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Metallurgical and Materials Transactions B 09/2003; 34(5):741-744. · 0.90 Impact Factor
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ABSTRACT: 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 RING-like 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 (Sua1.Uba2 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.
Journal of Biological Chemistry 01/2002; 276(52):48973-7. · 4.77 Impact Factor
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ABSTRACT: SUMO1/Smt3, a ubiquitin-like protein modifier, is known to be conjugated to other proteins and modulate their functions in various important processes. Similar to the ubiquitin system, SUMO1/Smt3 is activated in an ATP-dependent reaction by thioester bond formation with E1 (activating enzyme), transferred to E2 (conjugating enzyme), and passed to a substrate lysine. It remained unknown, however, whether any SUMO1/Smt3 ligases (E3s) are involved in the final transfer of this modifier. Here we report a novel factor Siz1 (YDR409w) required for septin-sumoylation of budding yeast, possibly acting as E3. Siz1 is a member of a new family (Miz1, PIAS3, etc.) containing a conserved domain with a similarity to a zinc-binding RING-domain, often found in ubiquitin ligases. In the siz1 mutant septin-sumoylation was completely abolished. A conserved cysteine residue in the domain was essential for this conjugation. Furthermore, Siz1 was localized at the mother-bud neck in the M-phase and physically bound to both E2 and the target proteins.
Gene 10/2001; 275(2):223-31. · 2.34 Impact Factor
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ABSTRACT: Using a two-hybrid screening with TOM1, a putative ubiquitin-ligase gene of Saccharomyces cerevisiae, we isolated KRR1, a homologue of human HRB2 (for human immunodeficiency virus type 1 Rev-binding protein 2). To characterize the gene function, we constructed temperature-sensitive krr1 mutants and isolated two multicopy suppressors. One suppressor is RPS14A, encoding a 40S ribosomal protein. The C-terminal-truncated rpS14p, which was reported to have diminished binding activity to 18S rRNA, failed to suppress the krr1 mutant. The other suppressor is a novel gene, KRI1 (for KRR1 interacting protein; YNL308c). KRI1 is essential for viability, and Kri1p is localized to the nucleolus. We constructed a galactose-dependent kri1 strain by placing KRI1 under control of the GAL1 promoter, so that expression of KRI1 was shut off when transferring the culture to glucose medium. Polysome and 40S ribosome fractions were severely decreased in the krr1 mutant and Kri1p-depleted cells. Pulse-chase analysis of newly synthesized rRNAs demonstrated that 18S rRNA is not produced in either mutant. However, wild-type levels of 25S rRNA are made in either mutant. Northern analysis revealed that the steady-state levels of 18S rRNA and 20S pre-rRNAs were reduced in both mutants. Precursors for 18S rRNA were detected but probably very unstable in both mutants. A myc-tagged Kri1p coimmunoprecipitated with a hemagglutinin-tagged Krr1p. Furthermore, the krr1 mutant protein was defective in its interaction with Kri1p. These data lead us to conclude that Krr1p physically and functionally interacts with Kri1p to form a complex which is required for 40S ribosome biogenesis in the nucleolus.
Molecular and Cellular Biology 12/2000; 20(21):7971-9. · 5.53 Impact Factor
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ABSTRACT: Yeast Smt3 is a ubiquitin-like protein similar to the mammalian SUMO-1. Cdc3, a septin component, is known to be modified by Smt3. The level of this modification was affected by Smt3-specific protease mutation ulp1-ts or overexpression of ULP1. By two-hybrid screening, we isolated 5 UIP (Ulp1 interacting protein) genes. UIP1 was identical to NUP42 encoding a component of the nuclear pore complex (NPC). Gle1, another NPC-associating component, also interacted with Ulp1 in the two-hybrid system and co-immunoprecipitation experiment. Thus Ulp1 associates with nucleoporins and may interact with septin rings in the telophase.
Journal of Biochemistry 12/2000; 128(5):723-5. · 2.37 Impact Factor
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ABSTRACT: The deletion of the TOM1 gene encoding a putative ubiquitin ligase causes a temperature sensitive cellular growth in Saccharomyces cerevisiae. The arrested cells exhibit pleiotropic defects in nuclear division, maintenance of nuclear structure and heat stress responses. We previously identified a zuo1 mutation as an extragenic suppressor of the tom1 mutant. ZUO1 encodes a DnaJ-related Hsp40. Here we show that a recessive cold sensitive mutation in PDR13 coding for an Hsp70 suppressed the tom1 mutation. The pdr13 deletion mutant was sensitive to high osmolarity, just like the zuo1 deletion mutant. A zuo1 pdr13 double deletion mutant did not show additive phenotypes. Furthermore, a tagged-Zuo1p was co-immunoprecipitated with a tagged-Pdr13p. Taken together, we propose that Pdr13p and Zuo1p are a new pair of Hsp70:Hsp40 molecular chaperones. In addition, Pdr13p co-sedimented with translating ribosomes and this association was independent of the presence of Zuo1p.
