Sem1p Is a Novel Subunit of the 26 S Proteasome from Saccharomyces cerevisiae

Abstract

The 26 S proteasome, which catalyzes degradation of polyubiquitinated proteins, is composed of the 20 S proteasome and the 19 S regulatory particle (RP). The RP is composed of the lid and base subcomplexes and regulates the catalytic activity of the 20 S proteasome. In this study, we carried out affinity purification of the lid and base subcomplexes from the tagged strains of Saccharomyces cerevisiae, and we found that the lid contains a small molecular mass protein, Sem1. The Sem1 protein binds with the 26 S proteasome isolated from a mutant with deletion of SEM1 but not with the 26 S proteasome from the wild type. The lid lacking Sem1 is unstable at a high salt concentration. The 19 S RP was immunoprecipitated together with Sem1 by immunoprecipitation using hemagglutinin epitope-tagged Sem1 as bait. Degradation of polyubiquitinated proteins in vivo or in vitro is impaired in the Sem1-deficient 26 S proteasome. In addition, genetic interaction between SEM1 and RPN10 was detected. The human Sem1 homologue hDSS1 was found to be a functional homologue of Sem1 and capable of interacting with the human 26 S proteasome. The results suggest that Sem1, possibly hDSS1, is a novel subunit of the 26 S proteasome and plays a role in ubiquitindependent proteolysis.In eukaryotic cells, the ubiquitin-proteasome system regulates various cellular processes (1-3). In this pathway, target proteins are polyubiquitinated by E1 1 /E2/E3 enzymes, and the thus formed polyubiquitin chain is recognized by the 26 S proteasome, and the protein portion is degraded in an ATP-dependent manner. The 26 S proteasome is composed of a core particle (CP, also known as the 20 S proteasome), which contains proteolytic active sites in its cavity, and the 19 S regulatory particle (RP), which regulates the catalytic activity of the CP (2-4). The CP, built of two copies each of seven distinct ␣ and seven distinct ␤ subunits as four stacked rings (␣ 1-7 ␤ 1-7 ␤ 1-7 ␣ 1-7 ), has three catalytic activities, namely chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities (2, 4). The RP consists of at least 17 distinct subunits (3, 5) and is composed of lid and base subcomplexes. The lid subcomplex consists of eight non-ATPase subunits (Rpn3, Rpn5 to Rpn9, Rpn11, and Rpn12), whereas the base subcomplex consists of six ATPase subunits (Rpt1 to Rpt6) and two non-ATPase subunits (Rpn1 and Rpn2) (6, 7). Recently, additional proteasome-associated proteins have been identified and characterized (8, 9). It has been reported that Rpn13 is a new subunit of the RP and that a mutant with deletion of RPN13 shows a defect in the ubiquitin fusion degradation (UFD) pathway (8).With the exception of RPN9, RPN10, and RPN13 genes, all of the regulatory subunit genes are essential. Rpn10 binds the polyubiquitin chain both in its free form and when incorporated into the 26 S proteasome (10 -13). Rpn10 positions at the interface between the lid and base and strengthens the lid-base interaction (6). The base is thought to promote translocation of subst...

DSS1 is required for the stability of BRCA2

“…Characterization of the endogenous DSS1 protein Previous studies have shown that endogenous human and mouse DSS1 cannot be detected by Western blotting, although both recombinant and yeast DSS1 are detectable by this method (Jantti et al, 1999;Marston et al, 1999;Wei et al, 2003;Gudmundsdottir et al, 2004;Isono et al, 2004;Sone et al, 2004). To detect endogenous human DSS1, we generated an anti-DSS1 antiserum against a full-length DSS1 GST-fusion protein.…”

“…It is also possible that binding of DSS1 to BRCA2 is required to prevent the exposure of a protein-degradation signal. Notably, the potential role of DSS1 as a promoter of BRCA2 protein stability stands in apparent contradiction to the presence of DSS1 as a component of the lid subcomplex of the 26S proteasome (Isono et al, 2004;Sone et al, 2004). It is likely that the function of DSS1 to stabilize BRCA2 molecules is independent of its role in proteasome function.…”

“…In addition, the yeast ortholog of DSS1, sem1p, regulates exocytosis and pseudohyphal differentiation (Jantti et al, 1999). Recently, DSS1 (sem1p/RPN7) has been found to form a structural subunit of the 26S proteasome and is required for the structural integrity of the 26S proteasome (Isono et al, 2004;Sone et al, 2004). DSS1 has also been identified as a 12-O-tetradecanoylphorbol-13-acetate-responsive gene expressed in keratinocyte progenitor cells that may be involved in early skin tumorigenesis (Wei et al, 2003).…”

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

“…The RP can be biochemically further divided into two substructures, the base and the lid. The base consists of six AAA-ATPase subunits, regulatory particle triple-A ATPase (Rpt)1p-Rpt6p, and three non-ATPase subunits, regulatory particle non-ATPase (Rpn)1p, Rpn2p, and Rpn13p, whereas the lid is made of nine non-ATPases, Rpn3p, Rpn5p-Rpn9p, Rpn11p, Rpn12p, and Sem1p (Rpn15p) (Glickman et al, 1998b;Leggett et al, 2002;Funakoshi et al, 2004;Sone et al, 2004). Rpn10p, a non-ATPase subunit that binds polyubiquitin (Ub) chains (van Nocker et al, 1996;Saeki et al, 2002;Elsasser et al, 2004), has been suggested to exist in the interface between the base and the lid (Fu et al, 2001).…”

Sem1

“…16,17 Genetic screens and proteomic approaches identified Sem1 as a component of the lid sub-complex of the regulatory particle (RP) of the proteasome in both budding yeast and humans. [18][19][20][21] Loss of Sem1 impairs the functional integrity of the RP. 18 Consistent with this, sem1∆ mutants show impaired ubiquitin-dependent protein degradation and accumulate polyubiquitinated proteins.…”