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

Takahashi, Maiko; Iwasaki, Hideki; Inoue, Hiroo; Takahashi, Kenji

doi:10.1515/bc.2002.140

Cited by 39 publications

(58 citation statements)

References 17 publications

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“…In various systems, RPN-12 has been shown to function in the regulatory particle of the proteasome and was proposed to share some activities with the subunit RPN-10 ( Wilkinson et al 2000;Glickman and Ciechanover 2002;Takahashi et al 2002). In C. elegans, only 3 of the 30 genes shown so far to encode proteasome subunits (Davy et al 2001) do not result in embryonic lethality upon disruption by RNAi: rpn-9, rpn-10, and rpn-12 (Takahashiet al 2002;Kamath et al 2003;Sonnichsen et al 2005).…”

Section: Resultsmentioning

confidence: 99%

A Genomewide Screen for Suppressors of par-2 Uncovers Potential Regulators of PAR Protein-Dependent Cell Polarity in Caenorhabditis elegans

Labbé

Pacquelet²,

Marty³

et al. 2006

Genetics

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The PAR proteins play an essential role in establishing and maintaining cell polarity. While their function is conserved across species, little is known about their regulators and effectors. Here we report the identification of 13 potential components of the C. elegans PAR polarity pathway, identified in an RNAi-based, systematic screen to find suppressors of par-2(it5ts) lethality. Most of these genes are conserved in other species. Phenotypic analysis of double-mutant animals revealed that some of the suppressors can suppress lethality associated with the strong loss-of-function allele par-2(lw32), indicating that they might impinge on the PAR pathway independently of the PAR-2 protein. One of these is the gene nos-3, which encodes a homolog of Drosophila Nanos. We find that nos-3 suppresses most of the phenotypes associated with loss of par-2 function, including early cell division defects and maternal-effect sterility. Strikingly, while PAR-1 activity was essential in nos-3; par-2 double mutants, its asymmetric localization at the posterior cortex was not restored, suggesting that the function of PAR-1 is independent of its cortical localization. Taken together, our results identify conserved components that regulate PAR protein function and also suggest a role for NOS-3 in PAR protein-dependent cell polarity.

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Section: Resultsmentioning

confidence: 99%

A Genomewide Screen for Suppressors of par-2 Uncovers Potential Regulators of PAR Protein-Dependent Cell Polarity in Caenorhabditis elegans

Labbé

Pacquelet²,

Marty³

et al. 2006

Genetics

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“…To date, genetic analysis of Rpn10 has been conducted on yeast, moss, plant, and fly (6,9,36,38,40,41,43). In the present study, we extended the genetic analysis of Rpn10 to vertebrates, employing gene-targeting techniques on mice.…”

Section: Discussionmentioning

confidence: 99%

“…Although Rpn10 is not essential for life or for overall ubiquitin-mediated protein turnover in yeast and worm (6,36,41,43), Rpn10-deficient mutants of Physcomitrella patens, Arabidopsis thaliana, and Drosophila melanogaster showed moresevere phenotypes such as developmental arrest and lethality (9,38,40). These results suggest that higher eukaryotes depend on Rpn10-mediated degradation of polyubiquitinated proteins for their development.…”

mentioning

confidence: 96%

Rpn10-Mediated Degradation of Ubiquitinated Proteins Is Essential for Mouse Development

Hamazaki

Sasaki

Kawahara

et al. 2007

Molecular and Cellular Biology

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Rpn10 is a subunit of the 26S proteasome that recognizes polyubiquitinated proteins. The importance of Rpn10 in ubiquitin-mediated proteolysis is debatable, since a deficiency of Rpn10 causes different phenotypes in different organisms. To date, the role of mammalian Rpn10 has not been examined genetically. Moreover, vertebrates have five splice variants of Rpn10 whose expressions are developmentally regulated, but their biological significance is not understood. To address these issues, we generated three kinds of Rpn10 mutant mice. Rpn10 knockout resulted in early-embryonic lethality, demonstrating the essential role of Rpn10 in mouse development. Rpn10a knock-in mice, which exclusively expressed the constitutive type of Rpn10 and did not express vertebrate-specific variants, grew normally, indicating that Rpn10 diversity is not essential for conventional development. Mice expressing the N-terminal portion of Rpn10, which contained a von Willebrand factor A (VWA) domain but lacked ubiquitin-interacting motifs (Rpn10⌬UIM), also exhibited embryonic lethality, suggesting the important contribution of UIM domains to viability, but survived longer than Rpn10-null mice, consistent with a "facilitator" function of the VWA domain. Biochemical analysis of the Rpn10⌬UIM liver showed specific impairment of degradation of ubiquitinated proteins. Our results demonstrate that Rpn10-mediated degradation of ubiquitinated proteins, catalyzed by UIMs, is indispensable for mammalian life.

