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Biomedical subjects

B Futcher

Publications and source records attributed to B Futcher.

At least 37 records · Page 2Linked to original sources
PubMed1996

Association of human fas (CD95) with a ubiquitin-conjugating enzyme (UBC-FAP).

A novel human ubiquitin conjugating enzyme (UBC) was found to associate with Fas (CD95). The mRNA for this UBC Fas-associated protein (FAP) was widely expressed in human tissues, and the protein was identified in several mammalian cell lines. UBC-FAP shows strong homology to two recently identified UBCs, Hus5 and Ubc9, which control yeast cell cycle progression. UBC-FAP, but not an active site mutant, complemented ubc9-1(ts) mutants. This suggests that UBC-FAP is a human homologue of Ubc9, possesses ubiquitin conjugating activity, and may play an important role in mammalian cell cycle regulation. A single amino acid substitution in the death domain of Fas that abolishes Fas-mediated apoptosis also abolished Fas association with UBC-FAP, suggesting that UBC-FAP may play a role in Fas signal transduction. The sequence of UBC-FAP is identical to that of HsUbc9, a UBC recently shown to interact with Rad51.

Animals↗
PubMed1996

Association of the Est1 protein with telomerase activity in yeast.

The est1 mutant was previously identified because it is defective in telomere maintenance and displays a senescent phenotype. To see if Est1 might be a component of yeast telomerase, we examined immunoprecipitated Est1. The yeast telomerase RNA Tlc1 specifically coprecipitated with Est1. Furthermore, the Est1 immunoprecipitates contained a telomerase-like activity. As expected for yeast telomerase, the activity elongated telomeric primers, it required dGTP and dTTP but not dATP or dCTP, and it was sensitive to RNase A. Further evidence suggesting that the activity was telomerase was obtained from experiments using a TLC1-1 mutant strain, which has a mutant telomerase template containing dG residues. The activity immunoprecipitated from TLC1-1 mutant strains incorporated 32P-labeled dCTP, while activity from TLC1 strains did not. Use of different telomeric primer substrates revealed two distinguishable telomerase-like activities: one was dependent on TLC1, and one was not. The TLC1-independent activity may be due to a second yeast telomerase RNA, or it may be some other kind of activity.

Amino Acid Sequence↗
PubMed1996

Synergy between trehalose and Hsp104 for thermotolerance in Saccharomyces cerevisiae.

We isolated a mutant strain unable to acquire heat shock resistance in stationary phase. Two mutations contributed to this phenotype. One mutation was at the TPS2 locus, which encodes trehalose-6-phosphate phosphatase. The mutant fails to make trehalose and accumulates trehalose-6-phosphate. The other mutation was at the HSP104 locus. Gene disruptions showed that tps2 and hsp104 null mutants each produced moderate heat shock sensitivity in stationary phase cells. The two mutations were synergistic and the double mutant had little or no stationary phase-induced heat shock resistance. The same effect was seen in the tps1 (trehalose-6-phosphate synthase) hsp104 double mutant, suggesting that the extreme heat shock sensitivity was due mainly to a lack of trehalose rather than to the presence of trehalose-6-phosphate. However, accumulation of trehalose-6-phosphate did cause some phenotypes in the tps2 mutant, such as temperature sensitivity for growth. Finally, we isolated a high copy number suppressor of the temperature sensitivity of tps2, which we call PMU1, which reduced the levels of trehalose-6-phosphate in tps2 mutants. The encoded protein has a region homologous to the active site of phosphomutases.

Amino Acid Sequence↗
PubMed1995

Role of a ubiquitin-conjugating enzyme in degradation of S- and M-phase cyclins.

Cell cycle progression in eukaryotes is controlled by the p34cdc2/CDC28 protein kinase and its short-lived, phase-specific regulatory subunits called cyclins. In Xenopus oocytes, degradation of M-phase (B-type) cyclins is required for exit from mitosis and is mediated by the ubiquitin-dependent proteolytic system. Here we show that B-type-cyclin degradation in yeast involves an essential nuclear ubiquitin-conjugating enzyme, UBC9. Repression of UBC9 synthesis prevents cell cycle progression at the G2 or early M phase, causing the accumulation of large budded cells with a single nucleus, a short spindle and replicated DNA. In ubc9 mutants both CLB5, an S-phase cyclin, and CLB2, an M-phase cyclin, are stabilized. In wild-type cells the CLB5 protein is unstable throughout the cell cycle, whereas CLB2 turnover occurs only at a specific cell-cycle stage. Thus distinct degradation signals or regulated interaction with the ubiquitin-protein ligase system may determine the cell-cycle specificity of cyclin proteolysis.

