Polyubiquitinated proteins tagged with multi-ubiquitin chains are substrates preferred by the 26 S proteasome (a ubiquitin/ATP-dependent proteolytic complex). Here, we developed a simple method for the efficient preparation of polyubiquitinated proteins which are degraded by the 26 S proteasome in an ATP-dependent manner. Our efficient method enabled us to produce ten monoclonal antibodies that recognized the multi-ubiquitin chains of the polyubiquitinated proteins, but not free ubiquitin or the protein moieties. Eight of the antibodies recognized only the multi-ubiquitin chains of the polyubiquitinated proteins, while the other two antibodies cross-reacted with mono-ubiquitin and methyl-ubiquitin, both of which are linked to proteins via an isopeptide bond, as well as with the multi-ubiquitin chains. Thus these antibodies are novel and useful tools for the identification and quantification of polyubiquitinated proteins in various cells and tissues under physiological and pathological conditions.

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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.

It is known that Lassa virus Z protein is sufficient for the release of virus-like particles (VLPs) and that it has two L domains, PTAP and PPPY, in its C terminus. However, little is known about the cellular factor for Lassa virus budding. We examined which cellular factors are used in Lassa virus Z budding. We demonstrated that Lassa Z protein efficiently produces VLPs and uses cellular factors, Vps4A, Vps4B, and Tsg101, in budding, suggesting that Lassa virus budding uses the multivesicular body pathway functionally. Our data may provide a clue to develop an effective antiviral strategy for Lassa virus.Lassa virus belongs to the family Arenaviridae, which includes Lymphocytic choriomeningitis virus (LCMV), Guanarito virus, Junin virus, and Machupo virus. Lassa virus is the etiological agent of a hemorrhagic fever, Lassa fever, that is endemic in West Africa.Arenaviruses are enveloped viruses with a bisegmented negative-strand RNA genome. Lassa virus has two genomic RNA segments designated L and S, with approximate sizes of 7.2 and 3.4 kb, respectively. Each RNA segment directs the synthesis of two proteins in opposite orientations, separated by an intergenic region. The S segment directs synthesis of the nucleoprotein and the two virion glycoproteins, GP1 and GP2, which are derived by posttranslational cleavage of a precursor polypeptide, GP-C. The oligomeric structures of GP1 and GP2 make up the spikes on the virion envelope and mediate virus interaction with the host cell surface receptor (2). The L segment codes for the virus RNA-dependent RNA polymerase (L) and a small RING finger protein (Z), which is a viral matrix protein.Recent studies have revealed that viral matrix proteins play critical roles during a late stage of virus budding in many enveloped RNA viruses, including retroviruses, rhabdoviruses, filoviruses, and orthomyxoviruses; when expressed alone in cells, they are released in the form of virus-like particles (VLPs). These viral proteins possess a so-called L domain containing PT/SAP, PPXY, and YPXL, which are critical motifs for efficient budding (6, 9, 10, 12,17,19,(26)(27)(28)31). Lassa virus Z protein is sufficient for the release of VLPs and contains PTAP and PPXY motifs near its carboxy terminus (18,24).The PTAP motif was first identified in human immunodeficiency virus (HIV) p6 gag and has been reported to bind Tsg101, which is a ubiquitin-conjugating E2 variant with a component of the vesicular protein-sorting machinery. The interaction between p6 gag and Tsg101 is required for HIV budding, and Tsg101 appears to facilitate this budding by linking the p6 late domain to the vacuolar protein-sorting (Vps) pathway (5). Recent reports have shown that targeting of Tsg101 by RNA interference causes a strong reduction in Z-mediated LCMV budding and that the Z protein is colocalized with Tsg101 (18). Another L-domain motif, PPXY, has also been determined to be the principal sequence that binds to the WW domain, consisting of 38 to 40 amino acids containing two widely spaced tryptophans. In fa...

The ubiquitin-like protein BAG-6 protects cells from newly synthesized misfolded proteins by tethering them to the proteasome.

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...

The 26S proteasome is a 2.5-MDa multisubunit protease complex that degrades polyubiquitylated proteins. Although its functions and structure have been extensively characterized, little is known about its dynamics in living cells. Here, we investigate the absolute concentration, spatio-temporal dynamics and complex formation of the proteasome in living cells using fluorescence correlation spectroscopy. We find that the 26S proteasome complex is highly mobile, and that almost all proteasome subunits throughout the cell are stably incorporated into 26S proteasomes. The interaction between 19S and 20S particles is stable even in an importin-a mutant, suggesting that the 26S proteasome is assembled in the cytoplasm. Furthermore, a genetically stabilized 26S proteasome mutant is able to enter the nucleus. These results suggest that the 26S proteasome completes its assembly process in the cytoplasm and translocates into the nucleus through the nuclear pore complex as a holoenzyme.

The gene for Tom1 was initially identified as a specific target of the oncogene v-myb. The Tom1 protein belongs to the VHS domain-containing protein family, and it has a GAT domain in a central part as well as an N-terminal VHS domain. VHS domain-containing proteins, including Hrs/Vps27, STAM, and GGA proteins, have been implicated in intracellular trafficking and sorting, but the role of Tom1 has not yet been elucidated. In this study, we found that Tom1 binds directly with ubiquitin chains and Tollip, which was initially isolated as a mediator of interleukin-1 signaling and has a capacity to bind ubiquitin chains. Gel filtration and subsequent Western blot analysis showed that endogenous Tom1 associates with Tollip to form a complex. In addition, Tom1 was found to be capable of binding to clathrin heavy chain through a typical clathrin-binding motif. Fluorescence microscopic analysis revealed that green fluorescent proteinTom1 was localized predominantly in the cytoplasm, whereas its mutant with deletion of the clathrin-binding motif had a diffuse localization throughout the cell. Thus, we propose that a Tom1-Tollip complex functions as a factor that links polyubiquitinated proteins to clathrin.