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.

No abstract

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.