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M Jacquet

Publications and source records attributed to M Jacquet.

At least 37 records · Page 2Linked to original sources
PubMed1998

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. Thirty-nine gene products (including those of ALD3, GDH3, GLK1, GPP2, HSP104, HXK1, PGM2, SOD2, SSA3, SSA4, TKL2, TPS1, and YBR149W) are dependent upon Msn2/4p for their induction at the diauxic transition. The expression of all these genes is repressed by cAMP. Thirty other genes identified during this study are still inducible in the mutant. A subset of these genes were found to be superinduced at the diauxic transition, and others were subject to cAMP repression (including ACH1, ADH2, ALD6, ATP2, GPD1, ICL1, and KGD2). We conclude from this analysis that Msn2/4p control a large number of genes induced at the diauxic transition but that other, as-yet-uncharacterized regulators, also contribute to this response. In addition, we show here that cAMP repression applies to both Msn2/4p-dependent and -independent control of gene expression at the diauxic shift. Furthermore, the fact that all the Msn2/4p gene targets are subject to cAMP repression suggests that these regulators could be targets for the cAMP-signaling pathway.

Cyclic AMP↗
PubMed1997

Dimerization of Cdc25p, the guanine-nucleotide exchange factor for Ras from Saccharomyces cerevisiae, and its interaction with Sdc25p.

The oligomerization state of Cdc25p, the guanine nucleotide exchange factor for ras from yeast, was analyzed using different complementary approaches. The two-hybrid system showed that the C-terminal part of Cdc25p (Cdc25-Ct) can interact with itself but also with Sdc25p-Ct, the corresponding part of Sdc25p, the other guanine exchange factor from yeast. The homotropic interaction of Cdc25p-Ct has been confirmed in yeast using immunoprecipitation experiments with epitope-tagged and beta-galactosidase-fused polypeptides. No other component was required for this interaction, since dimerization was shown to occur with material synthesized in vitro. The size of Cdc25-Ct produced in Escherichia coli has been directly measured on gel filtration columns and corresponds to a dimer. The dimerization domain is localized in the same part of the molecule as the catalytic domain and the portion responsible for membrane localization. The biological relevance of dimerization is still an open question, however by allowing heterodimerization with Sdc25p it could permit a more complex combinatorial regulation of ras in yeast.

Binding Sites↗
PubMed1997

The Ktr1p, Ktr3p, and Kre2p/Mnt1p mannosyltransferases participate in the elaboration of yeast O- and N-linked carbohydrate chains.

We have determined a role for Ktr1p and Ktr3p as mannosyltransferases in the synthesis of the carbohydrate chains attached to Saccharomyces cerevisiae O- and N-modified proteins. KTR1 and KTR3 encode related proteins that are highly similar to the Kre2p/Mnt1p Golgi alpha1,2-mannosyltransferase (Lussier, M., Camirand, A., Sdicu, A.-M., and Bussey, H. (1993) Yeast 9, 1057-1063; Mallet, L., Bussereau, F., and Jacquet, M. (1994) Yeast 10, 819-831). Examination of the electrophoretic mobility of a specifically O-linked protein from mutants and an analysis of their total O-linked mannosyl chains demonstrates that Ktr1p, Ktr3p, and Kre2p/Mnt1p have overlapping roles and collectively add most of the second and the third alpha1,2-linked mannose residues on O-linked oligosaccharides. Determination of the mobility of the specifically N-linked glycoprotein invertase in different null strains indicates that Ktr1p, Ktr3p, and Kre2p are also jointly involved in N-linked glycosylation, possibly in establishing some of the outer chain alpha1,2-linkages.

Glycosylation↗
PubMed1997

The nucleotide sequence of Saccharomyces cerevisiae chromosome XIV and its evolutionary implications.