Amino Acid Sequence↗
PubMed1995

A Saccharomyces cerevisiae Internet protein resource now available.

The QUEST Protein Database Center is now making available two Saccharomyces cerevisiae protein databases via the Internet. The yeast electrophoretic protein database (YEPD) is a database of approximately one hundred protein identifications on two-dimensional gels. The yeast protein database (YPD) is a database of gene names and properties of over 3500 yeast proteins of known sequence. These databases can be accessed via a World-Wide Web (WWW) server (URL http:@siva.cshl.org). YPD is available via public ftp (isis.cshl.org) as well, in a spreadsheet format, and in ASCII format. When accessed via WWW, both of these databases have hypertext links to other biological data, such as the SWISS-PROT protein sequence database and the Saccharomyces Genome Database (SacchDB), and to each other.

Computer Communication Networks↗
PubMed1995

Specialization of B-type cyclins for mitosis or meiosis in S. cerevisiae.

The CLB1, CLB2, and CLB3 genes encode B-type cyclins important for mitosis in Saccharomyces cerevisiae, while a fourth B-type cyclin gene, CLB4, has no clear role. The effects of homozygous clb mutations on meiosis were examined. Mutants homozygous for clb1 clb3, or for clb1 clb4, gave high levels of sporulation, but produced mainly two-spored asci instead of four-spored asci. The cells had completed meiosis I but not meiosis II, producing viable diploid ascospores. CLB1 and CLB4 seem to be much more important for meiosis than for mitosis and may play some special role in meiosis II. In contrast, CLB2 is important for mitosis but not meiosis. The level of Cdc28-Clb activity may be important in determining whether meiosis II will occur.

Centromere↗
PubMed1995

p34Cdc28-mediated control of Cln3 cyclin degradation.

Cln3 cyclin of the budding yeast Saccharomyces cerevisiae is a key regulator of Start, a cell cycle event in G1 phase at which cells become committed to division. The time of Start is sensitive to Cln3 levels, which in turn depend on the balance between synthesis and rapid degradation. Here we report that the breakdown of Cln3 is ubiquitin dependent and involves the ubiquitin-conjugating enzyme Cdc34 (Ubc3). The C-terminal tail of Cln3 functions as a transferable signal for degradation. Sequences important for Cln3 degradation are spread throughout the tail and consist largely of PEST elements, which have been previously suggested to target certain proteins for rapid turnover. The Cln3 tail also appears to contain multiple phosphorylation sites, and both phosphorylation and degradation of Cln3 are deficient in a cdc28ts mutant at the nonpermissive temperature. A point mutation at Ser-468, which lies within a Cdc28 kinase consensus site, causes approximately fivefold stabilization of a Cln3-beta-galactosidase fusion protein that contains a portion of the Cln3 tail and strongly reduces the phosphorylation of this protein. These data indicate that the degradation of Cln3 involves CDC28-dependent phosphorylation events.

Amino Acid Sequence↗
PubMed1994

Inhibition of G1 cyclin activity by the Ras/cAMP pathway in yeast.

In the yeast Saccharomyces cerevisiae, commitment to cell division (Start) requires growth to a critical cell size. The G1 cyclins Cln1, Cln2 and Cln3 activate the Cdc28 protein kinase and are rate-limiting activators of Start. When glucose is added to cells growing in a poor carbon source, the critical cell size required for Start is reset from a small to a large size. In yeast, glucose acts through Ras proteins to stimulate adenylyl cyclase, activating the three cyclic AMP-dependent protein kinases Tpk1, Tpk2 and Tpk3 (refs 8, 9). We find that stimulation of the Ras/cAMP pathway represses expression of CLN1, CLN2 and co-regulated genes, inhibiting Start. This helps explain the increase in critical size when cells are shifted from poor to rich medium. This connection between the molecules controlling growth (Ras/cAMP) and those controlling division (cyclins) helps explain how division is co-ordinated with growth.