Gene 11/2000; 257(1):131-7. · 2.34 Impact Factor
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Y Watanabe,
A Haugen-Strano,
A Umar,
K Yamada,
H Hemmi, Y Kikuchi,
S Takano,
Y Shibata,
J C Barrett,
T A Kunkel,
M Koi
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ABSTRACT: The human colorectal tumor cell line LoVo has a homozygous deletion in the hMSH2 gene from exon 3 to exon 8, is deficient in mismatch repair (MMR) activity, and exhibits microsatellite instability. To determine whether the introduction of a wild type hMSH2 into LoVo restores MMR activity and stabilizes microsatellite loci, we transferred a chromosome 2 fragment containing hMSH2 into a homologous recombination-proficient chicken DT40/human hybrid (DT40 2C) and a human chromosome 2 in a mouse A9 hybrid to LoVo. Transfers of these chromosomes into LoVo resulted in LoVo both with and without a wild-type hMSH2. Complete correlation was found between the presence of the wild-type hMSH2 and hMSH2 expression, an increased stability in microsatellite loci, and competency in MMR. The hMSH2-positive LoVo hybrids also showed an increased sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine. This enhanced toxicity is associated with G(2) cell-cycle arrest followed by premature mitosis and cell death. These results suggest that hMSH2 may be responsible for complementing mutator and drug-resistant phenotypes in chromosome 2-transferred LoVo cells. To test whether the hMSH2 in DT40 2C cells can be modified by homologous recombination, we transfected DT40 2C with a targeting vector containing an hMSH2 exon 4 disrupted by the zeocin-resistant gene. The results showed that the hMSH2 locus in DT40 2C was efficiently targeted by an exogeneously transfected homologous sequence, suggesting that transfer of a modified hMSH2 from DT40 2C to LoVo via chromosome transfer could be used to determine the function of hMSH2.
Molecular Carcinogenesis 10/2000; 29(1):37-49. · 3.16 Impact Factor
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ABSTRACT: The TOM1 gene codes for a so-called HECT protein, a putative ubiquitin ligase, in Saccharomyces cerevisiae. Deletion of the entire gene (tom1-10) or the sequence encoding the HECT domain (tom1-2) causes temperature sensitivity for growth. Here we report the isolation of extragenic, recessive suppressors of tom1-2, which were designated tmr (for tom1 revertant) mutations. These were classified into eight complementation groups and six of the genes were identified: tmr1/cyr1, tmnr2/sch9, tmr3/zuo1, tmr4, tmr5/mot1, tmr6/sse1, tmr7 and tmr8/kre6. These results suggested that the tom1 phenotype can be rescued by down-regulating the cAMP/PKA pathway. It was found that the temperature sensitivity of the tom1-2 mutant is indeed suppressed by multiple copies of PDE2 or BCY1, which encode negative regulators of the cAMP/PKA pathway. The MSN2 gene, which encodes a zinc-finger transcription factor involved in the general stress response is also a multicopy suppressor of tom1. It was found that induction levels of both STRE-mediated (general stress response) and HSE-mediated gene expression (heat shock response) upon shift to high temperature are reduced by more than half in the tom1 mutant. Most of the isolated tmr mutations rescued one of the defects seen in both types of heat stress response in the tom1 mutant.
MGG - Molecular and General Genetics 02/2000; 262(6):940-8.
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ABSTRACT: SMT3 of Saccharomyces cerevisiae is an essential gene encoding a ubiquitin-like protein similar to mammalian SUMO-1. When a tagged Smt3 or human SUMO-1 was expressed from GAL1 promoter, either gene rescued the lethality of the smt3 disruptant. By indirect-immunofluorescent microscopy, the HA-tagged Smt3 was detected mostly in nuclei and also at the mother-bud neck just like septin fibers. Indeed immunoprecipitation experiments revealed that Cdc3, one of septin components, was modified with Smt3. Furthermore, the protein level of the Cdc3-Smt3 conjugate was reduced and the septin rings disappeared in a ubc9-1 mutant at a restrictive temperature, where the Smt3 conjugation system should be defective. Thus, we conclude that Smt3 was conjugated to Cdc3 in septin rings localized at the mother-bud neck. Around the time of cytokinesis the Cdc3-Smt3 conjugate disappeared. We discuss the biological significance of this Smt3 conjugation to a septin component.