In 1992 we started assembling an ordered library of cosmid clones from chromosome XIV of the yeast Saccharomyces cerevisiae. At that time, only 49 genes were known to be located on this chromosome and we estimated that 80% to 90% of its genes were yet to be discovered. In 1993, a team of 20 European laboratories began the systematic sequence analysis of chromosome XIV. The completed and intensively checked final sequence of 784,328 base pairs was released in April, 1996. Substantial parts had been published before or had previously been made available on request. The sequence contained 419 known or presumptive protein-coding genes, including two pseudogenes and three retrotransposons, 14 tRNA genes, and three small nuclear RNA genes. For 116 (30%) protein-coding sequences, one or more structural homologues were identified elsewhere in the yeast genome. Half of them belong to duplicated groups of 6-14 loosely linked genes, in most cases with conserved gene order and orientation (relaxed interchromosomal synteny). We have considered the possible evolutionary origins of this unexpected feature of yeast genome organization.

Base Sequence↗
PubMed1997

Yeast RAS2 mutations modulating the ras-guanine exchange factor interaction.

We have used a two-hybrid approach to test various forms of Saccharomyces cerevisiae Ras2p for their ability to interact with the human guanine nucleotide exchange factor HGRF55. We have previously shown that a strong two-hybrid interaction is found between the HGRF55p and the dominant negative Ras2p(G22A) form of ras [Camus et al. (1995) Oncogene 11, 951-959]. We show here that the substitution N123I which weakens the guanine nucleotide binding also promotes ras-GEF interaction. We demonstrate that the R80D substitution alone completely abolishes the interaction of Ras2p(G22A) with GEF, whereas substitutions at positions 81, 82 and 73 have only small effects. Since residue 73 is involved in the response of ras to GEF, we propose that it plays a role in the conformational change induced by the GEF rather than in its binding. Those results emphasize the role of the alpha2 helix of the switch II region in the recognition of the GEF family.

Alleles↗
PubMed1997

Mitogen-activated lymphocytes: a good model for characterising lung CYP1A1 inducibility.

The CYP1A1 hyperinducibility phenotype occurring in some 10% of the human population corresponds to a higher risk of developing lung cancer. This study was undertaken to assess whether the inducibility factor, generally evaluated on mitogen-activated lymphocytes after PAH induction, represents correctly the lung situation. Optimal experimental conditions were determined for evaluating, on both lymphocytes and lung tissue explants, the inducibility factor, defined as the ratio of EROD activity (CYP1A1-specific) to cytochrome c reductase activity (unaffected by PAH induction). Paired results for lymphocytes and lung tissue samples from 10 lung cancer patients were compared. A good correlation was observed between lymphocyte and lung tissue inducibilities (R = 0.809; p = 0.005). In conclusion, mitogen-activated lymphocyte inducibility is indicative of lung tissue inducibility and constitutes a good marker for evaluating individual PAH inducibilities.

Aged↗
PubMed1997

[Ras proteins in Saccharomyces cerevisiae, their partners and their activation].

Ras proteins play the role of molecular switches by conformational change between a GTP and a GDP-bound state. In the yeast Saccharomyces cerevisiae, they are encoded by two partially redundant genes RAS1 and RAS2 with a different pattern of gene expression. They are essential for growth because they are required for the activation of the adenylate cyclase and thus the protein kinase A pathway. Other possible biological functions remains to be established. To achieve their biological function, they need to be processed after their synthesis, they are modified farnesylated and palmitoylated at their C-terminal end at their CaaX box. Palmitoylation, involved in membrane localization, is not essential for growth but required for glucose signaling whereas farnesylation appears to participate in adenylate cyclase activation. In the GTP-bound state ras proteins interact through their conserved effector domain with the adenylate cyclase, the product of the CYR1/CDC35 gene. They also interact with GTPase activating proteins encoded by IRA1 and IRA2. These proteins are specific for yeast ras. It has been shown that Ira2p recognizes specific residues of yeast ras not shared by mammalian ras. The interaction with the guanine nucleotide exchange factor (GEF) of the CDC25 family is enhanced by dominant negative mutations such as RAS2ala22. Using the two hybrid approach, we have showed the key role of position 80 in Ras2p and confirmed the involvement of the a2 helix, the other switching part of ras, in this interaction and the induced effect. As a counterpart we have identified positions in HGRF55 conserved in other GEF involved in ras interaction. The triggering elements of ras activation: the GEF Cdc25p and Sdc25p are limiting components of the ras system. Cdc25p is part of a multimolecular complex associated with the membrane. We have shown that it can form homodimers and heterodimers with Sdc25p. It is an unstable protein containing a cyclin destruction box. Therefore its activity on ras could be regulated by controlling its cellular content.

