Biomedical subjects

C De Virgilio

Publications and source records attributed to C De Virgilio.

At least 19 recordsLinked to original sources

PubMed2006

Cell growth control: little eukaryotes make big contributions.

The story of rapamycin is a pharmaceutical fairytale. Discovered as an antifungal activity in a soil sample collected on Easter Island, this macrocyclic lactone and its derivatives are now billion dollar drugs, used in, and being evaluated for, a number of clinical applications. Taking advantage of its antifungal property, the molecular Target Of Rapamycin, TOR, was first described in the budding yeast Saccharomyces cerevisiae. TORs encode large, Ser/Thr protein kinases that reside in two distinct, structurally and functionally conserved, multi-protein complexes. In yeast, these complexes coordinate many different aspects of cell growth. TOR complex 1, TORC1, promotes protein synthesis and other anabolic processes, while inhibiting macroautophagy and other catabolic and stress-response processes. TORC2 primarily regulates cell polarity, although additional readouts of this complex are beginning to be characterized. TORC1 appears to be activated by nutrient cues and inhibited by stresses and rapamycin; however, detailed mechanisms are not known. In contrast, TORC2 is insensitive to rapamycin and physiological regulators of this complex have yet to be defined. Given the unsurpassed resources available to yeast researchers, this simple eukaryote continues to contribute to our understanding of eukaryotic cell growth in general and TOR function in particular.

Animals↗

PubMed2002

Saccharomyces cerevisiae Ccr4-not complex contributes to the control of Msn2p-dependent transcription by the Ras/cAMP pathway.

The Ccr4-Not complex is a global regulator of transcription that affects genes positively and negatively and is thought to modulate the activity of TFIID. In the present work, we provide evidence that the Ccr4-Not complex may contribute to transcriptional regulation by the Ras/cAMP pathway. Several observations support this model. First, Msn2/4p-dependent transcription, which is known to be under negative control of cAMP-dependent protein kinase (PKA), is derepressed in all ccr4-not mutants. This phenotype is paralleled by specific post-translational modification defects of Msn2p in ccr4-not mutants relative to wild-type cells. Secondly, mutations in various NOT genes result in a synthetic temperature-sensitive growth defect when combined with mutations that compromise cells for PKA activity and at least partially suppress the effects of both a dominant-active RAS2Val-19 allele and loss of Rim15p. Thirdly, Not3p and Not5p, which are modified and subsequently degraded by stress signals that also lead to increased Msn2/4p-dependent activity, show a specific two-hybrid interaction with Tpk2p. Together, our results suggest that the Ccr4-Not complex may function as an effector of the Ras/cAMP pathway that contributes to repress basal, stress- and starvation-induced transcription by Msn2/4p.

Culture Media↗

PubMed2001

Bud8p and Bud9p, proteins that may mark the sites for bipolar budding in yeast.

The bipolar budding pattern of a/alpha Saccharomyces cerevisiae cells appears to depend on persistent spatial markers in the cell cortex at the two poles of the cell. Previous analysis of mutants with specific defects in bipolar budding identified BUD8 and BUD9 as potentially encoding components of the markers at the poles distal and proximal to the birth scar, respectively. Further genetic analysis reported here supports this hypothesis. Mutants deleted for BUD8 or BUD9 grow normally but bud exclusively from the proximal and distal poles, respectively, and the double-mutant phenotype suggests that the bipolar budding pathway has been totally disabled. Moreover, overexpression of these genes can cause either an increased bias for budding at the distal (BUD8) or proximal (BUD9) pole or a randomization of bud position, depending on the level of expression. The structures and localizations of Bud8p and Bud9p are also consistent with their postulated roles as cortical markers. Both proteins appear to be integral membrane proteins of the plasma membrane, and they have very similar overall structures, with long N-terminal domains that are both N- and O-glycosylated followed by a pair of putative transmembrane domains surrounding a short hydrophilic domain that is presumably cytoplasmic. The putative transmembrane and cytoplasmic domains of the two proteins are very similar in sequence. When Bud8p and Bud9p were localized by immunofluorescence and tagging with GFP, each protein was found predominantly in the expected location, with Bud8p at presumptive bud sites, bud tips, and the distal poles of daughter cells and Bud9p at the necks of large-budded cells and the proximal poles of daughter cells. Bud8p localized approximately normally in several mutants in which daughter cells are competent to form their first buds at the distal pole, but it was not detected in a bni1 mutant, in which such distal-pole budding is lost. Surprisingly, Bud8p localization to the presumptive bud site and bud tip also depends on actin but is independent of the septins.

