Recombinant expression, reconstitution and structure of human Anaphase Promoting Complex (APC/C).

Mechanistic and structural studies of large multi-subunit assemblies are greatly facilitated by their reconstitution in heterologous recombinant systems. Here, we describe the generation of recombinant human anaphase promoting complex (APC/C), 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 dependent on the APC/C coactivator Cdh1, and its 3-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 S. cerevisiae APC/C homology model, and in contrast to endogenous human APC/C, no evidence for conformational flexibility of the TPR lobe is observed. Additional density present in the human APC/C structure, proximal to Apc3/Cdc27 of the TPR lobe, is assigned to the tetratricopeptide repeat subunit Apc7, a subunit specific to vertebrate APC/C.
Biochem. J.
Date: Oct. 19, 2012
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