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Biomedical subjects

Yasushi Saeki

Publications and source records attributed to Yasushi Saeki.

12 recordsLinked to original sources
PubMed2007

Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullin-based E3 ligase activity.

Although cullin-1 neddylation is crucial for the activation of SCF ubiquitin E3 ligases, the underlying mechanisms for NEDD8-mediated activation of SCF remain unclear. Here we demonstrate by NMR and mutational studies that NEDD8 binds the ubiquitin E2 (UBC4), but not NEDD8 E2 (UBC12). Our data imply that NEDD8 forms an active platform on the SCF complex for selective recruitment of ubiquitin-charged E2s in collaboration with RBX1, and thereby upregulates the E3 activity.

Carrier Proteins↗
PubMed2006

The assembly pathway of the 19S regulatory particle of the yeast 26S proteasome.

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 DeltaN 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 DeltaN 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.

Active Transport, Cell Nucleus↗
PubMed2005

Link between the ubiquitin conjugation system and the ISG15 conjugation system: ISG15 conjugation to the UbcH6 ubiquitin E2 enzyme.

ISG15 is a ubiquitin-like protein that is upregulated on treatment with interferon. ISG15 is considered to be covalently conjugated to cellular proteins through a sequential reaction similar to that of the ubiquitin conjugation system consisting of E1/E2/E3 enzymes: UBE1L and UbcH8 have been reported to function as E1 and E2 enzymes, respectively, for ISG15 conjugation. Several cellular proteins have been identified as targets for ISG15 conjugation, but the roles of ISG15 conjugation remain unclear. In this study, we found that UbcH6 and UbcH8, E2 enzymes for ubiquitin conjugation, are covalently modified by ISG15. We also found that UbcH6 is capable of forming a thioester intermediate with ISG15 through Cys131. We determined that the Lys136 residue near the catalytic site Cys131 is the ISG15 conjugation site in UbcH6. We isolated ISG15-modified and unmodified UbcH6 proteins, and analyzed their abilities to form thioester intermediates with ubiquitin. A ubiquitin thioester intermediate was detected in the case of unmodified UbcH6, but not in that of ISG15-modified UbcH6, strongly suggesting that ISG15 conjugation to UbcH6 suppresses its ubiquitin E2 enzyme activity. Thus, we provide evidence for a link between the ubiquitin conjugation system and the ISG15 conjugation system.

Cell Line↗
PubMed2004

Functional analysis of Rpn6p, a lid component of the 26 S proteasome, using temperature-sensitive rpn6 mutants of the yeast Saccharomyces cerevisiae.

Rpn6p is a component of the lid of the 26 S proteasome. We isolated and analyzed two temperature-sensitive rpn6 mutants in the yeast, Saccharomyces cerevisiae. Both mutants showed defects in protein degradation in vivo. However, the affinity-purified 26 S proteasome of the rpn6 mutants grown at the permissive temperature degraded polyubiquitinated Sic1p efficiently, even at a higher temperature. Interestingly, their enzyme activity was even higher at a higher temperature, indicating that once made mutant proteasomes are stable and have little defect in the proteolytic function. These results suggest that the deficiency in protein degradation observed in vivo is rather due to a defect in the assembly of a holoenzyme at the restrictive temperature. Indeed, both rpn6 mutants grown at the restrictive temperature were defective in assembling the 26 S proteasome. A striking feature of the rpn6 mutants at the restrictive temperature was that there appeared a protein complex composed of only four of the nine lid components, Rpn5p, Rpn8p, Rpn9p, and Rpn11p. Altogether, we conclude that Rpn6p is essential for the integrity/assembly of the lid in the sense that it is necessary for the incorporation of Rpn3p, Rpn7p, Rpn12p, and Sem1p (Rpn15p) into the lid, thereby playing an essential role in the proper function of the 26 S proteasome.

Cell Cycle Proteins↗
PubMed2004

Definitive evidence for Ufd2-catalyzed elongation of the ubiquitin chain through Lys48 linkage.

Saccharomyces cerevisiae Ufd2 is a ubiquitin chain elongation factor in the ubiquitin fusion degradation (UFD) pathway and functions in stress tolerance. A recent study has suggested that the mammalian Ufd2 homologue UFD2a catalyzes formation of Lys27- and Lys33-linked polyubiquitin chains rather than the Lys48-linked chain, but the linkage type of the polyubiquitin chain formed by yeast Ufd2 remains unclear. To determine the property of Ufd2, we reconstituted the UFD pathway using purified enzymes from yeast. Direct determination of the ubiquitin chain linkage type in polyubiquitinated UFD substrates by MALDI-TOF mass spectrometry revealed that Ufd2 catalyzes elongation of the ubiquitin chain through Lys48 linkage.

Binding Sites↗
PubMed2004

Sem1p is a novel subunit of the 26 S proteasome from Saccharomyces cerevisiae.

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 ubiquitin-dependent proteolysis.

Blotting, Western↗
PubMed2004

Rpn7 Is required for the structural integrity of the 26 S proteasome of Saccharomyces cerevisiae.

