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  Electron microscopy and in vitro deneddylation reveal similar architectures and biochemistry of isolated human and Flag-mouse COP9 signalosome complexes

Rockel, B., Schmaler, T., Huang, X., & Dubiel, W. (2014). Electron microscopy and in vitro deneddylation reveal similar architectures and biochemistry of isolated human and Flag-mouse COP9 signalosome complexes. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 450(2), 991-997. doi:10.1016/j.bbrc.2014.06.093.

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 Creators:
Rockel, Beate1, Author              
Schmaler, Tilo2, Author
Huang, Xiaohua2, Author
Dubiel, Wolfgang2, Author
Affiliations:
1Baumeister, Wolfgang / Molecular Structural Biology, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565142              
2external, ou_persistent22              

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Free keywords: 26S PROTEASOME; STRUCTURAL INSIGHTS; NEDD8; SUBUNIT; SCF; CUL1; METALLOPROTEASE; DEGRADATION; PARTICLE; TOOLBOXCOP9 signalosome; Lid; Deneddylation; Cryo-electron microscopy; 3D-reconstruction; Single-particle analysis;
 Abstract: The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases conferring substrate specificity. The CSN controls a large family of Ub ligases called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators, transcription factors and DNA damage response proteins. The CSN possesses structural similarities with the 265 proteasome Lid complex and the translation initiation complex 3 (eIF3) indicating similar ancestry and function. Initial structures were obtained 14 years ago by 2D electron microscopy (EM). Recently, first 3D molecular models of the CSN were created on the basis of negative-stain EM and single-particle analysis, mostly with recombinant complexes. Here, we compare deneddylating activity and structural features of CSN complexes purified in an elaborate procedure from human erythrocytes and efficiently pulled down from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates. 3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN were generated by negative stain EM and by cryo-EM. Both complexes show a central U-shaped segment from which several arms emanate. This structure, called the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away from the horseshoe. Compared to 3D models of negatively stained CSN complexes, densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because biochemical and structural results obtained with CSN complexes isolated from human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts are very similar, Flag-CSN pulldowns are a proper alternative to CSN preparation from erythrocytes. (C) 2014 Elsevier Inc. All rights reserved.

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Language(s): eng - English
 Dates: 2014
 Publication Status: Published in print
 Pages: 7
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Degree: -

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Title: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Source Genre: Journal
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Publ. Info: 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA : ACADEMIC PRESS INC ELSEVIER SCIENCE
Pages: - Volume / Issue: 450 (2) Sequence Number: - Start / End Page: 991 - 997 Identifier: ISSN: 0006-291X