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-Structure paper
タイトル | Recombinant expression, reconstitution and structure of human anaphase-promoting complex (APC/C). |
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ジャーナル・号・ページ | Biochem J, Vol. 449, Issue 2, Page 365-371, Year 2013 |
掲載日 | 2013年1月15日 |
著者 | Ziguo Zhang / Jing Yang / Eric H Kong / William C H Chao / Edward P Morris / Paula C A da Fonseca / David Barford / |
PubMed 要旨 | 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 ...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. |
リンク | Biochem J / PubMed:23078409 |
手法 | EM (単粒子) |
解像度 | 20.0 Å |
構造データ | EMDB-2226: |
由来 |
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