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.

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SUMO1/Smt3, a ubiquitin-like protein modifier, is known to conjugate to other proteins and modulate their functions in various important processes. Similar to the ubiquitin conjugation system, SUMO/Smt3 is transferred to substrate lysine residues through the thioester cascade of E1 (activating enzyme) and E2 (conjugating enzyme). In our previous report (Takahashi, Y., Toh-e, A., and Kikuchi, Y. (2001) Gene 275, 223-231), we showed that Siz1/Ull1 (YDR409w) of budding yeast, a member of the human PIAS family containing a RINGlike domain, is a strong candidate for SUMO1/Smt3 ligase because the SUMO1/Smt3 modification of septin components was abolished in the ull1 mutant and Ull1 associated with E2 (Ubc9) and the substrates (septin components) in immunoprecipitation experiments. Here we have developed an in vitro Smt3 conjugation system for a septin component (Cdc3) using purified recombinant proteins. In this system, Ull1 is additionally required as well as E1 (Sua1⅐Uba2 complex), E2 (Ubc9), and ATP. A cysteine residue of the RING-like domain was essential for the conjugation both in vivo and in vitro. Furthermore, a region containing the RING-like domain directly interacted with Ubc9 and Cdc3. Thus, this SUMO/Smt3 ligase functions as an adaptor between E2 and the target proteins.SUMO 1 (small ubiquitin-like modifier)/Smt3 is a member of a growing family of ubiquitin-related proteins and is known to be conjugated to RanGAP1, PML (promyelocytic leukemia), I B␣, p53, septins, etc. (1-4). Not only are the amino acid sequences and the three-dimensional structures similar between SUMO/ Smt3 and ubiquitin, but their conjugation systems and the enzymes involved are highly related (5-9). In the ubiquitin pathway a third enzyme, ubiquitin ligase (E3), is often required for the final transfer of this modifier and plays a crucial role by recognizing target proteins and by promoting their conjugation (10). It remains unknown, however, whether any SUMO1/Smt3 ligases (E3s) are involved in this conjugation pathway.In the ubiquitin pathway, some E3 components such as Apc11 of the anaphase-promoting complex and Rbx1 of the SCF (Skp1, cullin, F-box protein)⅐ubiquitin ligase complex contain a zinc-binding RING domain with an octet of ordered cysteine and histidine residues forming a cross-brace around two zinc atoms (11,12). This type of ubiquitin ligases (E3s) has to interact with E2 and the substrate at the same time because apparently they do not form the thioester bond with ubiquitin. In the case of c-Cbl proto-oncoprotein, its RING domain interacts with UbcH7 (E2), and the tyrosine kinase binding domain, a region close to the RING domain, recognizes its substrate. Thus, c-Cbl proto-oncoprotein functions as a bridging molecule between E2 and its substrate (13).In budding yeast, Smt3 is the only member of the SUMO family, and the Smt3 conjugation system is essential for mitotic growth. The lethality of the smt3 deletion mutant can be suppressed by expressing human SUMO1, suggesting that SUMO1 is a functional homologue of yeast Smt...

The 26S proteasome consists of the 20S proteasome (core particle) and the 19S regulatory particle made of the base and lid substructures, and it is mainly localized in the nucleus in yeast. To examine how and where this huge enzyme complex is assembled, we performed biochemical and microscopic characterization of proteasomes produced in two lid mutants, rpn5-1 and rpn7-3, and a base mutant ⌬N rpn2, of the yeast Saccharomyces cerevisiae. We found that, although lid formation was abolished in rpn5-1 mutant cells at the restrictive temperature, an apparently intact base was produced and localized in the nucleus. In contrast, in ⌬N rpn2 cells, a free lid was formed and localized in the nucleus even at the restrictive temperature. These results indicate that the modules of the 26S proteasome, namely, the core particle, base, and lid, can be formed and imported into the nucleus independently of each other. Based on these observations, we propose a model for the assembly process of the yeast 26S proteasome.