Actins↗

PubMed2000

Saccharomyces cerevisiae Ras/cAMP pathway controls post-diauxic shift element-dependent transcription through the zinc finger protein Gis1.

The Saccharomyces cerevisiae protein kinase Rim15 was identified previously as a component of the Ras/cAMP pathway acting immediately downstream of cAMP-dependent protein kinase (cAPK) to control a broad range of adaptations in response to nutrient limitation. Here, we show that the zinc finger protein Gis1 acts as a dosage-dependent suppressor of the rim15Delta defect in nutrient limitation-induced transcriptional derepression of SSA3. Loss of Gis1 results in a defect in transcriptional derepression upon nutrient limitation of various genes that are negatively regulated by the Ras/cAMP pathway (e.g. SSA3, HSP12 and HSP26). Tests of epistasis as well as transcriptional analyses of Gis1-dependent expression indicate that Gis1 acts in this pathway downstream of Rim15 to mediate transcription from the previously identified post-diauxic shift (PDS) element. Accordingly, deletion of GIS1 partially suppresses, and overexpression of GIS1 exacerbates the growth defect of mutant cells that are compromised for cAPK activity. Moreover, PDS element-driven expression, which is negatively regulated by the Ras/cAMP pathway and which is induced upon nutrient limitation, is almost entirely dependent on the presence of Gis1.

Bacterial Proteins↗

PubMed2000

The riboflavin/FAD cycle in rat liver mitochondria.

Here we provide evidence that mitochondria isolated from rat liver can synthesize FAD from riboflavin that has been taken up and from endogenous ATP. Riboflavin uptake takes place via a carrier-mediated process, as shown by the inverse relationship between fold accumulation and riboflavin concentration, the saturation kinetics [riboflavin Km and Vmax values were 4.4+/-1.3 microM and 35+/-5 pmol x min(-1) (mg protein)(-1), respectively] and the inhibition shown by the thiol reagent mersalyl, which cannot enter the mitochondria. FAD synthesis is due to the existence of FAD synthetase (EC 2.7.7.2), localized in the matrix, which has as a substrate pair mitochondrial ATP and FMN synthesized from taken up riboflavin via the putative mitochondrial riboflavin kinase. In the light of certain features, including the protein thermal stability and molecular mass, mitochondrial FAD synthetase differs from the cytosolic isoenzyme. Apparent Km and apparent Vmax values for FMN were 5.4+/-0.9 microM and 22.9+/-1.4 pmol x min(-1) x (mg matrix protein)(-1), respectively. Newly synthesized FAD inside the mitochondria can be exported from the mitochondria in a manner sensitive to atractyloside but insensitive to mersalyl. The occurrence of the riboflavin/FAD cycle is proposed to account for riboflavin uptake in mitochondria biogenesis and riboflavin recovery in mitochondrial flavoprotein degradation; both are prerequisites for the synthesis of mitochondrial flavin cofactors.

Animals↗

PubMed1999

The thermophilic yeast Hansenula polymorpha does not require trehalose synthesis for growth at high temperatures but does for normal acquisition of thermotolerance.

The TPS1 gene from Hansenula polymorpha, which encodes trehalose-6-phosphate (Tre6P) synthase, has been isolated and characterized. The deletion of TPS1 rendered H. polymorpha cells incapable of trehalose synthesis under conditions where wild-type cells normally accumulate high levels of trehalose. Interestingly, the loss of Tre6P synthase did not cause any obvious growth defects on a glucose-containing medium, even at high temperatures, but seriously compromised the cells' ability to acquire thermotolerance.

