Three members of the yeast N-BAR proteins family form heterogeneous lattices in vivo and interact differentially with two RabGAP proteins.

The yeast N-BAR (Bin/Amphiphysin/Rvs167) protein Rvs167 is recruited by the Rab GTPase Activating Proteins (RabGAP) Gyp5 and Gyl1 to the tip of small buds to act in exocytosis. Investigating other N-BAR proteins involved in Gyp5/Gyl1/Rvs167 complexes, we found that Rvs161, an Rvs167 paralog, is absent from the complexes formed at the tip of small buds. Immunoprecipitation and Bimolecular Fluorescence Complementation (BiFC) analysis show that both Rvs167 and Rvs161 interact in vivo with Gvp36, an N-BAR protein.

The RabGAP proteins Gyp5p and Gyl1p recruit the BAR domain protein Rvs167p for polarized exocytosis.

The Rab GTPase-activating proteins (GAP) Gyp5p and Gyl1p are involved in the control of polarized exocytosis at the small-bud stage in Saccharomyces cerevisiae. Both Gyp5p and Gyl1p interact with the N-Bin1/Amphiphysin/Rvs167 (BAR) domain protein Rvs167p, but the biological function of this interaction is unclear. We show here that Gyp5p and Gyl1p recruit Rvs167p to the small-bud tip, where it plays a role in polarized exocytosis.

Differential localization to cytoplasm, nucleus or P-bodies of yeast PKA subunits under different growth conditions.

Our aim in this work was to further characterize the complexity and specificity of the three different isoforms (Tpk1, Tpk2 and Tpk3) of the catalytic and regulatory (Bcy1) subunits of PKA in Saccharomyces cerevisiae. We thus analyzed the subcellular localization of the PKA subunits in living cells by using strains carrying GFP (green fluorescent protein) fused to each PKA subunit. During exponential growth on glucose, both Bcy1 and Tpk2 localized in the nucleus, whereas Tpk1 and Tpk3 showed a mixed pattern of nucleo-cytoplasmic localization.

H2O2 activates the nuclear localization of Msn2 and Maf1 through thioredoxins in Saccharomyces cerevisiae.

The cellular response to hydrogen peroxide (H(2)O(2)) is characterized by a repression of growth-related processes and an enhanced expression of genes important for cell defense. In budding yeast, this response requires the activation of a set of transcriptional effectors. Some of them, such as the transcriptional activator Yap1, are specific to oxidative stress, and others, such as the transcriptional activators Msn2/4 and the negative regulator Maf1, are activated by a wide spectrum of stress conditions.

Interdependence of the Ypt/RabGAP Gyp5p and Gyl1p for recruitment to the sites of polarized growth.

Gyp5p and Gyl1p are two members of the Ypt/Rab guanosine triphosphatases-activating proteins involved in the control of polarized exocytosis in Saccharomyces cerevisiae. We had previously shown that Gyp5p and Gyl1p colocalize at the sites of polarized growth and belong to the same complex in subcellular fractions enriched in plasma membrane or secretory vesicles. Here, we investigate the interaction between Gyp5p and Gyl1p as well as the mechanism of their localization to the sites of polarized growth.

High hydrostatic pressure activates gene expression through Msn2/4 stress transcription factors which are involved in the acquired tolerance by mild pressure precondition in Saccharomyces cerevisiae.

Msn2 and Msn4 transcription factors activate expression of stress-responsive element (STRE) controlled genes in response to various stresses triggering the environmental stress response in Saccharomyces cerevisiae. Although high hydrostatic pressure is known to induce gene expression modification in yeast, the transcription factors involved in this response are currently uncharacterized. In this work, we show that elevated pressure activates STRE dependent transcription through Msn2/4, which are also required for cell resistance and cell adaptation to high pressure.

Role of Gal11, a component of the RNA polymerase II mediator in stress-induced hyperphosphorylation of Msn2 in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the Msn2 transcription factor is a key element in mediating the environmental stress response (ESR), leading to the induction of 100-200 genes through the cis-acting Stress Response Element (STRE) in response to various physico-chemical stresses and nutritional variations. This activation is accompanied by a stress-induced hyperphosphorylation of Msn2. By a systematic screening we identified two proteins essential in this process: (i) the cyclin-dependent Ssn3/Srb10 protein kinase, part of a module of the RNA polymerase II mediator, which has already been shown to be involved in hyperphosphorylation and degradation of Msn2 upon stress, and (ii) Gal11, a component of the mediator.

The transcriptional activation region of Msn2p, in Saccharomyces cerevisiae, is regulated by stress but is insensitive to the cAMP signalling pathway.

