Chaperone-driven proteasome assembly

Assembly of the 34-subunit, 2.5 MDa 26S proteasome is a carefully choreographed intricate process. It starts with formation of a seven-membered α-ring that serves as a template for assembly of the complementary β-ring-forming ‘half-proteasomes’. Dimerization results in a latent 20S core particle that can serve further as a platform for 19S regulatory particle attachment and formation of the biologically active 26S proteasome for ubiquitin-dependent proteolysis. Both general and dedicated proteasome assembly chaperones regulate the efficiency and outcome of critical steps in proteasome biogenesis, and in complex association.

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In-depth Analysis of the Lid Subunits Assembly Mechanism in Mammals

Biomolecules ◽

10.3390/biom9060213 ◽

2019 ◽

Vol 9 (6) ◽

pp. 213 ◽

Cited By ~ 3

Author(s):

Minghui Bai ◽

Xian Zhao ◽

Kazutaka Sahara ◽

Yuki Ohte ◽

Yuko Hirano ◽

...

Keyword(s):

26S Proteasome ◽

Core Particle ◽

Proteasome Subunit ◽

Ubiquitinated Proteins ◽

Assembly Pathway ◽

Assembly Mechanism ◽

Depth Analysis ◽

Regulatory Particle ◽

Assembly Intermediate ◽

Assembly Pathways

The 26S proteasome is a key player in the degradation of ubiquitinated proteins, comprising a 20S core particle (CP) and a 19S regulatory particle (RP). The RP is further divided into base and lid subcomplexes, which are assembled independently from each other. We have previously demonstrated the assembly pathway of the CP and the base by observing assembly intermediates resulting from knockdowns of each proteasome subunit and the assembly chaperones. In this study, we examine the assembly pathway of the mammalian lid, which remains to be elucidated. We show that the lid assembly pathway is conserved between humans and yeast. The final step is the incorporation of Rpn12 into the assembly intermediate consisting of two modular complexes, Rpn3-7-15 and Rpn5-6-8-9-11, in both humans and yeast. Furthermore, we dissect the assembly pathways of the two modular complexes by the knockdown of each lid subunit.

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The Assembly Pathway of the 19S Regulatory Particle of the Yeast 26S Proteasome

Molecular Biology of the Cell ◽

10.1091/mbc.e06-07-0635 ◽

2007 ◽

Vol 18 (2) ◽

pp. 569-580 ◽

Cited By ~ 78

Author(s):

Erika Isono ◽

Kiyoshi Nishihara ◽

Yasushi Saeki ◽

Hideki Yashiroda ◽

Naoko Kamata ◽

...

Keyword(s):

26S Proteasome ◽

Restrictive Temperature ◽

Enzyme Complex ◽

Core Particle ◽

Yeast Saccharomyces Cerevisiae ◽

The Core ◽

Assembly Pathway ◽

Regulatory Particle ◽

Mutant Cells

The 26S proteasome consists of the 20S proteasome (core particle) and the 19S regulatory particle made of the base and lid substructures, and it is mainly localized in the nucleus in yeast. To examine how and where this huge enzyme complex is assembled, we performed biochemical and microscopic characterization of proteasomes produced in two lid mutants, rpn5-1 and rpn7-3, and a base mutant ΔN rpn2, of the yeast Saccharomyces cerevisiae. We found that, although lid formation was abolished in rpn5-1 mutant cells at the restrictive temperature, an apparently intact base was produced and localized in the nucleus. In contrast, in ΔN rpn2 cells, a free lid was formed and localized in the nucleus even at the restrictive temperature. These results indicate that the modules of the 26S proteasome, namely, the core particle, base, and lid, can be formed and imported into the nucleus independently of each other. Based on these observations, we propose a model for the assembly process of the yeast 26S proteasome.

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Genetic analyses of the Arabidopsis 26S proteasome regulatory particle reveal its importance during light stress and a specific role for the N-Terminus of RPT2 in development.

Plant Signaling & Behavior ◽

10.4161/psb.20934 ◽

2012 ◽

Vol 7 (8) ◽

pp. 973-978 ◽

Cited By ~ 4

Author(s):

Kwang-Hee Lee ◽

Richard S. Marshall ◽

Lucas M. Slivicke ◽

Richard D. Vierstra

Keyword(s):

Light Stress ◽

Specific Role ◽

26S Proteasome ◽

Genetic Analyses ◽

N Terminus ◽

Regulatory Particle

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26S proteasome regulatory particle mutants have increased oxidative stress tolerance

The Plant Journal ◽

10.1111/j.1365-313x.2007.03322.x ◽

2008 ◽

Vol 53 (1) ◽

pp. 102-114 ◽

Cited By ~ 95

Author(s):

Jasmina Kurepa ◽

Akio Toh-e ◽

Jan A. Smalle

Keyword(s):

Oxidative Stress ◽

Stress Tolerance ◽

26S Proteasome ◽

Oxidative Stress Tolerance ◽

Regulatory Particle

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Maturation of the proteasome core particle induces an affinity switch that controls regulatory particle association

Nature Communications ◽

10.1038/ncomms7384 ◽

2015 ◽

Vol 6 (1) ◽

Cited By ~ 23

Author(s):

Prashant S. Wani ◽

Michael A. Rowland ◽

Alex Ondracek ◽

Eric J. Deeds ◽

Jeroen Roelofs

Keyword(s):

