Laura Frontali | Università degli Studi "La Sapienza" di Roma (original) (raw)

Papers by Laura Frontali

Research paper thumbnail of Role of Hog1 and Yaf9 in the transcriptional response of Saccharomyces cerevisiae to cesium chloride

Physiological Genomics, 2008

We analysed the global transcriptional response of S. cerevisiae cells exposed to different conce... more We analysed the global transcriptional response of S. cerevisiae cells exposed to different concentrations of CsCl in the growth medium and at different times from addition. Early responsive genes were mainly involved in cell wall structure and biosynthesis. About half of the induced genes were previously shown to respond to other alkali metal cations in a Hog1-dependent fashion.

Research paper thumbnail of Transformation of the yeast Kluyveromyces lactis by new vectors derived from the 1.6 μm circular plasmid pKD1

The circular plasmid pKD1 (or 1.6 gm DNA) has recently been isolated from Kluyveromyces drosophil... more The circular plasmid pKD1 (or 1.6 gm DNA) has recently been isolated from Kluyveromyces drosophilarum. This plasmid appears to have a functional organization analogous to that of the 2 g DNA of Saccharomyces cerevisiae, although the respective nucleotide sequences show little homology, pKD1 can be transferred toKluyverornyces lactis where it is replicated stably. Using recombinant molecules derived from pKD1, a practical transformation system has been developed for Kluyveromyces lactis, with an efficiency and stability comparable to the 2#-based Saccharomyces cerevisiae transformation system.

Research paper thumbnail of The yeast model suggests the use of short peptides derived from mt LeuRS for the therapy of diseases due to mutations in several mt tRNAs

We have previously established a yeast model of mitochondrial (mt) diseases. We showed that defec... more We have previously established a yeast model of mitochondrial (mt) diseases. We showed that defective respiratory phenotypes due to point-mutations in mt tRNA Leu(UUR) , tRNA Ile and tRNA Val could be relieved by overexpression of both cognate and non-cognate nuclearly encoded mt aminoacyl-tRNA synthetases (aaRS) LeuRS, IleRS and ValRS. More recently, we showed that the isolated carboxy-terminal domain (Cterm) of yeast mt LeuRS, and even short peptides derived from the human Cterm, have the same suppressing abilities as the whole enzymes. In this work, we extend these results by investigating the activity of a number of mt aaRS from either class I or II towards a panel of mt tRNAs. The Cterm of both human and yeast mt LeuRS has the same spectrum of activity as mt aaRS belonging to class I and subclass a, which is the most extensive among the whole enzymes. Yeast Cterm is demonstrated to be endowed with mt targeting activity. Importantly, peptide fragments β30_31 and β32_33, derived from the human Cterm, have even higher efficiency as well as wider spectrum of activity, thus opening new avenues for therapeutic intervention. Bind-shifting experiments show that the β30_31 peptide directly interacts with human mt tRNA Leu(UUR) and tRNA Ile , suggesting that the rescuing activity of isolated peptide fragments is mediated by a chaperone-like mechanism. Wide-range suppression appears to be idiosyncratic of LeuRS and its fragments, since it is not shared by Cterminal regions derived from human mt IleRS or ValRS, which are expected to have very different structures and interactions with tRNAs.

Research paper thumbnail of Disruption of six novel genes from chromosome VII ofSaccharomyces cerevisiae reveals one essential gene and one gene which affects the growth rate

