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

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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Faculty Opinions recommendation of A reverse genetic analysis of components of the Toll signaling pathway in Caenorhabditis elegans.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1002561.28554 ◽

2001 ◽

Author(s):

Jonathan Hodgkin

Keyword(s):

Caenorhabditis Elegans ◽

Genetic Analysis ◽

Signaling Pathway ◽

Reverse Genetic ◽

Toll Signaling

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JAMP Optimizes ERAD to Protect Cells from Unfolded Proteins

Molecular Biology of the Cell ◽

10.1091/mbc.e08-08-0839 ◽

2008 ◽

Vol 19 (11) ◽

pp. 5019-5028 ◽

Cited By ~ 9

Author(s):

Marianna Tcherpakov ◽

Limor Broday ◽

Agnes Delaunay ◽

Takayuki Kadoya ◽

Ashwani Khurana ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Er Stress ◽

Transmembrane Protein ◽

26S Proteasome ◽

Misfolded Proteins ◽

Unfolded Proteins ◽

Cellular Homeostasis ◽

Channel Proteins ◽

Proteasome Subunits ◽

Opposite Response

Clearance of misfolded proteins from the ER is central for maintenance of cellular homeostasis. This process requires coordinated recognition, ER-cytosol translocation, and finally ubiquitination-dependent proteasomal degradation. Here, we identify an ER resident seven-transmembrane protein (JAMP) that links ER chaperones, channel proteins, ubiquitin ligases, and 26S proteasome subunits, thereby optimizing degradation of misfolded proteins. Elevated JAMP expression promotes localization of proteasomes at the ER, with a concomitant effect on degradation of specific ER-resident misfolded proteins, whereas inhibiting JAMP promotes the opposite response. Correspondingly, a jamp-1 deleted Caenorhabditis elegans strain exhibits hypersensitivity to ER stress and increased UPR. Using biochemical and genetic approaches, we identify JAMP as important component for coordinated clearance of misfolded proteins from the ER.

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Reverse genetic analysis of Caenorhabditis elegans presenilins reveals redundant but unequal roles for sel-12 and hop-1 in Notch-pathway signaling

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.96.5.2497 ◽

1999 ◽

Vol 96 (5) ◽

pp. 2497-2502 ◽

Cited By ~ 52

Author(s):

B. Westlund ◽

D. Parry ◽

R. Clover ◽

M. Basson ◽

C. D. Johnson

Keyword(s):

Caenorhabditis Elegans ◽

Genetic Analysis ◽

Notch Pathway ◽

Reverse Genetic

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Use of RNA Interference and Complementation To Study the Function of the Drosophila and Human 26S Proteasome Subunit S13

Molecular and Cellular Biology ◽

10.1128/mcb.23.15.5320-5330.2003 ◽

2003 ◽

Vol 23 (15) ◽

pp. 5320-5330 ◽

Cited By ~ 49

Author(s):

Josefin Lundgren ◽

Patrick Masson ◽

Claudio A. Realini ◽

Patrick Young

Keyword(s):

Cell Cycle ◽

Rna Interference ◽

Cell Cycle Progression ◽

Degradation Pathway ◽

26S Proteasome ◽

Wild Type ◽

Proteasome Subunit ◽

Rnai Treatment ◽

Regulatory Complex

ABSTRACT The S13 subunit (also called Pad1, Rpn11, and MPR1) is a component of the 19S complex, a regulatory complex essential for the ubiquitin-dependent proteolytic activity of the 26S proteasome. To address the functional role of S13, we combined double-stranded RNA interference (RNAi) against the Drosophila proteasome subunit DmS13 with expression of wild-type and mutant forms of the homologous human gene, HS13. These studies show that DmS13 is essential for 26S function. Loss of the S13 subunit in metazoan cells leads to increased levels of ubiquitin conjugates, cell cycle defects, DNA overreplication, and apoptosis. In vivo assays using short-lived proteasome substrates confirmed that the 26S ubiquitin-dependent degradation pathway is compromised in S13-depleted cells. In complementation experiments using Drosophila cell lines expressing HS13, wild-type HS13 was found to fully rescue the knockdown phenotype after DmS13 RNAi treatment, while an HS13 containing mutations (H113A-H115A) in the proposed isopeptidase active site was unable to rescue. A mutation within the conserved MPN/JAMM domain (C120A) abolished the ability of HS13 to rescue the Drosophila cells from apoptosis or DNA overreplication. However, the C120A mutant was found to partially restore normal levels of ubiquitin conjugates. The S13 subunit may possess multiple functions, including a deubiquitinylating activity and distinct activities essential for cell cycle progression that require the conserved C120 residue.