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Yeast GST1 gene, whose product is a GTP-binding protein structurally related to polypeptide chain elongation factor-1␣ (EF1␣), was first described to be essential for the G 1 to S phase transition (GSPT) of the cell cycle, and the product was recently reported to function as a polypeptide chain release factor 3 (eRF3) in yeast. Although we previously cloned a human homologue (renamed as GSPT1) of the yeast gene, it has remained to be determined whether GSPT1 also functions as eRF3 or if another GSPT may have such a function in mammalian cells. In the present study, we isolated two mouse GSPT genes, the counterpart of human GSPT1 and a novel member of the GSPT gene family, GSPT2. Both the mouse GSPTs had a two-domain structure characterized as an amino-terminal no-homologous region (approximately 200 amino acids) and a carboxyl-terminal conserved eukaryotic elongation factor-1␣-like domain (428 amino acids). Messenger RNAs of the two GSPTs could be detected in all mouse tissues surveyed, although the level of GSPT2 message appeared to be relatively abundant in the brain. The mouse GSPT1 was expressed in a proliferation-dependent manner in Swiss 3T3 cells, whereas the expression of GSPT2 was constant during the cell-cycle progression. Immunoprecipitation assays in COS-7 cells expressing flag epitope-tagged proteins demonstrated that not only GSPT1 but also GSPT2 was capable of interacting with eRF1. Such interaction between GSPT2 and eRF1 was also confirmed by yeast two-hybrid analysis. Taken together, these data indicated that the novel GSPT2 may interact with eRF1 to function as eRF3 in mammalian cells.

The SSD1 gene has been isolated as a single copy suppressor of many mutants, such as sit4, slk1/bck1, pde2, and rpc31, in the yeast Saccharomyces cerevisiae. Ssd1p has domains showing weak but significant homology with RNase II-related proteins, Cyt4p, Dss1p, VacB, and RNase II, which are involved in the modification of RNA. We found that Ssd1p had the ability to bind RNA, preferably poly(rA), as well as single-stranded DNA. Interestingly, the most conserved domain among the RNase II-related proteins was not necessary for interaction with RNA. Indirect immunofluorescence staining with anti-Ssd1p antibody revealed that Ssd1p was detected mainly in the cytoplasm. Furthermore, sucrose gradient sedimentation analysis demonstrated that Ssd1p was not cofractionated with polyribosomes, suggesting that Ssd1p is not particularly bound to a translationally active subpopulation of mRNA in the cytoplasm.Cellular RNAs do not exist as a free form but as an RNAprotein complex. The proteins that directly associate with RNA are thought to play important roles in the regulation of gene expression at the post-transcriptional level (1, 2). In eukaryotic cells, proteins that bind to RNA polymerase II transcripts include both heterogeneous nuclear RNA-binding proteins and cytoplasmic mRNA-binding proteins. Heterogeneous nuclear RNA-binding proteins bind pre-mRNAs and are associated with them during the processing events required for the formation of mature mRNA (1). Once mRNAs are transported to the cytoplasm, they form cytoplasmic mRNA-binding protein complexes (2). Cytoplasmic mRNA-binding proteins seem to regulate translation, localization, or stability of mRNA (3). At present, many RNA-binding proteins have been isolated and characterized (4, 5), but their functions have not been fully understood.In Saccharomyces cerevisiae, the SSD1 gene has been first characterized to suppress the sit4 mutation defective in a protein phosphatase subunit (6). Not only in this case, but also in many other cases, SSD1 has been isolated as a single copy suppressor of mutation defective in RPC31 encoding a subunit of RNA polymerase III (7), in PDE2 encoding the cyclic AMP phosphodiesterase (8), in BCK1 encoding mitogen-activated protein kinase kinase kinase (9), in MPK1 encoding mitogenactivated protein kinase (10), or in G 1 cyclin (11). These reports indicate that SSD1 is involved in many systems. Sutton et al.also reported that there are two alleles of the SSD1 gene; one is called ssd1-d (dead) and the other is called SSD1-V (viable). They described that SSD1-V could suppress the double mutations of ssd1-d and sit4 (6). We have also isolated the SSD1 gene as the MCS1 gene involved in stable maintenance of the minichromosome (12). The SSD1/MCS1 gene product was detected as a ϳ160-kDa protein in certain wild type strains bearing SSD1-V, such as KA31 or RAY-3A, whereas a protein of this size was not detected in another wild type strain bearing ssd1-d, such as YPH499 (7). These findings indicate that SSD1-V is simply a wild type gene and ssd1-d is a ...

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