Yeast, 2000

Six ORFs of unknown function located on chromosome VII of Saccharomyces cerevisiae were disrupted... more Six ORFs of unknown function located on chromosome VII of Saccharomyces cerevisiae were disrupted in two different genetic backgrounds, and the phenotype of the generated mutants was analysed. Disruptions of ORFs YGR256w, YGR272c, YGR273c, YGR275w and YGR276c were carried out using the disruption marker kanMX4 anked by short homology regions, whereas ORF YGR255c was inactivated with a long¯anking homology (LFH) disruption cassette . Tetrad analysis of the heterozygous disruptants revealed that ORF YGR255c, previously identi®ed as COQ6 and encoding a protein involved in the biosynthesis of coenzime Q (Tzagoloff and Dieckmann, 1990), is an essential gene. The same analysis also revealed that sporulation of the ygr272cD heterozygous diploid produced two small colonies per ascus that were also G418-resistant, indicating that the inactivation of ORF YGR272c could result in a slower growth rate. This result was con®rmed by growth tests of the haploid disruptants and by complementation of the phenotype after transformation with a plasmid carrying the cognate gene. No phenotypes could be associated to the inactivation of ORFs YGR256w, YGR273c, YGR275w and YGR276c. Two of these genes have recently been further characterized: ORF YGR255w, renamed RTT102, encodes a regulator of the Ty1-element transposition, whereas ORF YGR276c was found to encode the 70 kDa RNase H activity and was renamed RNH70 (Frank et al., 1999). a Calculated as the nucleotide position of each ORF in reference to the complete sequence of chromosome VII .

Research paper thumbnail of Regulation of the expression of theKluyveromyces lactis PDC1 gene: carbon source-responsive elements and autoregulation

Yeast, 1999

The yeast Kluyveromyces lactis has a single structural gene coding for pyruvate decarboxylase (Kl... more The yeast Kluyveromyces lactis has a single structural gene coding for pyruvate decarboxylase (KlPDC1). In order to study the regulation of the expression of KlPDC1, we have sequenced (EMBL Accession No. Y15435) its promoter and have fused the promoter to the reporter gene lacZ from E. coli. Transcription analysis in a Klpdc1 strain showed that KlPDC1 expression is subject to autoregulation. The PDC1 gene from Saccharomyces cerevisiae was able to complement the Rag phenotype of the Klpdc1 mutant strain and it could also repress transcription of the KlPDC1-lacZ fusion on glucose. A deletion analysis of the promoter region was performed to study carbon source-dependent regulation and revealed that at least two cis-acting regions are necessary for full induction of gene expression on glucose. Other cis-elements mediate repression on ethanol.

Research paper thumbnail of Molecular characterization of a plasmid from Pseudomonas fluorescens involved in styrene degradation

Plasmid, 1984

In this paper evidence is given that in a strain of Pseudomonas jluorescens able to grow on styre... more In this paper evidence is given that in a strain of Pseudomonas jluorescens able to grow on styrene as the sole carbon source, the degradation pathway of styrene is inducible and plasmid dependent. The plasmid, which we have called PEG is self-transmissible between Pseudomonas strains and has a size of 37 kb. A restriction map has been constructed and evidence for an inducible transcription of two separate regions of the plasmid has been obtained. io 1981 Academic Press. Inc.

Research paper thumbnail of Action of Rifamycin on Incorporation of Amino-acids into Protein in Cell-free Systems from B. subtilis

Research paper thumbnail of The complete DNA sequence of yeast chromosome III

Nature, 1992

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined... more The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map

Research paper thumbnail of Effect of Rifamycin on Protein Synthesis

Research paper thumbnail of Heterogeneity of mitochondrial DNA from Saccharomyces cerevisiae and genetic information for tRNA

Nucleic Acids Research, 1975

Mitochondrial DNA from wild-type Saccharomyces cerevisiae and from an "extreme" petite mutant wer... more Mitochondrial DNA from wild-type Saccharomyces cerevisiae and from an "extreme" petite mutant were analyzed by hybridization of several tRNAs on DNA fragments of different buoyant den sity, obtained by sonication and fractionation on a CsCl gradient. The hybridization patterns show that the genes for tRNAser, tRNAphe, tRNAhisv tRNAvalstRNAileu are present on wild-type mitochondrial DNA, while only genes for tRNAser and tRNAhis are present on petite mitochondrial DNA; moreover the hybridization patterns indicate that these genes are not clustered and suggest that more than one gene might exist for tRNAser and tRNAhis.