Cell Division↗
PubMed1994

Protein identifications for a Saccharomyces cerevisiae protein database.

The rapid progress in understanding the genes of the yeast Saccharomyces cerevisiae can be supplemented by two-dimensional (2-D) gel studies to understand global patterns of protein synthesis, protein modification, and protein degradation. The first step in building a protein database for yeast is to identify many of the spots on 2-D gels. We are using protein sequencing, overexpression of genes on high-copy number plasmids, and amino acid analysis to identify the proteins from 2-D gels of yeast. The amino acid analysis technique involves labeling yeast samples with different amino acids and using quantitative image analysis to determine the relative amino acid abundances. The observed amino acid abundances are then searched against the current database of 2600 known yeast protein sequences. At present about 90 proteins on our yeast maps have been identified, and the number is rising rapidly. With many known proteins on the map, it will soon be possible to use 2-D gel analysis to study regulatory pathways in normal and mutant yeast, with knowledge of many the protein products that respond to each genetic or environmental manipulation.

Amino Acid Sequence↗
PubMed1993

Mechanisms that help the yeast cell cycle clock tick: G2 cyclins transcriptionally activate G2 cyclins and repress G1 cyclins.

In budding yeast, G1 cyclins such as CLN1 and CLN2 are expressed in G1 and S phases, while mitotic cyclins such as CLB1 and CLB2 are expressed in G2 and M phases. We find that the CLBs play a central role in the transition from CLNs to CLBs: the CLBs stimulate their own expression while repressing that of CLNs. This negative regulation of CLNs may occur via the transcription factor SWI4, because CLBs are necessary for G2 repression of SCB-regulated genes like CLN1 and CLN2 but not for repression of MCB-regulated genes like DNA polymerase and CLB5. Furthermore, SW14 associates with CLB2 protein and is a substrate for the CLB2-associated CDC28 kinase in vitro.

Activity Cycles↗
PubMed1993

Comparison of the Saccharomyces cerevisiae G1 cyclins: Cln3 may be an upstream activator of Cln1, Cln2 and other cyclins.

In the budding yeast Saccharomyces cerevisiae, the G1 cyclins Cln1, Cln2 and Cln3 regulate entry into the cell cycle (Start) by activating the Cdc28 protein kinase. We find that Cln3 is a much rarer protein than Cln1 or Cln2 and has a much weaker associated histone H1 kinase activity. Unlike Cln1 and Cln2, Cln3 is not significantly cell cycle regulated, nor is it down-regulated by mating pheromone-induced G1 arrest. An artificial burst of CLN3 expression early in G1 phase accelerates Start and rapidly induces at least five other cyclin genes (CLN1, CLN2, HCS26, ORFD and CLB5) and the cell cycle-specific transcription factor SWI4. In similar experiments, CLN1 is less efficient than CLN3 at activating Start. Strikingly, expression of HCS26, ORFD and CLB5 is dependent on CLN3 in a cln1 cln2 strain, possibly explaining why CLN3 is essential in the absence of CLN1 and CLN2. To explain the potent ability of Cln3 to activate Start, despite its apparently weak biochemical activity, we propose that Cln3 may be an upstream activator of the G1 cyclins which directly catalyze Start. Given the large number of known cyclins, such cyclin cascades may be a common theme in cell cycle control.

Base Sequence↗
PubMed1993

Stress resistance of yeast cells is largely independent of cell cycle phase.

Rapidly growing cells of Saccharomyces cerevisiae are sensitive to heat shock, while non-growing stationary phase cells are highly resistant. We find that slowly growing cells have an intermediate degree of heat shock resistance that can be nearly as great as that of stationary phase cells. This resistance is correlated both with slow growth and with carbon catabolite derepression. Slowly growing cells also showed resistance to Zymolyase digestion of their cell walls. The stress resistance is a property of all the cells in the culture, and cell cycle position makes little difference to the degree of stress resistance. At least some of the properties normally associated with stationary phase cells do not require residence in stationary phase or any other particular compartment of the cell cycle. Stress resistance may be due to a diverse set of physiological adaptations available to cells regardless of their position in the cell cycle. That is, although stress resistance and stationary phase are often correlated, neither is the cause of the other.

