期刊
BIOCHEMICAL JOURNAL
卷 449, 期 -, 页码 365-371出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20121374
关键词
anaphase-promoting complex/cyclosome (APC/C); cell cycle; recombinant expression; single-particle electron microscopy; three-dimensional structure; ubiquitination
资金
- Cancer Research UK [C576/A14109]
- Cancer Research UK [14109] Funding Source: researchfish
- Medical Research Council [MC_UP_1201/6] Funding Source: researchfish
- MRC [MC_UP_1201/6] Funding Source: UKRI
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 similar to 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.
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