Recruitment of substrates to the 26S proteasome usually requires covalent attachment of the Lys48-linked polyubiquitin chain. In contrast, modifications with the Lys63-linked polyubiquitin chain and/or monomeric ubiquitin are generally thought to function in proteasome-independent cellular processes. Nevertheless, the ubiquitin chaintype specificity for the proteasomal targeting is still poorly understood, especially in vivo. Using mass spectrometry, we found that Rsp5, a ubiquitin-ligase in budding yeast, catalyzes the formation of Lys63-linked ubiquitin chains in vitro. Interestingly, the 26S proteasome degraded well the Lys63-linked ubiquitinated substrate in vitro. To examine whether Lys63-linked ubiquitination serves in degradation in vivo, we investigated the ubiquitination of Mga2-p120, a substrate of Rsp5. The polyubiquitinated p120 contained relatively high levels of Lys63-linkages, and the Lys63-linked chains were sufficient for the proteasome-binding and subsequent p120-processing. In addition, Lys63-linked chains as well as Lys48-linked chains were detected in the 26S proteasome-bound polyubiquitinated proteins. These results raise the possibility that Lys63-linked ubiquitin chain also serves as a targeting signal for the 26S proteaseome in vivo.

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...

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A series of chiral 2,3- and 3,4-methanoamino acid equivalents of stereochemical diversity were designed and synthesized from our chiral cyclopropane units, using a diastereoselective Grignard addition with ( R)- or ( S)- t-butanesulfinyl imines as the key step. These equivalents were converted into the proteasome inhibitor belactosin A and its cis-cyclopropane stereoisomer. The unnatural cis-isomer was shown to be more than twice as potent as belactosin A as a proteasome inhibitor.