Amino Acids↗
PubMed1993

Far1 and Fus3 link the mating pheromone signal transduction pathway to three G1-phase Cdc28 kinase complexes.

In the yeast Saccharomyces cerevisiae, the Cdc28 protein kinase controls commitment to cell division at Start, but no biologically relevant G1-phase substrates have been identified. We have studied the kinase complexes formed between Cdc28 and each of the G1 cyclins Cln1, Cln2, and Cln3. Each complex has a specific array of coprecipitated in vitro substrates. We identify one of these as Far1, a protein required for pheromone-induced arrest at Start. Treatment with alpha-factor induces a preferential association and/or phosphorylation of Far1 by the Cln1, Cln2, and Cln3 kinase complexes. This induced interaction depends upon the Fus3 protein kinase, a mitogen-activated protein kinase homolog that functions near the bottom of the alpha-factor signal transduction pathway. Thus, we trace a path through which a mitogen-activated protein kinase regulates a Cdc2 kinase.

Amino Acid Sequence↗
PubMed1992

The Cln3-Cdc28 kinase complex of S. cerevisiae is regulated by proteolysis and phosphorylation.

In Saccharomyces cerevisiae, several of the proteins involved in the Start decision have been identified; these include the Cdc28 protein kinase and three cyclin-like proteins, Cln1, Cln2 and Cln3. We find that Cln3 is a very unstable, low abundance protein. In contrast, the truncated Cln3-1 protein is stable, suggesting that the PEST-rich C-terminal third of Cln3 is necessary for rapid turnover. Cln3 associates with Cdc28 to form an active kinase complex that phosphorylates Cln3 itself and a co-precipitated substrate of 45 kDa. The cdc34-2 allele, which encodes a defective ubiquitin conjugating enzyme, dramatically increases the kinase activity associated with Cln3, but does not affect the half-life of Cln3. The Cln--Cdc28 complex is inactivated by treatment with non-specific phosphatases; prolonged incubation with ATP restores kinase activity to the dephosphorylated kinase complex. It is thus possible that phosphate residues essential for Cln-Cdc28 kinase activity are added autocatalytically. The multiple post-translational controls on Cln3 activity may help Cln3 tether division to growth.

Base Sequence↗
PubMed1992

Characterization of four B-type cyclin genes of the budding yeast Saccharomyces cerevisiae.

The previously described CLB1 and CLB2 genes encode a closely related pair of B-type cyclins. Here we present the sequences of another related pair of B-type cyclin genes, which we term CLB3 and CLB4. Although CLB1 and CLB2 mRNAs rise in abundance at the time of nuclear division, CLB3 and CLB4 are turned on earlier, rising early in S phase and declining near the end of nuclear division. When all possible single and multiple deletion mutants were constructed, some multiple mutations were lethal, whereas all single mutants were viable. All lethal combinations included the clb2 deletion, whereas the clb1 clb3 clb4 triple mutant was viable, suggesting a key role for CLB2. The inviable multiple clb mutants appeared to have a defect in mitosis. Conditional clb mutants arrested as large budded cells with a G2 DNA content but without any mitotic spindle. Electron microscopy showed that the spindle pole bodies had duplicated but not separated, and no spindle had formed. This suggests that the Clb/Cdc28 kinase may have a relatively direct role in spindle formation. The two groups of Clbs may have distinct roles in spindle formation and elongation.

Amino Acid Sequence↗
PubMed1991

Human D-type cyclin.

A cDNA library prepared from a human glioblastoma cell line has been introduced into a budding yeast strain that lacks CLN1 and CLN2 and is conditionally deficient for CLN3 function. We rescued a gene that we call cyclin D1. It is related to A-, B-, and CLN-type cyclins, but appears to define a new subclass within the cyclin gene family. Transcription of the cyclin D1 gene gives rise to two major transcripts through alternative polyadenylation. The cyclin D1 gene transcript and its 34 kd product are both abundant in the glioblastoma cell line of origin.

Amino Acid Sequence↗