Msn2p is a transcription factor that mediates a transient cellular response to multiple stresses and to changes in the nutritional environment. It was previously shown that the C-terminal half of Msn2p contains the DNA binding domain, a nuclear localization signal and nuclear export determinants which are activated by stress. In this report, we demonstrate that the N-terminal half of Msn2p contains the transcriptional activation domain(s).

Heat shock-induced degradation of Msn2p, a Saccharomyces cerevisiae transcription factor, occurs in the nucleus.

In the yeast Saccharomyces cerevisiae, the zinc finger transcription factor Msn2p is a central component of the general stress response. It is activated in response to a wide variety of environmental changes, including physicochemical stresses as well as nutritional starvation, and induces the expression of a large set of genes required for cellular adaptation. The transcriptional activity of Msn2p in response to stresses is transient, and must therefore be strictly controlled.

The control of the yeast H2O2 response by the Msn2/4 transcription factors.

We have analysed the contribution of the Msn2/4 transcription factors and the Ras-cAMP-protein kinase A (PKA) pathway to the control of the yeast H2O2 response. Strains deleted for MSN2 and MSN4 are hypersensitive to H2O2, although they can still adapt to this oxidant. They are also unable to induce 27 proteins of the H2O2 stimulon as shown by quantitative two-dimensional gel analysis.

Hyperphosphorylation of Msn2p and Msn4p in response to heat shock and the diauxic shift is inhibited by cAMP in Saccharomyces cerevisiae.

In response to various stresses, as well as during the diauxic transition, the Msn2p and Msn4p transcription factors of Saccharomyces cerevisiae are activated and induce a large set of genes. This activation is inhibited by the Ras/cAMP/PKA (cAMP-dependent protein kinase) pathway. Here we show by immunoblotting experiments that Msn2p and Msn4p are phosphorylated in vivo during growth on glucose, and become hyperphosphorylated at the diauxic transition and upon heat shock.

Selection of genes repressed by cAMP that are induced by nutritional limitation in Saccharomyces cerevisiae.

DNA-lacZ fusion libraries of yeast Saccharomyces cerevisiae were used to select genes coordinately regulated by the Ras-cAMP-cAPK signalling pathway. Sixteen new genes (AGP1, APE2, APE3, FPS1, GUT2, MDH2, PLB2, PYK2, RNR3, SUR1, UGA1, YHR033w, YBR006w, YHR143w, YMR086w and YOR173w) were found to be repressed by cAMP. Most of these genes encode for metabolic enzymes and are induced by nutritional limitations.

The heat shock response in yeast: differential regulations and contributions of the Msn2p/Msn4p and Hsf1p regulons.

The heat shock transcription factor Hsf1p and the stress-responsive transcription factors Msn2p and Msn4p are activated by heat shock in the yeast Saccharomyces cerevisiae. Their respective contributions to heat shock protein induction have been analysed by comparison of mutants and wild-type strains using [35S]-methionine labelling and two-dimensional gel electrophoresis. Among 52 proteins induced by a shift from 25 degrees C to 38 degrees C, half of them were found to be dependent upon Msn2p and/or Msn4p (including mostly antioxidants and enzymes involved in carbon metabolism), while the other half (including mostly chaperones and associated proteins) were dependent upon Hsf1p.

Msn2p and Msn4p control a large number of genes induced at the diauxic transition which are repressed by cyclic AMP in Saccharomyces cerevisiae.

The multicopy suppressors of the snf1 defect, Msn2p and Msn4p transcription factors (Msn2/4p), activate genes through the stress-responsive cis element (CCCCT) in response to various stresses. This cis element is also the target for repression by the cyclic AMP (cAMP)-signaling pathway. We analyzed the two-dimensional gel electrophoresis pattern of protein synthesis of the msn2 msn4 double mutant and compared it with that of the wild-type strain during exponential growth phase and at the diauxic transition.

The MBR1 gene from Saccharomyces cerevisiae is activated by and required for growth under sub-optimal conditions.

The MBR1 gene was isolated as a multicopy suppressor of the phenotype on glycerol medium of a Saccharomyces cerevisiae strain mutant for the Hap2/3/4/5 transactivator complex. In this paper, we show that Mbr1p is a limiting factor for growth on glycerol medium under the following sub-optimal culture conditions: in late growth phase, at low temperature, at high external pH or in the presence of 1,10-phenanthroline. Moreover, deletion of MBR1 protects cells against stress, whilst overexpression of this gene has the opposite effect.

SDC25, a dispensable Ras guanine nucleotide exchange factor of Saccharomyces cerevisiae differs from CDC25 by its regulation.

