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Zhang XD Jenkins JN Callahan FE Creech RG Si Y McCarty JC Saha S Ma DP 《Biochimica et biophysica acta》2003,1625(3):269-279
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UBC1 encodes a novel member of an essential subfamily of yeast ubiquitin-conjugating enzymes involved in protein degradation 总被引:23,自引:4,他引:19
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The covalent attachment of ubiquitin to cellular proteins is catalyzed by members of a family of ubiquitin-conjugating enzymes. These enzymes participate in a variety of cellular processes, including selective protein degradation, DNA repair, cell cycle control, and sporulation. In the yeast Saccharomyces cerevisiae, two closely related ubiquitin-conjugating enzymes, UBC4 and UBC5, have recently been shown to mediate the selective degradation of short-lived and abnormal proteins. We have now identified a third distinct member of this class of ubiquitin-conjugating enzymes, UBC1. UBC1, UBC4 and UBC5 are functionally overlapping and constitute an enzyme family essential for cell growth and viability. All three mediate selective protein degradation, however, UBC1 appears to function primarily in the early stages of growth after germination of spores. ubc1 mutants generated by gene disruption display only a moderate slow growth phenotype, but are markedly impaired in growth following germination. Moreover, yeast carrying the ubc1ubc4 double mutation are viable as mitotic cells, however, these cells fail to survive after undergoing sporulation and germination. This specific requirement for UBC1 after a state of quiescence suggests that degradation of certain proteins may be crucial at this transition point in the yeast life cycle. 相似文献
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Ubiquitin-conjugating enzymes UBC4 and UBC5 mediate selective degradation of short-lived and abnormal proteins. 总被引:67,自引:15,他引:52
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Ubiquitin-conjugating enzymes catalyse the covalent attachment of ubiquitin to target proteins. Members of this enzyme family are involved in strikingly diverse cellular functions: UBC2 (RAD6) is central to DNA repair, UBC3 (CDC34) is involved in cell cycle control. We have cloned the genes for two novel ubiquitin-conjugating enzymes, UBC4 and UBC5, from the yeast Saccharomyces cerevisiae. These enzymes mediate selective degradation of short-lived and abnormal proteins. UBC4 and UBC5 are closely related in sequence and complementing in function. Expression of UBC4 and UBC5 genes is heat inducible. UBC4 and UBC5 enzymes generate high mol. wt ubiquitin-protein conjugates in vivo consistent with previous studies which suggested that attachment of multiple ubiquitin molecules to proteolytic substrates is required for their selective degradation. UBC4 and UBC5 enzymes comprise a major part of total ubiquitin-conjugation activity in stressed cells. Turnover of short-lived proteins and canavanyl-peptides but not of long-lived proteins is markedly reduced in ubc4ubc5 mutants. Loss of UBC4 and UBC5 activity impairs cell growth, leads to inviability at elevated temperatures or in the presence of an amino acid analog, and induces the stress response. 相似文献
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Ubc9 is an enzyme involved in the conjugation of small ubiquitin related modifier (SUMO) to target proteins. A Saccharomyces cerevisiae ubc9 temperature sensitive (ts) mutant showed higher sensitivity to various DNA damaging agents such as methylmethanesulfonate (MMS) and UV at a semi-permissive temperature than wild-type cells. The sensitivity of ubc9ts cells was not suppressed by the introduction of a mutated UBC9 gene, UBC9-C93S, whose product is unable to covalently bind to SUMO and consequently fails to conjugate SUMO to target proteins. Diploid ubc9ts cells were more sensitive to various DNA damaging agents than haploid ubc9ts cells suggesting the involvement of homologous recombination in the sensitivity of ubc9ts cells. The frequency of interchromosomal recombination between heteroalleles, his1-1/his1-7 loci, in wild-type cells was remarkably increased upon exposure to MMS or UV. Although the frequency of spontaneous interchromosomal recombination between the heteroalleles in ubc9ts cells was almost the same as that of wild-type cells, no induction of interchromosomal recombination was observed in ubc9ts cells upon exposure to MMS or UV. 相似文献
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The Ubc3 (Cdc34) ubiquitin-conjugating enzyme is ubiquitinated and phosphorylated in vivo. 总被引:13,自引:5,他引:8
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The transition from G1 to S phase of the cell cycle in Saccharomyces cerevisiae requires the activity of the Ubc3 (Cdc34) ubiquitin-conjugating enzyme. S. cerevisiae cells lacking a functional UBC3 (CDC34) gene are able to execute the Start function that initiates the cell cycle but fail to form a mitotic spindle or enter S phase. The Ubc3 (Cdc34) enzyme has previously been shown to catalyze the attachment of multiple ubiquitin molecules to model substrates, suggesting that the role of this enzyme in cell cycle progression depends on its targeting an endogenous protein(s) for degradation. In this report, we demonstrate that the Ubc3 (Cdc34) protein is itself a substrate for both ubiquitination and phosphorylation. Immunochemical localization of the gene product to the nucleus renders it likely that the relevant substrates similarly reside within the nucleus. 相似文献