Research paper thumbnail of Dissection of the Carboxyl-Terminal Domain of the Proteasomal Subunit Rpn11 in Maintenance of Mitochondrial Structure and Function

Molecular Biology of the Cell, 2007

We have previously demonstrated that the C-terminal part of Rpn11, a deubiquitinating enzyme in t... more We have previously demonstrated that the C-terminal part of Rpn11, a deubiquitinating enzyme in the lid of the proteasome, is essential for maintaining a correct cell cycle and normal mitochondrial morphology and function. The two roles are apparently unlinked as the mitochondrial role is mapped to the Carboxy-terminus, whereas the catalytic deubiquitinating activity is found within the N-terminal region. The mitochondrial defects are observed in rpn11-m1 (originally termed mpr1-1), a mutation that generates Rpn11 lacking the last 31 amino acids. No mitochondrial phenotypes are recorded for mutations in the MPN؉/JAMM motif. In the present study, we investigated the participation of the last 31 amino acids of the Rpn11 protein by analysis of intragenic revertants and site-specific mutants. We identified a putative ␣-helix necessary for the maintenance of a correct cell cycle and determined that a very short region at the C-terminus of Rpn11 is essential for the maintenance of tubular mitochondrial morphology. Furthermore, we show that expression of the C-terminal part of Rpn11 is able to complement in trans all of the rpn11-m1 mitochondrial phenotypes. Finally, we investigate the mechanisms by which Rpn11 controls the mitochondrial shape and show that Rpn11 may regulate the mitochondrial fission and tubulation processes.

Research paper thumbnail of A Saccharomyces cerevisiae gene essential for viability has been conserved in evolution

Gene, 1995

To identify the gene coding for the endonuclease which processes the 3' end of mitochondrial (mt)... more To identify the gene coding for the endonuclease which processes the 3' end of mitochondrial (mt) tRNA transcripts in Saccharomyces cerevisiae, nuclear mutations able to complement a mt mutant (Ts932) defective for this process were isolated and analyzed. One of these mutants exhibited a growth defect both on respiratory and fermentable media. Complementation of this phenotype with a S. cerevisiae centromeric wild-type genomic library has allowed us to identify a new essential S. cerevisiae gene strongly conserved in various eukaryotic organisms.

Research paper thumbnail of Cesium chloride sensing and signaling in Saccharomyces cerevisiae : an interplay among the HOG and CWI MAPK pathways and the transcription factor Yaf9

FEMS Yeast Research, 2009

In yeast, many environmental stimuli are sensed and signaled by the MAP kinases pathways. In a pr... more In yeast, many environmental stimuli are sensed and signaled by the MAP kinases pathways. In a previous work, we showed that cesium chloride activates the HOG pathway and modulates the transcription of several genes, especially those involved in cell wall biosynthesis and organization. The response to cesium was largely overlapping with the response to salt and osmotic stress. However, when low cesium chloride concentrations were used, a specific response was eventually elicited. The cesium-specific response involved the Yaf9 protein and its activity of chromatin remodeling and transcription regulation. In this paper we show that the osmotic activity of cesium salt is detected and signaled by the two branches downstream of the Sln1 and Sho1 sensors of the HOG pathway, that seem to possess different but exchangeables functions in cesium signaling. However, the cesium-specific response mediated by Yaf9, that counteracts the efficiency of the HOG pathway, is not routed by these sensors. In addition, the cesium response also involves the cell wall integrity (CWI) pathway, which is activated by low concentration of cesium chloride. Mutations blocking the CWI pathway show sensitivity to this salt.

Research paper thumbnail of Regulation of primary carbon metabolism in Kluyveromyces lactis

Enzyme and Microbial Technology, 2000

In the recent past, through advances in development of genetic tools, the budding yeast Kluyverom... more In the recent past, through advances in development of genetic tools, the budding yeast Kluyveromyces lactis has become a model system for studies on molecular physiology of so-called "Nonconventional Yeasts." The regulation of primary carbon metabolism in K. lactis differs markedly from Saccharomyces cerevisiae and reflects the dominance of respiration over fermentation typical for the majority of yeasts. The absence of aerobic ethanol formation in this class of yeasts represents a major advantage for the "cell factory" concept and large-scale production of heterologous proteins in K. lactis cells is being applied successfully. First insight into the molecular basis for the different regulatory strategies is beginning to emerge from comparative studies on S. cerevisiae and K. lactis. The absence of glucose repression of respiration, a high capacity of respiratory enzymes and a tight regulation of glucose uptake in K. lactis are key factors determining physiological differences to S. cerevisiae. A striking discrepancy exists between the conservation of regulatory factors and the lack of evidence for their functional significance in K. lactis. On the other hand, structurally conserved factors were identified in K. lactis in a new regulatory context. It seems that different physiological responses result from modified interactions of similar molecular modules.