Adenylyl Cyclases↗
PubMed1996

Intergenic Flip Flop, a method for systematic gene disruption and cloning in yeast.

We have developed a strategy named Intergenic Flip Flop which, for each gene, allows us to produce in one experiment both a disrupting cassette and a plasmid for gap repair. The same method can also be used to insert a reporter gene downstream from the promoter. This approach extends the polymerase chain reaction (PCR)-based strategy proposed by Maftahi et al. 1996. Our method consists of PCR amplification of the two flanking intergenic regions of the open reading frame (ORF) of interest, using two sets of oligonucleotides. Each PCR product is flanked by two short defined nucleotidic sequences with a unique restriction site, allowing subsequent hybridization between them. The association of the two amplimers by the complementary sequences either in the same orientation as in genomic DNA or in the opposite orientation, allows the generation, after PCR, of two distinct cassettes which can be cloned into suitable vectors. When the amplimer in the head-to-tail orientation is cloned in a vector containing a selective marker for yeast such as G418 resistance, it provides a disrupting cassette after cleavage at the unique restriction site introduced by the PCR between the two intergenic amplimers. The amplimer with a direct orientation cloned into a yeast vector, after cleavage at the unique restriction site between the intergenic regions, permits cloning by gap repair of the gene of interest in yeast. Finally, a reporter gene can be inserted in the same plasmid. We report here the successful application of this strategy to an ORF of chromosome XIV of Saccharomyces cerevisiae: N1216.

Cloning, Molecular↗
PubMed1996

Membrane-anchoring domains of Cdc25p, a Saccharomyces cerevisiae ras exchange factor.

The CDC25 gene product from Saccharomyces cerevisiae, the prototype of the family of ras guanine nucleotide exchange factors, is expressed as a 180-kDa polypeptide, tightly bound to a membrane fraction. The ability to complement a cdc25 defect is located in the 3' part of the gene (codons 877-1589). Sequence analysis reveals only a short hydrophobic domain (residues 1459-1471) and no consensus sequence for post-translational acylation. The SH3 domain present in the N-terminal part of Cdc25p is not involved nor required for membrane localization, since the N-terminal part of Cdc25p did not fractionate with a membrane pellet. In contrast, the C-terminal part was attached to a 18000 g pellet after subcellular fractionation and immunoblotting. This subcellular localization was conserved in a ras1 ras2 double disruption mutant and in a ira2 disruption mutant. Immunofluorescence analysis showed a patchy staining, mainly at the periphery of the cells. These patches were quite distinct from actin patches by double immunolabeling. By analysing a set of truncated derivatives, the elements required for a particulate localization were restricted to residues 1441-1552.

Cell Cycle Proteins↗
PubMed1996

Correlation between P450 CYP1A1 inducibility, MspI genotype and lung cancer incidence.

The aim of this study was to verify a possible correlation between CYP1A1 induction, MspI genotype and lung cancer incidence. A case-control study was performed on 48 lung cancer patients and 81 healthy subjects to test the existence of a correlation, within a European population. The hyperinducible group exhibited a significantly higher risk of lung cancer (odds ratio = 3.41; P = 0.036), especially for adenocarcinoma (odds ratio = 5.29; P = 0.033). In contrast with the situation observed in Asian populations, the frequency of the M2 allele did not differ significantly in the total lung cancer population (7.82%) and the group of healthy subjects (10.71%). The median inducibility value was slightly higher among cancer patients with one or two M2 alleles than among patients homozygous for the wild-type allele (P = 0.09). However, the percentage of individuals possessing at least one mutated allele was not significantly higher among hyperinducible patients (37.5%) than among non-hyperinducible patients (16.0%). No significant correlation could be found between M2 allele and lung cancer or between M2 allele and CYP1A1 inducibility; the only positive correlation found was between CYP1A1 hyperinducibility and lung cancer incidence. Our observations do not support the view that the presence of the M2 allele at the MspI site of the CYP1A1 gene constitutes a significant lung cancer risk in Caucasians.

