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CDNA cloning of p112, the largest regulatory subunit of the human 26s proteasome, and functional analysis of its yeast homologue, sen3p.

ABSTRACT: The 26S proteasome is a large multisubunit protease complex, the largest regulatory subunit of which is a component named p112. Molecular cloning of cDNA encoding human p112 revealed a polypeptide predicted to have 953 amino acid residues and a molecular mass of 105,865. The human p112 gene was mapped to the q37.1-q37.2 region of chromosome 2. Computer analysis showed that p112 has strong similarity to the Saccharomyces cerevisiae Sen3p, which has been listed in a gene bank as a factor affecting tRNA splicing endonuclease. The SEN3 also was identified in a synthetic lethal screen with the nin1-1 mutant, a temperature-sensitive mutant of NIN1. NIN1 encodes p31, another regulatory subunit of the 26S proteasome, which is necessary for activation of Cdc28p kinase. Disruption of the SEN3 did not affect cell viability, but led to temperature-sensitive growth. The human p112 cDNA suppressed the growth defect at high temperature in a SEN3 disruptant, indicating that p112 is a functional homologue of the yeast Sen3p. Maintenance of SEN3 disruptant cells at the restrictive temperature resulted in a variety of cellular dysfunctions, including defects in proteolysis mediated by the ubiquitin pathway, in the N-end rule system, in the stress response upon cadmium exposure, and in nuclear protein transportation. The functional abnormality induced by SEN3 disruption differs considerably from various phenotypes shown by the nin1-1 mutation, suggesting that these two regulatory subunits of the 26S proteasome play distinct roles in the various processes mediated by the 26S proteasome.

SUBMITTER: Yokota K

PROVIDER: S-EPMC275938 | biostudies-other | 1996 Jun

REPOSITORIES: biostudies-other

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CDNA cloning of p112, the largest regulatory subunit of the human 26s proteasome, and functional analysis of its yeast homologue, sen3p.

Yokota K K Kagawa S S Shimizu Y Y Akioka H H Tsurumi C C Noda C C Fujimuro M M Yokosawa H H Fujiwara T T Takahashi E E Ohba M M Yamasaki M M DeMartino G N GN Slaughter C A CA Toh-e A A Tanaka K K

Molecular biology of the cell 19960601 6

The 26S proteasome is a large multisubunit protease complex, the largest regulatory subunit of which is a component named p112. Molecular cloning of cDNA encoding human p112 revealed a polypeptide predicted to have 953 amino acid residues and a molecular mass of 105,865. The human p112 gene was mapped to the q37.1-q37.2 region of chromosome 2. Computer analysis showed that p112 has strong similarity to the Saccharomyces cerevisiae Sen3p, which has been listed in a gene bank as a factor affecting ...[more]

PMID: 8816993

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Project description:Phytophthora root rot, caused by Phytophthora sojae is a destructive disease of soybean (Glycine max) worldwide. We previously confirmed that the bHLH transcription factor GmPIB1 (P. sojae-inducible bHLH transcription factor) reduces accumulation of reactive oxygen species (ROS) in cells by inhibiting expression of the peroxidase-related gene GmSPOD thus improving the resistance of hairy roots to P. sojae. To identify proteins interacting with GmPIB1 and assess their participation in the defense response to P. sojae, we obtained transgenic soybean hairy roots overexpressing GmPIB1 by Agrobacterium rhizogenes mediated transformation and examined GmPIB1 protein-protein interactions using immunoprecipitation combined with mass spectrometry. We identified 392 proteins likely interacting with GmPIB1 and selected 20 candidate genes, and only 26S proteasome regulatory subunit GmPSMD (Genbank accession no. XP_014631720) interacted with GmPIB1 in luciferase complementation and pull-down experiments and yeast two-hybrid assays. Overexpression of GmPSMD (GmPSMD-OE) in soybean hairy roots remarkably improved resistance to P. sojae and RNA interference of GmPSMD (GmPSMD -RNAi) increased susceptibility. In addition, accumulation of total ROS and hydrogen peroxide (H2O2) in GmPSMD-OE transgenic soybean hairy roots were remarkably lower than those of the control after P. sojae infection. Moreover, in GmPSMD-RNAi transgenic soybean hairy roots, H2O2 and the accumulation of total ROS exceeded those of the control. There was no obvious difference in superoxide anion (O2 -) content between control and transgenic hairy roots. Antioxidant enzymes include peroxidase (POD), glutathione peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT) are responsible for ROS scavenging in soybean. The activities of these antioxidant enzymes were remarkably higher in GmPSMD-OE transgenic soybean hairy roots than those in control, but were reduced in GmPSMD-RNAi transgenic soybean hairy roots. Moreover, the activity of 26S proteasome in GmPSMD-OE and GmPIB1-OE transgenic soybean hairy roots was significantly higher than that in control and was significantly lower in PSMD-RNAi soybean hairy roots after P. sojae infection. These data suggest that GmPSMD might reduce the production of ROS by improving the activity of antioxidant enzymes such as POD, SOD, GPX, CAT, and GmPSMD plays a significant role in the response of soybean to P. sojae. Our study reveals a valuable mechanism for regulation of the pathogen response by the 26S proteasome in soybean.

Dataset Information

CDNA cloning of p112, the largest regulatory subunit of the human 26s proteasome, and functional analysis of its yeast homologue, sen3p.

Publications

CDNA cloning of p112, the largest regulatory subunit of the human 26s proteasome, and functional analysis of its yeast homologue, sen3p.

Similar Datasets

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