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A fission yeast B-type cyclin functioning early in the cell cycle. 总被引:24,自引:0,他引:24
We have cloned a fission yeast gene, cig1+, encoding a 48 kd product that is most similar to cyclin B proteins. The cig1+ protein has a "cyclin box" approximately 40% identical to B-type cyclins of other species, but lacks the "destruction box" required for proteolysis of mitotic cyclins. Deletion of cig1+ had no observable effect on cell viability or progression through G2 or M phase, but instead caused a marked lag in the progression from G1 to S phase. G1 constituted approximately 70% of the cell cycle in cig1 deletion strains, as compared with less than 10% in cig1+ strains. Constitutive cig1+ overexpression was lethal, causing cessation of growth and arrest in G1. Expression of cig1+ failed to rescue an S. cerevisiae strain lacking CLN Start cyclins. Thus, cig1+ identifies a new class of B-type cyclin acting in G1 or S phase that appears to be functionally distinct from all previously described cyclin proteins. 相似文献
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Ubiquitin-dependent proteolytic systems underlie many processes, including the cell cycle, cell differentiation and responses to stress. One such system is the N-end rule pathway, which targets proteins bearing destabilizing N-terminal residues. Here we report that Ubr1p, the main recognition component of this pathway, regulates peptide import in the yeast Saccharomyces cerevisiae through degradation of Cup9p, a 35 kDa homeodomain protein. Cup9p was identified using a screen for mutants that bypass the previously observed requirement for Ubr1p in peptide import. We show that Cup9p is a short-lived protein (t1/2 approximately 5 min) whose degradation requires Ubr1p. Cup9p acts as a repressor of PTR2, a gene encoding the transmembrane peptide transporter. In contrast to engineered N-end rule substrates, which are recognized by Ubr1p through their destabilizing N-terminal residues, Cup9p is targeted by Ubr1p through an internal degradation signal. The Ubr1p-Cup9p-Ptr2p circuit is the first example of a physiological process controlled by the N-end rule pathway. An earlier study identified Cup9p as a protein required for an aspect of resistance to copper toxicity in S.cerevisiae. Thus, one physiological substrate of the N-end rule pathway functions as both a repressor of peptide import and a regulator of copper homeostasis. 相似文献
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Drosophila UbcD1 encodes a highly conserved ubiquitin-conjugating enzyme involved in selective protein degradation.
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Ubiquitin-dependent selective protein degradation serves to eliminate abnormal proteins and provides controlled short half-lives to certain cellular proteins, including proteins of regulatory function such as phytochrome, yeast MAT alpha 2 repressor, p53 and cyclin. Moreover, ubiquitin-dependent proteolysis is thought to play an essential role during development and in programmed cell death. We have cloned a gene from Drosophila melanogaster, UbcD1, coding for a protein with striking sequence similarity to the yeast ubiquitin-conjugating enzymes UBC4 and UBC5. These closely related yeast enzymes are known to be central components of a major proteolytic pathway of Saccharomyces cerevisiae. By doing a precise open reading frame replacement in the yeast genome we could show that the Drosophila UbcD1 enzyme can functionally substitute for yeast UBC4. UbcD1 driven by the UBC4 promoter rescues growth defects and temperature sensitivity of yeast ubc4 ubc5 double mutant cells. Moreover, expression of UbcD1 restores proteolysis proficiency in the ubc4 ubc5 double mutant, indicating that the Drosophila enzyme also mediates protein degradation. This structural and functional conservation suggests that the UbcD1-UBC4-UBC5 class of enzymes defines a major proteolytic pathway in probably all eukaryotes. 相似文献
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Differentiation of Trypanosoma brucei may be stage non-specific and does not require progression of cell cycle 总被引:1,自引:0,他引:1