Age Factors↗
PubMed1996

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. We demonstrate that the SDC25 gene product is a functional GEF for Ras: the complete SDC25 gene functionally replaces CDC25 when overexpressed or when transcribed under CDC25 transcriptional control at the CDC25 locus. Chimeric proteins between Sdc25p and Cdc25p are also functional GEFs for Ras. We also show that the two genes are differentially regulated: SDC25 is not transcribed at a detectable level in growth conditions when glucose is the carbon source. It is transcribed at the end of growth when nutrients are depleted and in cells grown on nonfermentable carbon sources. In contrast, CDC25 accumulation is slightly reduced when glucose is replaced by a nonfermentable carbon source.

Base Sequence↗
PubMed1995

Properties and regulation of the catalytic domain of Ira2p, a Saccharomyces cerevisiae GTPase-activating protein of Ras2p.

This work describes the biochemical characterization of the catalytic domain of Ira2p, a Saccharomyces cerevisiae GTPase-activating protein (GAP) regulating the RAS gene products. A fragment of 383 residues (amino acids 1644-2026) was produced in Escherichia coli as glutathione S-transferase fusion protein (GST-Ira2p-383) and highly purified (> 90%) by affinity chromatography. The affinity of Ras2p for the GST-fused Ira2p-383 was 18 microM and the maximal stimulation of the Ras2p GTPase activity 6,000 times. The Ira2p activity was confirmed to be strictly specific for Ras2p, no stimulatory effect on human c-H-ras p21 GTPase being detectable. Comparison with the GAP-like domain of mammalian p120-GAP and neurofibromin using yeast Ras2p as substrate showed that Ira2p-383 has an affinity and turnover intermediary between GAP-334 and NF1-414. The activity of Ira2p-383 was strongly inhibited by monovalent and divalent salts. The simultaneous presence of the catalytic domains of Ira2p and the yeast GDP/GTP exchange factor Cdc25p induced on Ras2p a multiple-round reaction of GTP hydrolysis and GDP/GTP exchange, showing that it is possible to reconstitute in vitro a S. cerevisiae system suitable for the study of the regulation of the Ras2p GDP/GTP cycle. The tubulin partially inhibited (25%) the GAP activity of the Ira2p-383. A larger Ira2p catalytic fragment, Ira2p-505 (amino acids 1549-2053), that showed the same Km for Ras2p as Ira2p-383, was also inhibited by tubulin to the same extent but with a higher affinity than Ira2p-383.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1995

A 43.5 kb segment of yeast chromosome XIV, which contains MFA2, MEP2, CAP/SRV2, NAM9, FKB1/FPR1/RBP1, MOM22 and CPT1, predicts an adenosine deaminase gene and 14 new open reading frames.

A 43,481 bp fragment from the left arm of chromosome XIV of Saccharomyces cerevisiae was sequenced. A gene for tRNA(phe) and 23 non-overlapping open reading frames (ORFs) were identified, seven of which correspond to known yeast genes: MFA2, MEP2, CAP/SRV2, NAM9, FKB1/FPR1/RBP1, MOM22 and CPT1. One ORF may correspond to the yet unidentified yeast adenosine deaminase gene. Among the 15 other ORFs, four exhibit known signatures, which include a protein tyrosine phosphatase, a cytoskeleton-associated protein and two ATP-binding proteins, four have similarities with putative proteins of yeast or proteins from other organisms and seven exibit no significant similarity with amino acid sequences described in data banks. One ORF is identical to yeast expressed sequence tags (EST) and therefore corresponds to an expressed gene. Six ORFs present similarities to human dbESTs, thus identifying motifs conserved during evolution. Nine ORFs are putative transmembrane proteins. In addition, one overlapping and three antisense ORFs, which are not likely to be functional, were detected.

Adenosine Deaminase↗
PubMed1995

Identification of guanine exchange factor key residues involved in exchange activity and Ras interaction.