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Identification and Characterization of a Drosophila Proteasome Regulatory Network

Molecular and Cellular Biology ◽

10.1128/mcb.25.11.4662-4675.2005 ◽

2005 ◽

Vol 25 (11) ◽

pp. 4662-4675 ◽

Cited By ~ 60

Author(s):

Josefin Lundgren ◽

Patrick Masson ◽

Zahra Mirzaei ◽

Patrick Young

Keyword(s):

Regulatory Network ◽

Proteasome Inhibition ◽

26S Proteasome ◽

Deletion Mapping ◽

Proteasome Subunit ◽

Mrna Induction ◽

Proteasome Subunits ◽

Reporter Assays ◽

Reporter Mrna

ABSTRACT Maintaining adequate proteasomal proteolytic activity is essential for eukaryotic cells. For metazoan cells, little is known about the composition of genes that are regulated in the proteasome network or the mechanisms that modulate the levels of proteasome genes. Previously, two distinct treatments have been observed to induce 26S proteasome levels in Drosophila melanogaster cell lines, RNA interference (RNAi)-mediated inhibition of the 26S proteasome subunit Rpn10/S5a and suppression of proteasome activity through treatment with active-site inhibitors. We have carried out genome array profiles from cells with decreased Rpn10/S5a levels using RNAi or from cells treated with proteasome inhibitor MG132 and have thereby identified candidate genes that are regulated as part of a metazoan proteasome network. The profiles reveal that the majority of genes that were identified to be under the control of the regulatory network consisted of 26S proteasome subunits. The 26S proteasome genes, including three new subunits, Ubp6p, Uch-L3, and Sem1p, were found to be up-regulated. A number of genes known to have proteasome-related functions, including Rad23, isopeptidase T, sequestosome, and the genes for the segregase complex TER94/VCP-Ufd1-Npl4 were also found to be up-regulated. RNAi-mediated inhibition against the segregase complex genes demonstrated pronounced stabilization of proteasome substrates throughout the Drosophila cell. Finally, transcriptional reporter assays and deletion mapping studies in Drosophila demonstrate that proteasome mRNA induction is dependent upon the 5′ untranslated regions (UTRs). Transfer of the 5′ UTR from the proteasome subunit Rpn1/S2 to a noninducible promoter was sufficient to confer transcriptional upregulation of the reporter mRNA after proteasome inhibition.

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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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Structural characterization of the interaction of Ubp6 with the 26S proteasome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1510449112 ◽

2015 ◽

Vol 112 (28) ◽

pp. 8626-8631 ◽

Cited By ~ 72

Author(s):

Antje Aufderheide ◽

Florian Beck ◽

Florian Stengel ◽

Michaela Hartwig ◽

Andreas Schweitzer ◽

...

Keyword(s):

Intermediate Energy ◽

Catalytic Triad ◽

26S Proteasome ◽

Conformational Space ◽

Structural Basis ◽

Conformational Switching ◽

Interacting Protein ◽

Specific Protease ◽

Proteasome Subunits ◽

Regulatory Particle

In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6; ubiquitin-specific protease (USP) 14 in mammals] is the most abundant proteasome-interacting protein and has multiple roles in regulating proteasome function. Here, we investigate the structural basis of the interaction between Ubp6 and the 26S proteasome in the presence and absence of the inhibitor ubiquitin aldehyde. To this end we have used single-particle electron cryomicroscopy in combination with cross-linking and mass spectrometry. Ubp6 binds to the regulatory particle non-ATPase (Rpn) 1 via its N-terminal ubiquitin-like domain, whereas its catalytic USP domain is positioned variably. Addition of ubiquitin aldehyde stabilizes the binding of the USP domain in a position where it bridges the proteasome subunits Rpn1 and the regulatory particle triple-A ATPase (Rpt) 1. The USP domain binds to Rpt1 in the immediate vicinity of the Ubp6 active site, which may effect its activation. The catalytic triad is positioned in proximity to the mouth of the ATPase module and to the deubiquitylating enzyme Rpn11, strongly implying their functional linkage. On the proteasome side, binding of Ubp6 favors conformational switching of the 26S proteasome into an intermediate-energy conformational state, in particular upon the addition of ubiquitin aldehyde. This modulation of the conformational space of the 26S proteasome by Ubp6 explains the effects of Ubp6 on the kinetics of proteasomal degradation.

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