Gene Deletion↗

PubMed1998

Composition and functional analysis of the Saccharomyces cerevisiae trehalose synthase complex.

In the yeast Saccharomyces cerevisiae, trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), which convert glucose 6-phosphate plus UDP-glucose to trehalose, are part of the trehalose synthase complex. In addition to the TPS1 (previously also called GGS1, CIF1, BYP1, FDP1, GLC6, and TSS1) and TPS2 (also described as HOG2 and PFK3) gene products, this complex also contains a regulatory subunit encoded by TSL1. We have constructed a set of isogenic strains carrying all possible combinations of deletions of these three genes and of TPS3, a homologue of TSL1 identified by systematic sequencing. Deletion of TPS1 totally abolished TPS activity and measurable trehalose, whereas deletion of any of the other genes in most cases reduced both. Similarly, deletion of TPS2 completely abolished TPP activity, and deletion of any of the other genes resulted in a reduction of this activity. Therefore, it appears that all subunits are required for optimal enzymatic activity. Since we observed measurable trehalose in strains lacking all but the TPS1 gene, some phosphatase activity in addition to Tps2 can hydrolyze trehalose 6-phosphate. Deletion of TPS3, in particular in a tsl1Delta background, reduced both TPS and TPP activities and trehalose content. Deletion of TPS2, TSL1, or TPS3 and, in particular, of TSL1 plus TPS3 destabilized the trehalose synthase complex. We conclude that Tps3 is a fourth subunit of the complex with functions partially redundant to those of Tsl1. Among the four genes studied, TPS1 is necessary and sufficient for growth on glucose and fructose. Even when overproduced, none of the other subunits could take over this function of Tps1 despite the homology shared by all four proteins. A portion of Tps1 appears to occur in a form not bound by the complex. Whereas TPS activity in the complex is inhibited by Pi, Pi stimulates the monomeric form of Tps1. We discuss the possible role of differentially regulated Tps1 in a complex-bound or monomeric form in light of the requirement of Tps1 for trehalose production and for growth on glucose and fructose.

Blotting, Western↗

PubMed1998

Expression of a functional barley sucrose-fructan 6-fructosyltransferase in the methylotrophic yeast Pichia pastoris.

The cDNA encoding sucrose-fructan 6-fructosyltransferase (6-SFT) from barley (Hordeum vulgare) has been expressed in the methylotrophic yeast Pichia pastoris, using a translational fusion into vector pPICZ alphaC, containing the N-terminal signal sequence of Saccharomyces cerevisiae alpha-factor to allow entry into the secretory pathway. Transformed Pichia produced and secreted a functional 6-SFT which had characteristics similar to the barley enzyme, but had a pronounced additional 1-SST activity when incubated with sucrose.

5' Untranslated Regions↗

PubMed1998

Saccharomyces cerevisiae cAMP-dependent protein kinase controls entry into stationary phase through the Rim15p protein kinase.

The Saccharomyces cerevisiae protein kinase Rim15p was identified previously as a stimulator of meiotic gene expression. Here, we show that loss of Rim15p causes an additional pleiotropic phenotype in cells grown to stationary phase on rich medium; this phenotype includes defects in trehalose and glycogen accumulation, in transcriptional derepression of HSP12, HSP26, and SSA3, in induction of thermotolerance and starvation resistance, and in proper G1 arrest. These phenotypes are commonly associated with hyperactivity of the Ras/cAMP pathway. Tests of epistasis suggest that Rim15p may act in this pathway downstream of the cAMP-dependent protein kinase (cAPK). Accordingly, deletion of RIM15 suppresses the growth defect of a temperature-sensitive adenylate-cyclase mutant and, most importantly, renders cells independent of cAPK activity. Conversely, overexpression of RIM15 suppresses phenotypes associated with a mutation in the regulatory subunit of cAPK, exacerbates the growth defect of strains compromised for cAPK activity, and partially induces a starvation response in logarithmically growing wild-type cells. Biochemical analyses reveal that cAPK-mediated in vitro phosphorylation of Rim15p strongly inhibits its kinase activity. Taken together, these results place Rim15p immediately downstream and under negative control of cAPK and define a positive regulatory role of Rim15p for entry into both meiosis and stationary phase.