We have carried out a functional analysis of the human HGRF55 exchange factor in the yeast Saccharomyces cerevisiae. Twelve residues conserved among most of all known guanine exchange factors (GEFs) have been independently changed to alanine. Taking advantage of the ability of Hgrf55p to replace the yeast Cdc25p exchange factor, and using the two-hybrid system with RAS2ala22 allele, we have identified key residues for the interaction with Ras and/or its activation. Substitution of arginine 392 to alanine leads to a complete loss of interaction with Ras, though the protein remains stable. Substitution of Asp266 or Arg359 to alanine results in inactive proteins at 39 degrees C, still able however to interact with Ras. The other charged-to-alanine substitutions led to no detectable phenotype when present alone but most of them dramatically increased the temperature sensitive phenotype observed with [Asp266Ala] substitution. Surprisingly, the cysteine to alanine substitution in the highly conserved PCVPF/Y motif proved to be without effect, suggesting that the sulfhydryl group is not essential for stability or interaction with Ras.

Alanine↗
PubMed1995

The cellular content of Cdc25p, the Ras exchange factor in Saccharomyces cerevisiae, is regulated by destabilization through a cyclin destruction box.

The Cdc25p and Sdc25p proteins were the first members of the family of guanine nucleotide exchange factors to be identified. These proteins promote the formation of active Ras-GTP complex from inactive Ras-GDP complex by exchange of GDP for GTP. Therefore Cdc25p which is the main positive regulator of Ras, regulates through Ras the activity of adenylate cyclase in Saccharomyces cerevisiae. The amino-terminal part of Cdc25p has a sequence similar to the cyclin destruction box (CDB) of mitotic cyclins. This sequence has been reported to be required for ubiquitin-dependent proteolysis. In this study we show that Cdc25p is an unstable polypeptide with a half-life of 15-20 min. Its instability depends upon the presence of the CDB which can also confer instability to other proteins. Degradation of Cdc25p and CDB containing beta-galactosidase was found to be independent of various cell cycle arrest points. The fast degradation of Cdc25p opens the possibility that Ras and the cAMP cascade in yeast are directly modulated by the cellular content of the guanine nucleotide exchange factor rather than variation in activity or localization control.

Amino Acid Sequence↗
PubMed1995

The mitochondrial receptor complex: Mom22 is essential for cell viability and directly interacts with preproteins.

A multisubunit complex in the mitochondrial outer membrane is responsible for targeting and membrane translocation of nuclear-encoded preproteins. This receptor complex contains two import receptors, a general insertion pore and the protein Mom22. It was unknown if Mom22 directly interacts with preproteins, and two views existed about the possible functions of Mom22: a central role in transfer of preproteins from both receptors to the general insertion pore or a more limited function dependent on the presence of the receptor Mom19. For this report, we identified and cloned Saccharomyces cerevisiae MOM22 and investigated whether it plays a direct role in targeting of preproteins. A preprotein accumulated at the mitochondrial outer membrane was cross-linked to Mom22. The cross-linking depended on the import stage of the preprotein. Overexpression of Mom22 suppressed the respiratory defect of yeast cells lacking Mom19 and increased preprotein import into mom19 delta mitochondria, demonstrating that Mom22 can function independently of Mom19. Overexpression of Mom22 even suppressed the lethal phenotype of a double deletion of the two import receptors known so far (mom19 delta mom72 delta). Deletion of the MOM22 gene was lethal for yeast cells, identifying Mom22 as one of the few mitochondrial membrane proteins essential for fermentative growth. These results suggest that Mom22 plays an essential role in the mitochondrial receptor complex. It directly interacts with preproteins in transit and can perform receptor-like activities.

Amino Acid Sequence↗
PubMed1995

[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. Ras are signal transducing proteins involved in the activation of the adenylate cyclase in yeast. They are activated by Cdc25p which has been shown to contain a Guanine Exchange Factor domain (GEF). SDC25, a gene partially homologous to CDC25, also contains a GEF domain but seems to be under a different regulation. It has been used to demonstrate the first guanine exchange activity on ras in vitro and was shown to be active by gene transfer in mammalian cells. Both Cdc25p and Sdc25p are associated to membrane and contain SH3 domains which are supposed to bind still unidentified proteins. Cdc25p is an unstable protein which contains a cyclin destruction box. Therefore activating effect on ras could be regulated by its level of expression. We have contributed to the isolation of a mammalian CDC25 homolog and we are analysing by directed mutagenesis key positions for ras activation of the human homolog HGRF55. That was performed by complementation analysis of yeast mutants as well as by use of two hybrid system. These approaches led us to the discovery of residues involved in ras interaction.

Animals↗