubiquitin ligase that regulates cell-cycle progression. Human APC/C is composed of 14 distinct proteins that assemble into a complex of at least 19 subunits with a combined molecular mass of ~1.2 MDa. We show that recombinant human APC/C is correctly assembled, as judged by its capacity to ubiquitinate the budding yeast APC/C substrate Hsl1 (histone synthetic lethal 1) dependent on the APC/C co-activator Cdh1 [Cdc (cell division cycle) 20 homologue 1], and its three-dimensional reconstruction by electron microscopy and single-particle analysis. Successful reconstitution validates the subunit composition of human APC/C. The structure of human APC/C is compatible with the Saccharomyces cerevisiae APC/C homology model, and in contrast with endogenous human APC/C, no evidence for conformational flexibility of the TPR (tetratricopeptide repeat) lobe is observed. Additional density present in the human APC/C structure, proximal to Apc3/Cdc27 of the TPR lobe, is assigned to the TPR subunit Apc7, a subunit specific to vertebrate APC/C.

Deep Proteomics of the Xenopus laevis Egg using an mRNA-Derived Reference Database

“…At first glance, the anaphase promoting complex (APC) subunits are scattered relatively widely. However, some of the APC subunits are known to be dimeric, while some are monomeric [30]. Our precision is not good enough to separate these populations, but the dimeric subunits tend to have higher concentrations than the monomeric subunits (Fig.…”

biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes

“…12); CDH1 is an APC/C coactivator required to recruit substrates to the APC/C during mitotic exit and the G1 and G0 phase (13); and EMI1 and SKP1 form a heterodimeric inhibitor of APC/C-CDH1 (14). Earlier studies relied on the coexpression of human recombinant APC/C subunits from several baculoviruses (14)(15)(16). Using biGBac, we were able to assemble all 17 cDNAs previously cloned into pLIB vectors in a defined order into five different pBIG1 constructs and assembled the PGCs from these into one pBIG2 construct (FW_II-1; all pBIG1 and pBIG2 expression constructs used in this study are listed in Tables S3 and S4).…”

Recombinant expression and reconstitution of multiprotein complexes by the USER cloning method in the insect cell-baculovirus expression system

“…Our transfer vectors are integrated into MultiBac BAC by fusions at the Tn7 and Cre-LoxP integration sites for generation of the recombinant baculovirus genome. We have successfully used this method for the reconstitution of numerous multiprotein complexes including the mitotic checkpoint complex (MCC) [15] and various functional complexes of the APC/C [16,28], including human APC/C with its coactivator (Cdh1) and inhibitor Emi1 (a total of 17 subunits assembled into a complex of 22 subunits) [24]. We illustrate this approach with the reconstitution of recombinant APC/C from Sacchromyces cerevisiae.…”

“…This together with the insertion of a Cre-LoxP fusion site, created the MultiBac bacmid suitable for accepting the first generation pFBDM and pUCDM baculovirus transfer vectors through fusions at the Tn7 and Cre-LoxP integration sites, respectively [5]. This multiprotein expression strategy allows the production of large quantities of multisubunit complexes from the baculovirus-insect cell expression system, see for example [12][13][14][15][16][17][18].…”