Core Particle ◽

Regulatory Particle ◽

Particle Association

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Ubiquitinated proteins promote the association of proteasomes with the deubiquitinating enzyme Usp14 and the ubiquitin ligase Ube3c

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1701734114 ◽

2017 ◽

Vol 114 (17) ◽

pp. E3404-E3413 ◽

Cited By ~ 30

Author(s):

Chueh-Ling Kuo ◽

Alfred Lewis Goldberg

Keyword(s):

Ubiquitin Ligase ◽

Mammalian Cells ◽

Proteasome Activity ◽

Deubiquitinating Enzyme ◽

Core Particle ◽

Ubiquitinated Proteins ◽

Protein Ubiquitination ◽

Cell Extracts ◽

Regulatory Particle ◽

In Cells

In mammalian cells, the 26S proteasomes vary in composition. In addition to the standard 28 subunits in the 20S core particle and 19 subunits in each 19S regulatory particle, a small fraction (about 10–20% in our preparations) also contains the deubiquitinating enzyme Usp14/Ubp6, which regulates proteasome activity, and the ubiquitin ligase, Ube3c/Hul5, which enhances proteasomal processivity. When degradation of ubiquitinated proteins in cells was inhibited, levels of Usp14 and Ube3c on proteasomes increased within minutes. Conversely, when protein ubiquitination was prevented, or when purified proteasomes hydrolyzed the associated ubiquitin conjugates, Usp14 and Ube3c dissociated rapidly (unlike other 26S subunits), but the inhibitor ubiquitin aldehyde slowed their dissociation. Recombinant Usp14 associated with purified proteasomes preferentially if they contained ubiquitin conjugates. In cells or extracts, adding Usp14 inhibitors (IU-1 or ubiquitin aldehyde) enhanced Usp14 and Ube3c binding further. Thus, in the substrate- or the inhibitor-bound conformations, Usp14 showed higher affinity for proteasomes and surprisingly enhanced Ube3c binding. Moreover, adding ubiquitinated proteins to cell extracts stimulated proteasome binding of both enzymes. Thus, Usp14 and Ube3c cycle together on and off proteasomes, and the presence of ubiquitinated substrates promotes their association. This mechanism enables proteasome activity to adapt to the supply of substrates.

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Tagging the proteasome active site β5 causes tag specific phenotypes in yeast

Scientific Reports ◽

10.1038/s41598-020-75126-1 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Kenrick A. Waite ◽

Alicia Burris ◽

Jeroen Roelofs

Keyword(s):

Nitrogen Starvation ◽

Critical Role ◽

26S Proteasome ◽

Core Particle ◽

Poor Growth ◽

Physiological Conditions ◽

Cellular Processes ◽

Storage Granules ◽

Fluorescent Tags ◽

Gfp Tag

Abstract The efficient and timely degradation of proteins is crucial for many cellular processes and to maintain general proteostasis. The proteasome, a complex multisubunit protease, plays a critical role in protein degradation. Therefore, it is important to understand the assembly, regulation, and localization of proteasome complexes in the cell under different conditions. Fluorescent tags are often utilized to study proteasomes. A GFP-tag on the β5 subunit, one of the core particle (CP) subunits with catalytic activity, has been shown to be incorporated into proteasomes and commonly used by the field. We report here that a tag on this subunit results in aberrant phenotypes that are not observed when several other CP subunits are tagged. These phenotypes appear in combination with other proteasome mutations and include poor growth, and, more significantly, altered 26S proteasome localization. In strains defective for autophagy, β5-GFP tagged proteasomes, unlike other CP tags, localize to granules upon nitrogen starvation. These granules are reflective of previously described proteasome storage granules but display unique properties. This suggests proteasomes with a β5-GFP tag are specifically recognized and sequestered depending on physiological conditions. In all, our data indicate the intricacy of tagging proteasomes, and possibly, large complexes in general.

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Reverse Genetic Analysis of the Caenorhabditis elegans 26S Proteasome Subunits by RNA Interference

Biological Chemistry ◽

10.1515/bc.2002.140 ◽

2002 ◽

Vol 383 (7-8) ◽

pp. 1263-1266 ◽

Cited By ~ 23

Author(s):

M. Takahashi ◽

H. Iwasaki ◽

H. Inoue ◽

K. Takahashi

Keyword(s):

Rna Interference ◽

Caenorhabditis Elegans ◽

Genetic Analysis ◽

Postembryonic Development ◽

26S Proteasome ◽

Reverse Genetic ◽

Proteasome Subunit ◽

Proteasome Subunits ◽

Regulatory Particle

Abstract Reverse genetic analysis was performed on the Caenorhabditis elegans 26S proteasome subunit genes by doublestranded RNAmediated interference (RNAi). Embryonic and postembryonic lethality was caused by interference of all of the eight tested 20S core subunits and all of the 19S regulatory particle subunits except for CeRpn9, CeRpn10, and Ce Rpn12, where RNAi caused no abnormality. However, synthetic suppression of CeRpn10 and CeRpn12 was lethal, whereas neither the combination of Ce Rpn9 with CeRpn10 nor with CeRpn12 resulted in abnormalities in RNAi. These results indicate that the 26S proteasome is indispensable for embryogenesis and postembryonic development, although Ce Rpn9, CeRpn10, and CeRpn12 are not essential, at least under the conditions used. CeRpn10 and Ce Rpn12 are considered to compensate for the suppression of each other.

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