Biochemical and Biophysical Research Communications 07/1999; 259(3):582-7. · 2.48 Impact Factor
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ABSTRACT: The previously identified BUL1 gene was found to encode a protein bound to Rsp5-ubiquitin ligase in budding yeast. We have identified the BUL2 gene as a functional homologue of BUL1. The bul1 bul2 double disruptant was sensitive to various stresses, such as high temperature, salts, and a non-fermentable carbon source. Each Bul protein has a putative PY-motif that has been predicted to interact with one of three WW-domains of Rsp5. A mutant Bul1 containing an altered PY-motif was defective in ability to bind to Rsp5 in the two-hybrid system and hardly co-immunoprecipitated with Rsp5. Furthermore, the mutant was not able to overcome all growth defects of the double disruptant. Thus, Bul proteins are essential for growth in various stress conditions, and their functions are mediated through the PY-motif, probably by binding to Rsp5.
Gene 01/1999; 225(1-2):39-46. · 2.34 Impact Factor
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ABSTRACT: Yeast GST1 gene, whose product is a GTP-binding protein structurally related to polypeptide chain elongation factor-1alpha (EF1alpha), was first described to be essential for the G1 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-1alpha-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.
Journal of Biological Chemistry 09/1998; 273(35):22254-9. · 4.77 Impact Factor
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ABSTRACT: Four DNA mismatch repair genes have been identified as being susceptible genes for hereditary nonpolyposis colorectal cancer. Deficiency of one of the mismatch repair genes causes the replication error phenotype in more than 80 percent of patients with hereditary nonpolyposis colorectal cancer and in 10 to 30 percent of patients with sporadic colorectal cancer. To determine which mismatch repair gene is lacking the function in patients with replication error-positive colorectal cancer, several approaches have been used at the nucleic acid and protein levels. We studied replication error in 40 samples of randomly selected colorectal cancers and expression of hMSH2 and hMLH1 proteins analyzed by immunoblot in the tumor and normal tissues of the replication error-positive and replication error-negative samples.
Frozen tumor and normal tissues were obtained from 40 Japanese patients who had colorectal cancer. According to the Amsterdam criteria, those patients were classified as having 39 sporadic and 1 unknown colorectal cancers. Genomic DNA was extracted from tumor and normal tissues for determining replication error with eight microsatellite markers. Expression of hMSH2 and hMLH1 proteins in cell lysates of tumor and normal tissues of 16 patients was analyzed by immunoblot.
The replication error phenotype was found in 6 (15 percent) of the 39 sporadic cases. hMLH1 protein was not detected in two of the six replication error-positive tumor tissues and not in the normal tissues, indicating that the tumor cells of the two patients had severe mutations in both alleles of the hMLH1 gene. Another four replication error-positive and ten replication error-negative tumors and normal tissues expressed hMLH1 protein. hMSH2 protein was detected in all samples.
hMLH1 protein was undetectable in the two tumor tissues of the six replication error-positive samples of sporadic colorectal cancer. The detection procedure used here may have potential use for determining a dysfunctional mismatch repair gene product.
Diseases of the Colon & Rectum 11/1997; 40(10 Suppl):S23-8. · 3.13 Impact Factor
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Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 11/1997; 42(14 Suppl):2240-6.
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ABSTRACT: 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.
Journal of Biological Chemistry 07/1997; 272(26):16103-9. · 4.77 Impact Factor
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Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 10/1996; 41(12 Suppl):1826-32.
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ABSTRACT: We characterized a temperature-sensitive mutant of Saccharomyces cerevisiae in which a mini-chromosome was unstable at a high temperature and cloned a new gene which encodes a basic and hydrophilic protein (110 kDa). The disruption of this gene caused the same temperature-sensitive growth as the original mutation. By using the two-hybrid system, we further isolated RSP5 (reverses Spt- phenotype), which encodes a hect (homologous to E6-AP C terminus) domain, as a gene encoding a ubiquitin ligase. Thus, we named our gene BUL1 (for a protein that binds to the ubiquitin ligase). BUL1 seems to be involved in the ubiquitination pathway, since a high dose of UBI1, encoding a ubiquitin, partially suppressed the temperature sensitivity of the bul1 disruptant as well as that of a rsp5 mutant. Coexpression of RSP5 and BUL1 on a multicopy plasmid was toxic for mitotic growth of the wild-type cells. Pulse-chase experiments revealed that Bul1 in the wild-type cells remained stable, while the bands of Bul1 in the rsp5 cells were hardly detected. Since the steady-state levels of the protein were the same in the two strains as determined by immunoblotting analysis, Bul1 might be easily degraded during immunoprecipitation in the absence of intact Rsp5. Furthermore, both Bul1 and Rsp5 appeared to be associated with large complexes which were separated through a sucrose gradient centrifugation, and Rsp5 was coimmunoprecipitated with Bul1. We discuss the possibility that Bul1 functions together with Rsp5 in protein ubiquitination.