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“…In contrast to Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster, which have one RPT2 gene, the RPT2 subunit in Arabidopsis (Arabidopsis thaliana) is encoded by a two-member gene family (Rubin et al, 1998;Takahashi et al, 2002;Wó jcik and DeMartino, 2002;Shibahara et al, 2004;Ueda et al, 2004;Sö nnichsen et al, 2005;. AtRPT2a (At4g29040) and AtRPT2b (At2g20140; Fig.…”

Section: Isolation Of Arabidopsis Rpt2 Mutantsmentioning

confidence: 99%

Loss of 26S Proteasome Function Leads to Increased Cell Size and Decreased Cell Number in Arabidopsis Shoot Organs

et al. 2009

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Although the final size of plant organs is influenced by environmental cues, it is generally accepted that the primary size determinants are intrinsic factors that regulate and coordinate cell proliferation and cell expansion. Here, we show that optimal proteasome function is required to maintain final shoot organ size in Arabidopsis (Arabidopsis thaliana). Loss of function of the subunit regulatory particle AAA ATPase (RPT2a) causes a weak defect in 26S proteasome activity and leads to an enlargement of leaves, stems, flowers, fruits, seeds, and embryos. These size increases are a result of increased cell expansion that compensates for a reduction in cell number. Increased ploidy levels were found in some but not all enlarged organs, indicating that the cell size increases are not caused by a higher nuclear DNA content. Partial loss of function of the regulatory particle non-ATPase (RPN) subunits RPN10 and RPN12a causes a stronger defect in proteasome function and also results in cell enlargement and decreased cell proliferation. However, the increased cell volumes in rpn10-1 and rpn12a-1 mutants translated into the enlargement of only some, but not all, shoot organs. Collectively, these data show that during Arabidopsis shoot development, the maintenance of optimal proteasome activity levels is important for balancing cell expansion with cell proliferation rates.The 26S proteasome (26SP) is a multisubunit, multicatalytic, 2.4-MD protease responsible for the degradation of proteins involved in various biological processes (Varshavsky, 2005;DeMartino and Gillette, 2007;Hanna and Finley, 2007;. Prior to their degradation, most 26SP target proteins are covalently modified with a polyubiquitin chain in a three-step enzymatic reaction . In addition to its central function in recognizing and degrading polyubiquitinated proteins, the 26SP can also degrade proteins that were not modified by polyubiquitination (Benaroudj et al., 2001;Lee et al., 2006;Pande et al., 2007).The 26SP consists of a cylindrical 20S core complex and two 19S regulatory particles that cap the 20S core on both ends. The 20S proteasome (20SP) is composed of seven related a-subunits and seven related b-subunits arranged in a stack of four heptameric rings. The outer rings are composed of a-subunits and the inner rings are composed of b-subunits, of which three have proteolytic activities described as caspase-, trypsin-, and chymotrypsin-like . The regulatory particles (RPs) serve as highly restrictive gatekeepers for the core protease. Each RP is composed of a lid subcomplex, which contains at least nine non-ATPase subunits designated RPN3, RPN5 to RPN9, RPN11, RPN12, and RPN15 and a base subcomplex that contains RPN1, RPN2, RPN13, and RPT1 to RPT6 subunits. The RPN10 and RPN13 subunits have been shown to recognize polyubiquitinated proteins; thus, they define the main interaction points of the 26SP with its target proteins (Young et al., 1998;Smalle and Vierstra, 2004;Husnjak et al., 2008;Schreiner et al., 2008). Ubiquitinated proteins can also int...

show abstract

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

Cited by 39 publications

References 17 publications

A Genomewide Screen for Suppressors of par-2 Uncovers Potential Regulators of PAR Protein-Dependent Cell Polarity in Caenorhabditis elegans

A Genomewide Screen for Suppressors of par-2 Uncovers Potential Regulators of PAR Protein-Dependent Cell Polarity in Caenorhabditis elegans

Rpn10-Mediated Degradation of Ubiquitinated Proteins Is Essential for Mouse Development

Loss of 26S Proteasome Function Leads to Increased Cell Size and Decreased Cell Number in Arabidopsis Shoot Organs

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