Base Sequence↗

PubMed1998

Saccharomyces cerevisiae mitochondria can synthesise FMN and FAD from externally added riboflavin and export them to the extramitochondrial phase.

Evidence is given that mitochondria isolated from Saccharomyces cerevisiae can take up externally added riboflavin and synthesise from it both flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) probably due to the existence of the mitochondrial riboflavin kinase already reported and the novel mitochondria FAD synthetase. Moreover Saccharomyces cerevisiae mitochondria can export the newly synthesised flavin derivatives to the extramitochondrial phase. This has been proven to take place with 1:1 stoichiometry with riboflavin decrease outside mitochondria, thus showing that flavin traffic occurs across the mitochondrial membranes.

Flavin Mononucleotide↗

PubMed1997

Flavin adenine dinucleotide and flavin mononucleotide metabolism in rat liver--the occurrence of FAD pyrophosphatase and FMN phosphohydrolase in isolated mitochondria.

In order to gain some insight into mitochondrial flavin biochemistry, rat liver mitochondria essentially free of lysosomal and microsomal contamination were prepared and their capability to metabolise externally added and endogenous FAD and FMN tested both spectroscopically and via HPLC. The existence of two novel mitochondrial enzymes, namely FAD pyrophosphatase (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2), which catalyse FAD-->FMN and FMN-->riboflavin conversion, respectively, is shown. They differ from each other and from extramitochondrial enzymes, as judged by their pH profile and inhibitor sensitivity, and can be separated in a partial FAD pyrophosphatase purification. Digitonin titration and subfractionation experiments show that FAD pyrophosphatase is located in the outer mitochondrial membrane and FMN phosphohydrolase in the intermembrane space. Since these enzymes can metabolise endogenous FAD and FMN, which are made available by using both Triton X-100 and the effector oxaloacetate, a proposal is made that FAD pyrophosphatase and FMN phosphohydrolase play a major role in mitochondrial flavoprotein turnover.

Acid Phosphatase↗

PubMed1997

A septin-based hierarchy of proteins required for localized deposition of chitin in the Saccharomyces cerevisiae cell wall.

Just before bud emergence, a Saccharomyces cerevisiae cell forms a ring of chitin in its cell wall; this ring remains at the base of the bud as the bud grows and ultimately forms part of the bud scar marking the division site on the mother cell. The chitin ring seems to be formed largely or entirely by chitin synthase III, one of the three known chitin synthases in S. cerevisiae. The chitin ring does not form normally in temperature-sensitive mutants defective in any of four septins, a family of proteins that are constituents of the "neck filaments" that lie immediately subjacent to the plasma membrane in the mother-bud neck. In addition, a synthetic-lethal interaction was found between cdc12-5, a temperature-sensitive septin mutation, and a mutant allele of CHS4, which encodes an activator of chitin synthase III. Two-hybrid analysis revealed no direct interaction between the septins and Chs4p but identified a novel gene, BNI4, whose product interacts both with Chs4p and Cdc10p and with one of the septins, Cdc10p; this analysis also revealed an interaction between Chs4p and Chs3p, the catalytic subunit of chitin synthase III. Bni4p has no known homologues; it contains a predicted coiled-coil domain, but no other recognizable motifs. Deletion of BNI4 is not lethal, but causes delocalization of chitin deposition and aberrant cellular morphology. Overexpression of Bni4p also causes delocalization of chitin deposition and produces a cellular morphology similar to that of septin mutants. Immunolocalization experiments show that Bni4p localizes to a ring at the mother-bud neck that lies predominantly on the mother-cell side (corresponding to the predominant site of chitin deposition). This localization depends on the septins but not on Chs4p or Chs3p. A GFP-Chs4p fusion protein also localizes to a ring at the mother-bud neck on the mother-cell side. This localization is dependent on the septins, Bni4p, and Chs3p. Chs3p, whose normal localization is similar to that of Chs4p, does not localize properly in bni4, chs4, or septin mutant strains or in strains that accumulate excess Bni4p. In contrast, localization of the septins is essentially normal in bni4, chs4, and chs3 mutant strains and in strains that accumulate excess Bni4p. Taken together, these results suggest that the normal localization of chitin synthase III activity is achieved by assembly of a complex in which Chs3p is linked to the septins via Chs4p and Bni4p.