Molecular and Cellular Biology 08/1996; 16(7):3255-63. · 5.53 Impact Factor
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ABSTRACT: A new gene (STM1; suppressor of tom1) of Saccharomyces cerevisiae was isolated by the ability to suppress the temperature sensitivity of a tom1 mutant, by increasing its gene dosage. The gene could also suppress the temperature sensitivity of the htr1 disruptant (Kikuchi et al. (1994) Mol. Gen. Genet. 245, 107-116) and was physically mapped in the region near PEP3 on chromosome XII R. The predicted gene product (29,999 Da) is basic and partially homologous to various histone H1. The level of the gene expression increased 2-fold when exposed to mating pheromone.
Biochimica et Biophysica Acta 10/1995; 1263(3):285-8. · 4.66 Impact Factor
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ABSTRACT: A new gene (SMS1; serine-rich multi-copy suppressor) of Saccharomyces cerevisiae was isolated by the ability to suppress the temperature sensitivity of the htr1 disruptant (Kikuchi et al. (1994) Mol. Gen. Genetics, in press) by increasing its gene dosage. The predicted gene product contains a serine-rich domain followed by a putative transmembrane region. The SMS1 gene was physically and genetically mapped in the region near cdc3 on chromosome XII R.
Biochimica et Biophysica Acta 02/1995; 1260(1):94-6. · 4.66 Impact Factor
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ABSTRACT: A new temperature-sensitive mutant of Saccharomyces cerevisiae was isolated. Arrested cells grown at the nonpermissive temperature were of dumb-bell shape and contained large vacuoles. A DNA fragment was cloned based on its ability to complement this temperature sensitivity. The HTR1 gene encodes a putative protein of 93 kDa without significant homology to any known proteins. The gene was mapped between ade5 and lys5 on the left arm of chromosome VII. The phenotype of the gene disruptant appeared to be strain-specific; disruption of the gene in strain W303 caused the cells to become temperature sensitive. The arrested phenotype here was similar to that of the original ts mutant and cells in G2/M phase predominated at high temperature. Another disruptant in a strain YPH background grew slowly at high temperature due to slow progression through G2/M phase, and morphologically abnormal (elongated) cells accumulated. A single-copy suppressor that alleviated the temperature-sensitive defects in both strains was identified as MCS1/SSD1. The wild-type strains W303 and YPH are known to carry defective MCS1/SSD1 alleles; hence HTR1 may function redundantly with MCS1/SSD1 to suppress the temperature-sensitive phenotypes. In addition, based on a halo bioassay, the disruptant strains appeared to be defective in recovery from, or adaptive response to G1 arrest mediated by mating pheromone, even at the permissive temperature. Thus the gene has at least two functions and is designated HTR1 (required for high temperature growth and recovery from G1 arrest induced by mating pheromone).
MGG - Molecular and General Genetics 11/1994; 245(1):107-16.
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ABSTRACT: A temperature-sensitive (ts) mutant of Saccharomyces cerevisiae was isolated in which mini-chromosomes were unstable at high temperature. The MCS1 gene (Mini-Chromosome Stability 1) was cloned by the ability of complementing the temperature sensitivity, and was found to be identical to SSD1/SRK1/SSL1. When MCS1/SSD1 was disrupted in a certain wild-type (wt) strain, mini-chromosomes were unstable, even at 30 degrees C, indicating that the gene is involved in chromosome stability. The Mcs1/Ssd1 protein was detected as a 170-kDa protein by immuno-blotting analysis and this 170-kDa protein could not be detected in the ts mutant and certain wt strains. Our results are consistent with the genetic data that there are two polymorphic forms of the gene, SSD1-v and ssd1-d [Sutton et al., Mol. Cell. Biol. 11 (1991) 2133-2148]. Furthermore, genetic backgrounds other than MCS1/SSD1 caused strain-specific phenotype. The protein, precipitated by specific antibodies, was phosphorylated.
Gene 06/1994; 143(1):135-8. · 2.34 Impact Factor