Research paper thumbnail of Participation of the proteasomal lid subunit Rpn11 in mitochondrial morphology and function is mapped to a distinct C-terminal domain

Biochemical Journal, 2004

Substrates destined for degradation by the 26 S proteasome are labelled with polyubiquitin chains... more Substrates destined for degradation by the 26 S proteasome are labelled with polyubiquitin chains. Rpn11/Mpr1, situated in the lid subcomplex, partakes in the processing of these chains or in their removal from substrates bound to the proteasome. Rpn11 also plays a role in maintaining mitochondrial integrity, tubular structure and proper function. The recent finding that Rpn11 participates in proteasome-associated deubiquitination focuses interest on the MPN+ (Mpr1, Pad1, N-terminal)/JAMM (JAB1/MPN/Mov34) metalloprotease site in its N-terminal domain. However, Rpn11 damaged at its C-terminus (the mpr1-1 mutant) causes pleiotropic effects, including proteasome instability and mitochondrial morphology defects, resulting in both proteolysis and respiratory malfunctions. We find that overexpression of WT (wild-type) RPN8, encoding a paralogous subunit that does not contain the catalytic MPN+ motif, corrects proteasome conformations and rescues cell cycle phenotypes, but is unable to correct defects in the mitochondrial tubular system or respiratory malfunctions associated with the mpr1-1 mutation. Transforming mpr1-1 with various RPN8-RPN11 chimaeras or with other rpn11 mutants reveals that a WT C-terminal region of Rpn11 is necessary, and more surprisingly sufficient, to rescue the mpr1-1 mitochondrial phenotype. Interestingly, single-site mutants in the catalytic MPN+ motif at the N-terminus of Rpn11 lead to reduced proteasome-dependent deubiquitination connected with proteolysis defects. Nevertheless, these rpn11 mutants suppress the mitochondrial phenotypes associated with mpr1-1 by intragene complementation. Together, these results point to a unique role for the C-terminal region of Rpn11 in mitochondrial maintenance that may be independent of its role in proteasome-associated deubiquitination.

Research paper thumbnail of How to bring orphan genes into functional families

Yeast, 1999

In the framework of the B1 Consortium of the EUROFAN-1 project, we set up a series of simple phen... more In the framework of the B1 Consortium of the EUROFAN-1 project, we set up a series of simple phenotypic tests that can be performed on a large number of strains at a time. This methodological approach was intended to help assign functions of putative genes coding for unknown proteins to several specific aspects of cell biology. The tests were chosen to study phenotypes which should be affected by numerous genes. In this report, we examined the sensitivity/resistance or the adaptation of the cell to physical or chemical stresses (thermotolerance, osmotolerance and ethanol sensitivity), the effects of the alteration of the level of protein phosphorylation (sensitivity or resistance to compounds affecting the activity of protein kinases or phosphatases) and the effects of compounds interfering with synthesis of nucleic acids or proteins. Deletions in 66 genes of unknown function have been tested in 21 different conditions. In many deletant strains, phenotypes were observed and, for the most promising candidates, tetrad analysis was performed in order to verify co-segregation of the deletion marker with the phenotype.

Research paper thumbnail of Role of Hog1 and Yaf9 in the transcriptional response of Saccharomyces cerevisiae to cesium chloride

Physiological Genomics, 2008

We analysed the global transcriptional response of S. cerevisiae cells exposed to different conce... more We analysed the global transcriptional response of S. cerevisiae cells exposed to different concentrations of CsCl in the growth medium and at different times from addition. Early responsive genes were mainly involved in cell wall structure and biosynthesis. About half of the induced genes were previously shown to respond to other alkali metal cations in a Hog1-dependent fashion.

