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Published on: October 12, 2018
Recombinant expression, reconstitution and structure of human anaphase-promoting complex (APC/C)
Ziguo Zhang1, Jing Yang, Eric H Kong
1Division of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK.
The Biochemical Journal
|October 20, 2012
Summary
Researchers successfully reconstituted human anaphase-promoting complex/cyclosome (APC/C), a key cell-cycle regulator. This structural and functional validation confirms its subunit composition and provides insights into its molecular architecture.
Area of Science:
- Cell Biology
- Structural Biology
- Biochemistry
Background:
- Large multi-subunit protein assemblies are crucial for cellular processes.
- Reconstitution in heterologous systems aids mechanistic and structural studies.
- The anaphase-promoting complex/cyclosome (APC/C) is a vital E3 ubiquitin ligase regulating cell-cycle progression.
Purpose of the Study:
- To generate recombinant human APC/C in a heterologous system.
- To validate the subunit composition and structural integrity of human APC/C.
- To investigate the structure and potential conformational flexibility of human APC/C.
Main Methods:
- Recombinant expression and assembly of human APC/C.
- Functional assays using budding yeast APC/C substrates (Hsl1) and co-activators (Cdh1).
- Electron microscopy and single-particle analysis for 3D reconstruction.
Main Results:
- Successfully reconstituted functional human APC/C.
- 3D reconstruction revealed a structure compatible with the yeast homology model.
- Identified additional density assigned to the vertebrate-specific Apc7 subunit.
- Observed no conformational flexibility in the TPR lobe, unlike endogenous human APC/C.
Conclusions:
- Reconstitution validates the proposed subunit composition of human APC/C.
- The determined structure provides a high-resolution model for human APC/C.
- The findings highlight structural differences and specific subunits in vertebrate APC/C.
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