The SDC25 gene of Saccharomyces cerevisiae is homologous to CDC25. Its 3' domain encodes a guanine nucleotide exchange factor (GEF) for Ras. Nevertheless, the GEF encoded by CDC24 is determinant for the Ras/cAMP pathway activation in growth.

High cAMP levels antagonize the reprogramming of gene expression that occurs at the diauxic shift in Saccharomyces cerevisiae.

In order to analyse the involvement of the cAMP pathway in the regulation of gene expression in Saccharomyces cerevisiae, we have examined the effect of cAMP on protein synthesis by using two-dimensional gel electrophoresis. cAMP had only a minor effect on the protein pattern of cells growing exponentially on glucose. However, it interfered with the changes in gene expression normally occurring upon glucose exhaustion in yeast cultures, maintaining a protein pattern typical of cells growing on glucose.

[Yeast and the control of RAS by exchange factors].

Two isofunctional ras genes are present in the yeast Saccharomyces cerevisiae. Albeit their targets differ between mammals and yeast, they have conserved their regulators. The study of their positive regulators, guanine nucleotide exchange factors, have provided routes to the discovery of their regulatory elements in mammals.

Two subclasses of guanine exchange factor (GEF) domains revealed by comparison of activities of chimeric genes constructed from CDC25, SDC25 and BUD5 in Saccharomyces cerevisiae.

Guanine Exchange Factor (GEF) activity for Ras proteins has been associated with a conserved domain in Cdc25p, Sdc25p in Saccharomyces cerevisiae and several other proteins recently found in other eukaryotes. We have assessed the structure-function relationships between three different members of this family in S. cerevisiae, Cdc25p, Sdc25p and Bud5p.

The C-terminal part of the CDC25 gene product has Ras-nucleotide exchange activity when present in a chimeric SDC25-CDC25 protein.

The CDC25 gene from S. cerevisiae encodes an activator of Ras proteins. The C-terminal part of a structurally-related protein encoded by the SDC25 gene is characterised by a Ras-guanine nucleotide exchange activity in vitro whereas the C-terminal part of CDC25 gives no detectable exchange activity.

The CCS1 gene from Saccharomyces cerevisiae which is involved in mitochondrial functions is identified as IRA2 an attenuator of RAS1 and RAS2 gene products.

The ccs1-1 mutation of Saccharomyces cerevisiae, which has been previously described, is associated with an increase in cytochrome content, in respiration, and in ATP synthesis. In addition, this mutation leads to the same phenotype as cells de-regulated in the cAMP pathway. From a yeast genomic library, we have isolated a DNA fragment in a recombinant plasmid pCD1 which complements the ccs1-1 mutation.

Positive selection for Dictyostelium discoideum mutants lacking UMP synthase activity based on resistance to 5-fluoroorotic acid.

In the cellular slime mould Dictyostelium discoideum the two enzymatic activities of the pyrimidine pathway, orotidine-5'-phosphate decarboxylase (EC 4.1.1.

The COOH-domain of the product of the Saccharomyces cerevisiae SCD25 gene elicits activation of p21-ras proteins in mammalian cells.

The evidence presented here indicates that the domain containing the COOH-terminal part of the Saccharomyces cerevisiae SCD25 gene product (C-domain), which is homologous to the COOH-terminal part of CDC25 protein, can elicit activation of mammalian ras proteins in CHO cells. Transfection of expression vectors carrying the C-domain of SCD25, but not of CDC25, promotes the GTP-bound form of ras proteins as determined by analysis of the guanine nucleotides bound to ras proteins immunoprecipitated by Y13-259 mAb, and enhances transcription of a HIV-LTR-CAT construct. This is the first demonstration of the activation of ras proteins by transfection of a single heterologous gene.

SDC25, a CDC25-like gene which contains a RAS-activating domain and is a dispensable gene of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the CDC25 gene product activates adenylate cyclase through RAS1 and RAS2 gene products. We have recently described the cloning of a DNA fragment which suppresses the cdc25 mutation but not ras1, ras2, or cdc35 mutations. This fragment contains a 5'-truncated open reading frame which shares 47% identity with the C-terminal part of the CDC25 gene.

The C-terminal part of the CDC25 gene product plays a key role in signal transduction in the glucose-induced modulation of cAMP level in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, addition of glucose to cells grown under glucose-derepressed conditions induces a transient rise in the intracellular level of cAMP. This modulation requires functional elements of the cAMP-producing pathway, adenylate cyclase, ras proteins and the product of CDC25 gene. To determine whether or not the CDC25 gene product is a transducing element in the signal-transmission pathway leading from glucose to ras adenylate cyclase we have made use of the mutated allele RAS2Ile152 whose gene product uncouples the product of CDC25 from adenylate cyclase, but does not promotes other secondary phenotypes.