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Complete subunit architecture of the proteasome regulatory particle.

Lander GC, Estrin E, Matyskiela ME, Bashore C, Nogales E, Martin A - Nature (2012)

Bottom Line: Unexpectedly, the ATPase subunits within the base unfoldase are arranged in a spiral staircase, providing insight into potential mechanisms for substrate translocation through the central pore.Large conformational rearrangements of the lid upon holoenzyme formation suggest allosteric regulation of deubiquitination.We provide a structural basis for the ability of the proteasome to degrade a diverse set of substrates and thus regulate vital cellular processes.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720, USA.

ABSTRACT
The proteasome is the major ATP-dependent protease in eukaryotic cells, but limited structural information restricts a mechanistic understanding of its activities. The proteasome regulatory particle, consisting of the lid and base subcomplexes, recognizes and processes polyubiquitinated substrates. Here we used electron microscopy and a new heterologous expression system for the lid to delineate the complete subunit architecture of the regulatory particle from yeast. Our studies reveal the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes and the protein unfolding machinery at subnanometre resolution, outlining the substrate's path to degradation. Unexpectedly, the ATPase subunits within the base unfoldase are arranged in a spiral staircase, providing insight into potential mechanisms for substrate translocation through the central pore. Large conformational rearrangements of the lid upon holoenzyme formation suggest allosteric regulation of deubiquitination. We provide a structural basis for the ability of the proteasome to degrade a diverse set of substrates and thus regulate vital cellular processes.

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Related in: MedlinePlus

The lid subcomplex within the holoenzyme assemblya) Negative-stain 3D reconstruction at ~15 Å resolution shows resemblance between endogenous (left) and recombinant (right) lid. b) Locations of lid (yellow) and base (cyan) within the subnanometer holoenzyme reconstruction. c) Six copies of the crystal structure of a PCI domain (PDBid: 1RZ4) are docked into the lid electron density, showing a horseshoe-shaped arrangement of the winged-helix domains. Each domain is colored according to its respective lid subunit (Fig. 2).
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Figure 1: The lid subcomplex within the holoenzyme assemblya) Negative-stain 3D reconstruction at ~15 Å resolution shows resemblance between endogenous (left) and recombinant (right) lid. b) Locations of lid (yellow) and base (cyan) within the subnanometer holoenzyme reconstruction. c) Six copies of the crystal structure of a PCI domain (PDBid: 1RZ4) are docked into the lid electron density, showing a horseshoe-shaped arrangement of the winged-helix domains. Each domain is colored according to its respective lid subunit (Fig. 2).

Mentions: To compare the functionalities of recombinant and endogenous lid, we established conditions for their in-vitro reconstitution with base and 20S core subcomplexes from yeast to yield 26S holoenzyme. These reassembled particles were assayed for their activity in ubiquitin-dependent substrate degradation by using a poly-ubiquitinated GFP-cyclin fusion protein and following the decrease in GFP fluorescence. Proteasome reconstituted with E. coli-expressed lid supported robust substrate degradation (Fig. S3). Importantly, the 3D EM reconstructions from negative-stained samples of both lid subcomplexes are practically identical (Fig. 1a, S4), establishing this recombinant system as an ideal tool for our structural studies of the regulatory particle.


Complete subunit architecture of the proteasome regulatory particle.

Lander GC, Estrin E, Matyskiela ME, Bashore C, Nogales E, Martin A - Nature (2012)

The lid subcomplex within the holoenzyme assemblya) Negative-stain 3D reconstruction at ~15 Å resolution shows resemblance between endogenous (left) and recombinant (right) lid. b) Locations of lid (yellow) and base (cyan) within the subnanometer holoenzyme reconstruction. c) Six copies of the crystal structure of a PCI domain (PDBid: 1RZ4) are docked into the lid electron density, showing a horseshoe-shaped arrangement of the winged-helix domains. Each domain is colored according to its respective lid subunit (Fig. 2).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285539&req=5

Figure 1: The lid subcomplex within the holoenzyme assemblya) Negative-stain 3D reconstruction at ~15 Å resolution shows resemblance between endogenous (left) and recombinant (right) lid. b) Locations of lid (yellow) and base (cyan) within the subnanometer holoenzyme reconstruction. c) Six copies of the crystal structure of a PCI domain (PDBid: 1RZ4) are docked into the lid electron density, showing a horseshoe-shaped arrangement of the winged-helix domains. Each domain is colored according to its respective lid subunit (Fig. 2).
Mentions: To compare the functionalities of recombinant and endogenous lid, we established conditions for their in-vitro reconstitution with base and 20S core subcomplexes from yeast to yield 26S holoenzyme. These reassembled particles were assayed for their activity in ubiquitin-dependent substrate degradation by using a poly-ubiquitinated GFP-cyclin fusion protein and following the decrease in GFP fluorescence. Proteasome reconstituted with E. coli-expressed lid supported robust substrate degradation (Fig. S3). Importantly, the 3D EM reconstructions from negative-stained samples of both lid subcomplexes are practically identical (Fig. 1a, S4), establishing this recombinant system as an ideal tool for our structural studies of the regulatory particle.

Bottom Line: Unexpectedly, the ATPase subunits within the base unfoldase are arranged in a spiral staircase, providing insight into potential mechanisms for substrate translocation through the central pore.Large conformational rearrangements of the lid upon holoenzyme formation suggest allosteric regulation of deubiquitination.We provide a structural basis for the ability of the proteasome to degrade a diverse set of substrates and thus regulate vital cellular processes.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720, USA.

ABSTRACT
The proteasome is the major ATP-dependent protease in eukaryotic cells, but limited structural information restricts a mechanistic understanding of its activities. The proteasome regulatory particle, consisting of the lid and base subcomplexes, recognizes and processes polyubiquitinated substrates. Here we used electron microscopy and a new heterologous expression system for the lid to delineate the complete subunit architecture of the regulatory particle from yeast. Our studies reveal the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes and the protein unfolding machinery at subnanometre resolution, outlining the substrate's path to degradation. Unexpectedly, the ATPase subunits within the base unfoldase are arranged in a spiral staircase, providing insight into potential mechanisms for substrate translocation through the central pore. Large conformational rearrangements of the lid upon holoenzyme formation suggest allosteric regulation of deubiquitination. We provide a structural basis for the ability of the proteasome to degrade a diverse set of substrates and thus regulate vital cellular processes.

Show MeSH
Related in: MedlinePlus