Ubiquitination and proteasomal degradation of cell cycle regulatory proteins are known to play a pivotal role in controlling the progression of the eukaryotic cell cycle. Using the technique of RNA interference (RNAi) on the bloodstream form of Trypanosoma brucei, we were able to knock down expression of each of the 11 non-ATPase regulatory subunit proteins (Rpns) in the 19S regulatory complex of the 26S proteasome. In each case, the knock-down led to arrest of cells within the G1 and G2 phases, suggesting blockage of cell cycle progression at both G1/S and G2/M boundaries. This finding differs from that observed previously in the procyclic form of T. brucei, in which loss of individual Rpns blocks only passage across the G2/M boundary. Thus, proteasomal degradation of additional regulatory protein(s) may be required for exiting from G1 phase in the bloodstream form. In vitro differentiation of each of the 11 Rpn-depleted bloodstream form cell lines into the procyclic form was monitored. Each cell line proceeded to completion of the differentiation process like the wild-type cells with the total percentage of differentiated cells about equivalent to the sum of G1 and G2 cells. Thus, cells trapped in either G1 or G2 phase can apparently still enter and complete the process of differentiation, which is probably neither stage specific nor dependent on the progression of the T. brucei cell cycle. The process is probably a simple pattern change of gene expression in the trypanosome induced by a temperature decrease from 37 degrees C to 26 degrees C in the presence of citrate and cis-aconitate. 相似文献
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The ras-like yeast YPT1 gene is itself essential for growth, sporulation, and starvation response. 总被引:30,自引:9,他引:21
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The Saccharomyces cerevisiae gene YPT1 encodes a protein that exhibits significant homology to the mammalian ras proteins. Using gene disruption techniques, we have shown that the intact YPT1 gene is required for spore viability. Lethality caused by loss of YPT1 function, unlike that caused by loss of the yeast ras homologs RAS1 and RAS2 function, is not suppressed by the bcy1 mutation, suggesting that YPT1 does not act through the adenylate cyclase regulatory system. A cold-sensitive allele, ypt1-1, was constructed. At the nonpermissive temperature, mutants died, exhibiting aberrant nuclear morphology, as well as abnormal distribution of actin and tubulin. The mutant cells died without exhibiting classical cell-cycle-specific arrest; nevertheless, examination of cellular DNA content suggests that the YPT1 function is required, particularly after S phase. Cells carrying the ypt1-1 mutation died upon nitrogen starvation even at a temperature permissive for growth; diploid cells homozygous for ypt1-1 did not sporulate. The YPT1 gene is thus involved in nutritional regulation of the cell cycle as well as in normal progression through the mitotic cell cycle. 相似文献
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The ubiquitin proteasome system plays a pivotal role in controlling the cell cycle. The budding yeast F-box protein Dia2 is required for genomic stability and is targeted for ubiquitin-dependent degradation in a cell cycle–dependent manner, but the identity of the ubiquitination pathway is unknown. We demonstrate that the Hect domain E3 ubiquitin ligase Tom1 is required for Dia2 protein degradation. Deletion of DIA2 partially suppresses the temperature-sensitive phenotype of tom1 mutants. Tom1 is required for Dia2 ubiquitination and degradation during G1 and G2/M phases of the cell cycle, whereas the Dia2 protein is stabilized during S phase. We find that Tom1 binding to Dia2 is enhanced in G1 and reduced in S phase, suggesting a mechanism for this proteolytic switch. Tom1 recognizes specific, positively charged residues in a Dia2 degradation/NLS domain. Loss of these residues blocks Tom1-mediated turnover of Dia2 and causes a delay in G1–to–S phase progression. Deletion of DIA2 rescues a delay in the G1–to–S phase transition in the tom1Δ mutant. Together our results suggest that Tom1 targets Dia2 for degradation during the cell cycle by recognizing positively charged residues in the Dia2 degradation/NLS domain and that Dia2 protein degradation contributes to G1–to–S phase progression. 相似文献
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Combinations of different ubiquitin-conjugating (Ubc) enzymes and other factors constitute subsidiary pathways of the ubiquitin system, each of which ubiquitinates a specific subset of proteins. There is evidence that certain sequence elements or structural motifs of target proteins are degradation signals which mark them for ubiquitination by a particular branch of the ubiquitin system and for subsequent degradation. Our aim was to devise a way of searching systematically for degradation signals and to determine to which ubiquitin system subpathways they direct the proteins. We have constructed two reporter gene libraries based on the lacZ or URA3 genes which, in Saccharomyces cerevisiae, express fusion proteins with a wide variety of C-terminal extensions. From these, we have isolated clones producing unstable fusion proteins which are stabilized in various ubc mutants. Among these are 10 clones whose products are stabilized in ubc6, ubc7 or ubc6ubc7 double mutants. The C-terminal extensions of these clones, which vary in length from 16 to 50 amino acid residues, are presumed to contain degradation signals channeling proteins for degradation via the UBC6 and/or UBC7 subpathways of the ubiquitin system. Some of these C-terminal tails share similar sequence motifs, and a feature common to almost all of these sequences is a highly hydrophobic region such as is usually located inside globular proteins or inserted into membranes. 相似文献