Amino Acid Sequence↗

PubMed1997

Structural analysis of the subunits of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae and their function during heat shock.

Synthesis of trehalose in the yeast Saccharomyces cerevisiae is catalysed by the trehalose-6-phosphate (Tre6P) synthase/phosphatase complex, which is composed of at least three different subunits encoded by the genes TPS1, TPS2, and TSL1. Previous studies indicated that Tps1 and Tps2 carry the catalytic activities of trehalose synthesis, namely Tre6P synthase (Tps1) and Tre6P phosphatase (Tps2), while TsI1 was suggested to have regulatory functions. In this study two different approaches have been used to clarify the molecular composition of the trehalose synthase complex as well as the functional role of its potential subunits. Two-hybrid analyses of the in vivo interactions of Tps1, Tps2, TsI1, and Tps3, a protein with high homology to TsI1, revealed that both TsI1 and Tps3 can interact with Tps1 and Tps2; the latter two proteins also interact with each other. In addition, trehalose metabolism upon heat shock was analysed in a set of 16 isogenic yeast strains carrying deletions of TPS1, TPS2, TSL1, and TPS3 in all possible combinations. These results not only confirm the previously suggested roles for Tps1 and Tps2, but also provide, for the first time, evidence that TsI1 and Tps3 may share a common function with respect to regulation and/or structural stabilization of the Tre6P synthase/phosphatase complex in exponentially growing, heat-shocked cells.

Bacterial Proteins↗

PubMed1997

Trehalose synthesis is important for the acquisition of thermotolerance in Schizosaccharomyces pombe.

Yeast cells show an adaptive response to a mild heat shock, resulting in thermotolerance acquisition. This is accompanied by induction of heat-shock protein (hsp) synthesis and rapid accumulation of trehalose. Genetic approaches to determine the specific role of trehalose in heat-induced thermotolerance in Saccharomyces cerevisiae have been hampered by the finding that deletion of TPS1, the gene encoding trehalose-6-phosphate synthase, causes a variety of pleiotropic effects, including inability to grow on glucose-containing media. Here, we have studied a tps1 mutant of the yeast Schizosaccharomyces pombe that reportedly has no such growth defects. We show that tps1 mutants have a serious defect in heat shock-induced acquisition of thermotolerance if conditioned at highly elevated temperatures (40-42.5 degrees C), which, in wild-type cells, prevent hsp but not trehalose synthesis. In contrast, hsp synthesis appears to become particularly important under conditions in which trehalose synthesis is either absent (in tps1 mutant strains) or not fully induced (conditioning at moderately elevated temperatures, i.e. 35 degrees C). In addition, pka1 mutants deficient in cAMP-dependent protein kinase were examined. Unconditioned pka1 cells had low levels of trehalose but a high basal level of thermotolerance. It was found that pka1 mutant cells, contrary to wild-type cells, accumulated large amounts of trehalose, even during a 50 degrees C treatment. pka1 tps1 double mutants lacked this ability and showed reduced intrinsic thermotolerance, indicating a particularly important role for trehalose synthesis, which takes place during the challenging heat shock.

Adaptation, Physiological↗

PubMed1996

The septins: roles in cytokinesis and other processes.

The septins are a novel family of proteins that were first recognized in yeast as proteins associated with the neck filaments. Recent work has shown that septins are also present in other fungi, insects, and vertebrates. Despite the apparent differences in modes of cytokinesis amongst species, septins appear to be essential for this process in both fungal and animal cells. The septins also appear to be involved in various other aspects of the organization of the cell surface.