Rpn7 is one of the lid subunits of the 26 S proteasome regulatory particle. The RPN7 gene is known to be essential, but its function remains to be elucidated. To explore the function of Rpn7, we isolated and characterized temperature-sensitive rpn7 mutants. All of the rpn7 mutants obtained accumulated poly-ubiquitinated proteins when grown at the restrictive temperature. The N-end rule substrate (Ub-Arg-beta-galactosidase), the UFD pathway substrate (Ub-Pro-beta-galactosidase), and cell cycle regulators (Pds1 and Clb2) were found to be stabilized in experiments using one of the rpn7 mutants termed rpn7-3 at the restrictive temperature, indicating its defect in the ubiquitin-proteasome pathway. Subsequent analysis of the structure of the 26 S proteasome in rpn7-3 cells suggested that the defect was in the assembly of the 26 S holoenzyme. The most striking characteristic of the proteasome of the rpn7-3 mutant was that a lid subcomplex affinity-purified from the rpn7-3 cells grown at the restrictive temperature contained only 5 of the 8 lid components, a phenomenon that has not been reported in the previously isolated lid mutants. From these results, we concluded that Rpn7 is required for the integrity of the 26 S complex by establishing a correct lid structure.

Amino Acid Substitution↗
PubMed2004

Intracellularly inducible, ubiquitin hydrolase-insensitive tandem ubiquitins inhibit the 26S proteasome activity and cell division.

We constructed polyubiquitin derivatives that contain a tandem repeat of ubiquitins and were insensitive to ubiquitin hydrolases. They were designated tandem ubiquitin (tUb) with the number of repeats, such as tUb2. When tUbs were expressed under the control of the GAL1 promoter in the wild-type yeast strain, growth was strongly inhibited. Under these conditions, the degradation of N-end rule substrates, a UFD substrate and Gcn4 was inhibited, indicating that the tUb inhibits 26S proteasome activity. Consistent with this, tUb binds to the 26S proteasome. We showed that tUb inhibited the in vitro degradation of polyubiquitinylated Sic1 by the 26S proteasome. When tUB6 messenger RNA was injected into Xenopus embryos, cell division was inhibited, suggesting that tUb can be used as a versatile inhibitor of the 26S proteasome.

Cell Division↗
PubMed2003

N-Terminal modifications of the 19S regulatory particle subunits of the yeast proteasome.

The yeast (Saccharomyces cerevisiae) contains three N-acetyltransferases, NatA, NatB, and NatC, each of which acetylates proteins with different N-terminal regions. The 19S regulatory particle of the yeast 26S proteasome consists of 17 subunits, 12 of which are N-terminally modified. By using nat1, nat3, and mak3 deletion mutants, we found that 8 subunits, Rpt4, Rpt5, Rpt6, Rpn2, Rpn3, Rpn5, Rpn6, and Rpn8, were NatA substrates, and that 2 subunits, Rpt3 and Rpn11, were NatB substrates. Mass spectrometric analysis revealed that the initiator Met of Rpt2 precursor polypeptide was processed and a part of the mature Rpt2 was N-myristoylated. The crude extracts from the normal strain and the nat1 deletion mutant were similar in chymotrypsin-like activity in the presence of ATP in vitro and in the accumulation level of the 26S proteasome. These characteristics were different from those of the 20S proteasome: the chymotrypsin-like activity and accumulation level of 20S proteasome were appreciably higher from the nat1 deletion mutant than from the normal strain.

Acetylation↗
PubMed2002

Identification of ubiquitin-like protein-binding subunits of the 26S proteasome.

Ubiquitin-like proteins Rad23 and Dsk2 have recently been shown to be capable of binding both polyubiquitin chains and the 26S proteasome. The ubiquitin-like domains (Ubls) of Rad23 and Dsk2 are indispensable for their interaction with the 26S proteasome, but the proteasome subunits capable of binding the Ubl have not been identified. Here, we report that the Ubls of both Rad23 and Dsk2 can bind with the 19S regulatory particle (RP) of the 26S proteasome in vivo and in vitro. A competition assay using the respective Ubls of Rad23 and Dsk2 revealed that they bind to the RP in a competitive manner. The base subcomplex of the RP was found to have the ability to bind the Ubl. By cross-linking experiments, Rpn1 and Rpn2 were identified as Ubl-binding subunits. Taken together, the results suggest that the Rpn1 and Rpn2 in the base subcomplex form the receptor for the ubiquitin-like protein.

Binding, Competitive↗
PubMed2002

Ubiquitin-like proteins and Rpn10 play cooperative roles in ubiquitin-dependent proteolysis.

Rpn10, a subunit of the 26S proteasome, has been proposed to act as a receptor for multiubiquitin chains in ubiquitin-dependent proteolysis. However, studies on RPN10-deleted mutants in yeasts have suggested the presence of other multiubiquitin chain-binding factors functioning in ubiquitin-dependent proteolysis. Here, we report that a mutant with a triple deletion of RAD23, DSK2, and RPN10 genes accumulates large amounts of polyubiquitinated proteins, as is the case with a mutant with RAD23 and DSK2 deletions under restrictive conditions. Dsk2, Rad23, and Rpn10 have different capacities to bind multiubiquitin chains. Another ubiquitin-like protein, Ddi1, has similar activity to those of Rad23 and Dsk2. Taken together, the results suggest that ubiquitin-like proteins, Rad23, Dsk2, possibly Ddi1, and Rpn10 play cooperative roles in ubiquitin-dependent proteolysis, serving as multiubiquitin chain-binding proteins.

Carrier Proteins↗