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Control of the yeast cell cycle is associated with assembly/disassembly of the Cdc28 protein kinase complex 总被引:31,自引:0,他引:31
The Saccharomyces cerevisiae gene CDC28 encodes a protein kinase required for progression from G1 to S phase in the cell cycle. We present evidence that the active form of the Cdc28 protein kinase is a complex of approximately 160 kd containing an endogenous substrate, p40, and possibly other polypeptides. This complex phosphorylates p40 and exogenous histone H1 in vitro. Cell cycle arrest during G1 results in inactivation of the protein kinase accompanied by the disassembly of the complex. Furthermore, assembly of the complex is regulated during the cell cycle, reaching a maximum during G1. Partial complexes thought to be intermediates in the assembly process phosphorylate histone H1 but not p40. Addition of soluble factors to these partial complexes in vitro restores p40 phosphorylation and causes the complex to increase to the mature size. A model is presented in which p40 phosphorylation is required during G1 for cells to initiate a new cell cycle. 相似文献
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Characterization of a novel CDC gene (ORC1) partly homologous to CDC6 of Saccharomyces cerevisiae. 总被引:2,自引:0,他引:2
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A novel cell cycle gene was identified by a computer search for genes partly homologous to known CDC genes, CDC6 of Saccharomyces cerevisiae and CDC18 of Schizosaccharomyces pombe, using the nucleotide sequence data base for S. cerevisiae produced by the Yeast Sequencing Project. The protein sequence coded by the cloned gene was found to be identical to that of purified ORC1 protein. Disruption of the gene and subsequent tetrad analysis revealed that the gene was essential for growth. The function of the gene product was analyzed by depleting the protein from the cell using a mutant haploid strain containing the disrupted ORC1 gene on the chromosome and a galactose-inducible gene coding for HA-tagged ORC1 protein on a single copy plasmid. The HA-tagged protein was expressed during growth in the presence of galactose but began to decrease rapidly upon depletion of galactose. Analysis of the cell cycle progression of the mutant cells by FACS after the removal of galactose from the medium, and microscope observations of cells and their nuclei revealed that the normal progression of 2N cells was immediately impeded as the ORC1 protein started to decrease. This was blocked completely in the cells that had progressed to the S phase under conditions deficient in ORC1 protein followed by cell death. Two-dimensional gel analysis of the replication intermediates after the galactose removal revealed that the depletion of ORC1 protein caused a decrease in the frequency of initiation of chromosomal replication, eventually resulting in the inhibition of replication as a whole. The function of the ORC1 protein in the cell cycle progression of S. cerevisiae is discussed in light of current information on ORC. 相似文献
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The Morphogenesis Checkpoint in Saccharomyces cerevisiae: Cell Cycle Control of Swe1p Degradation by Hsl1p and Hsl7p
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John N. McMillan Mark S. Longtine Rey A. L. Sia Chandra L. Theesfeld Elaine S. G. Bardes John R. Pringle Daniel J. Lew 《Molecular and cellular biology》1999,19(10):6929-6939
In Saccharomyces cerevisiae, the Wee1 family kinase Swe1p is normally stable during G(1) and S phases but is unstable during G(2) and M phases due to ubiquitination and subsequent degradation. However, perturbations of the actin cytoskeleton lead to a stabilization and accumulation of Swe1p. This response constitutes part of a morphogenesis checkpoint that couples cell cycle progression to proper bud formation, but the basis for the regulation of Swe1p degradation by the morphogenesis checkpoint remains unknown. Previous studies have identified a protein kinase, Hsl1p, and a phylogenetically conserved protein of unknown function, Hsl7p, as putative negative regulators of Swe1p. We report here that Hsl1p and Hsl7p act in concert to target Swe1p for degradation. Both proteins are required for Swe1p degradation during the unperturbed cell cycle, and excess Hsl1p accelerates Swe1p degradation in the G(2)-M phase. Hsl1p accumulates periodically during the cell cycle and promotes the periodic phosphorylation of Hsl7p. Hsl7p can be detected in a complex with Swe1p in cell lysates, and the overexpression of Hsl7p or Hsl1p produces an effective override of the G(2) arrest imposed by the morphogenesis checkpoint. These findings suggest that Hsl1p and Hsl7p interact directly with Swe1p to promote its recognition by the ubiquitination complex, leading ultimately to its destruction. 相似文献