Cell Division↗

PubMed1996

SPR28, a sixth member of the septin gene family in Saccharomyces cerevisiae that is expressed specifically in sporulating cells.

The septins are a recently recognized family of proteins that are present in a wide variety of fungal and animal cells, where they are involved in cytokinesis and apparently in other processes involving the organization of the cell surface. Five previously described Saccharomyces cerevisiae septins are associated with the neck filaments of vegetative cells and/or with the developing prospore wall of sporulating cells. We report here the characterization of SPR28, a sixth member of the S. cerevisiae septin gene family whose existence was revealed by the yeast genome project. Analysis of mRNA levels showed that SPR28 is a new member of the group of "late genes' that are expressed at high levels during the meiotic divisions and ascospore formation. The septin it encodes, Spr28p, exhibited specific two-hybrid interactions with itself and with three other septins that are expressed in sporulating cells. Consistent with these results, an Spr28p-green fluorescent protein fusion was induced during meiosis I and appeared to be associated with the developing prospore walls. Deletion of SPR28 in either a wild-type or an spr3 delta background produced no obvious abnormalities in vegetative cells and had little or no effect on sporulation, suggesting that the septins have redundant roles during spore formation.

Amino Acid Sequence↗

PubMed1995

Mutation of RGA1, which encodes a putative GTPase-activating protein for the polarity-establishment protein Cdc42p, activates the pheromone-response pathway in the yeast Saccharomyces cerevisiae.

We have selected yeast mutants that exhibit a constitutively active pheromone-response pathway in the absence of the beta subunit of the trimeric G protein. Genetic analysis of one such mutant revealed that it contained recessive mutations in two distinct genes, both of which contributed to the constitutive phenotype. One mutation identifies the RGA1 locus (Rho GTPase activating protein), which encodes a protein with homology to GAP domains and to LIM domains. Deletion of RGA1 is sufficient to activate the pathway in strains lacking the G beta subunit. Moreover, in wild-type strains, deletion of RGA1 increases signaling in the pheromone pathway, whereas over-expression of RGA1 dampens signaling, demonstrating that Rga1p functions as a negative regulator of the pheromone response pathway. The second mutation present in the original mutant proved to be an allele of a known gene, PBS2, which encodes a putative protein kinase that functions in the high osmolarity stress pathway. The pbs2 mutation enhanced the rga1 mutant phenotype, but by itself did not activate the pheromone pathway. Genetic and two-hybrid analyses indicate that an important target of Rga1p is Cdc42p, a p21 GTPase required for polarity establishment and bud emergence. This finding coupled with recent experiments with mammalian and yeast cells indicating that Cdc42p can interact with and activate Ste20p, a protein kinase that operates in the pheromone pathway, leads us to suggest that Rga1p controls the activity of Cdc42p, which in turn controls the magnitude of signaling in the pheromone pathway via Ste20p.

Amino Acid Sequence↗

PubMed1995

Role for the Rho-family GTPase Cdc42 in yeast mating-pheromone signal pathway.

In the budding yeast Saccharomyces cerevisiae, the process of conjugation of haploid cells of genotype MATa and MAT alpha to form MATa/alpha diploids is triggered by pheromones produced by each mating type. These pheromones stimulate a cellular response by interaction with receptors linked to a heterotrimeric G protein. Although genetic analysis indicates that the pheromone signal is transmitted through the G beta gamma dimer, the initial target(s) of G protein activation remain to be determined. Temperature-sensitive cells with mutations of the CDC24 and CDC42 genes, which are incapable of budding and of generating cell polarity at the restrictive temperature, are also unable to mate. Cdc24 acts as a guanylyl-nucleotide-exchange factor for the Rho-type GTPase Cdc42, which has been shown to be a fundamental component of the molecular machinery controlling morphogenesis in eukaryotic cells. Therefore, the inability of cdc24 and cdc42 mutants to mate has been presumed to be due to a requirement for generation of cell polarity and related morphogenetic events during conjugation. But here we show that Cdc42 has a direct signalling role in the mating-pheromone response between the G protein and the downstream protein kinase cascade.

Cell Cycle Proteins↗