Research paper thumbnail of Transformation of the yeast Kluyveromyces lactis by new vectors derived from the 1.6 μm circular plasmid pKD1

The circular plasmid pKD1 (or 1.6 gm DNA) has recently been isolated from Kluyveromyces drosophil... more The circular plasmid pKD1 (or 1.6 gm DNA) has recently been isolated from Kluyveromyces drosophilarum. This plasmid appears to have a functional organization analogous to that of the 2 g DNA of Saccharomyces cerevisiae, although the respective nucleotide sequences show little homology, pKD1 can be transferred toKluyverornyces lactis where it is replicated stably. Using recombinant molecules derived from pKD1, a practical transformation system has been developed for Kluyveromyces lactis, with an efficiency and stability comparable to the 2#-based Saccharomyces cerevisiae transformation system.

Research paper thumbnail of The yeast model suggests the use of short peptides derived from mt LeuRS for the therapy of diseases due to mutations in several mt tRNAs

We have previously established a yeast model of mitochondrial (mt) diseases. We showed that defec... more We have previously established a yeast model of mitochondrial (mt) diseases. We showed that defective respiratory phenotypes due to point-mutations in mt tRNA Leu(UUR) , tRNA Ile and tRNA Val could be relieved by overexpression of both cognate and non-cognate nuclearly encoded mt aminoacyl-tRNA synthetases (aaRS) LeuRS, IleRS and ValRS. More recently, we showed that the isolated carboxy-terminal domain (Cterm) of yeast mt LeuRS, and even short peptides derived from the human Cterm, have the same suppressing abilities as the whole enzymes. In this work, we extend these results by investigating the activity of a number of mt aaRS from either class I or II towards a panel of mt tRNAs. The Cterm of both human and yeast mt LeuRS has the same spectrum of activity as mt aaRS belonging to class I and subclass a, which is the most extensive among the whole enzymes. Yeast Cterm is demonstrated to be endowed with mt targeting activity. Importantly, peptide fragments β30_31 and β32_33, derived from the human Cterm, have even higher efficiency as well as wider spectrum of activity, thus opening new avenues for therapeutic intervention. Bind-shifting experiments show that the β30_31 peptide directly interacts with human mt tRNA Leu(UUR) and tRNA Ile , suggesting that the rescuing activity of isolated peptide fragments is mediated by a chaperone-like mechanism. Wide-range suppression appears to be idiosyncratic of LeuRS and its fragments, since it is not shared by Cterminal regions derived from human mt IleRS or ValRS, which are expected to have very different structures and interactions with tRNAs.

Research paper thumbnail of Disruption of six novel genes from chromosome VII ofSaccharomyces cerevisiae reveals one essential gene and one gene which affects the growth rate

Yeast, 2000

Six ORFs of unknown function located on chromosome VII of Saccharomyces cerevisiae were disrupted... more Six ORFs of unknown function located on chromosome VII of Saccharomyces cerevisiae were disrupted in two different genetic backgrounds, and the phenotype of the generated mutants was analysed. Disruptions of ORFs YGR256w, YGR272c, YGR273c, YGR275w and YGR276c were carried out using the disruption marker kanMX4 anked by short homology regions, whereas ORF YGR255c was inactivated with a long¯anking homology (LFH) disruption cassette . Tetrad analysis of the heterozygous disruptants revealed that ORF YGR255c, previously identi®ed as COQ6 and encoding a protein involved in the biosynthesis of coenzime Q (Tzagoloff and Dieckmann, 1990), is an essential gene. The same analysis also revealed that sporulation of the ygr272cD heterozygous diploid produced two small colonies per ascus that were also G418-resistant, indicating that the inactivation of ORF YGR272c could result in a slower growth rate. This result was con®rmed by growth tests of the haploid disruptants and by complementation of the phenotype after transformation with a plasmid carrying the cognate gene. No phenotypes could be associated to the inactivation of ORFs YGR256w, YGR273c, YGR275w and YGR276c. Two of these genes have recently been further characterized: ORF YGR255w, renamed RTT102, encodes a regulator of the Ty1-element transposition, whereas ORF YGR276c was found to encode the 70 kDa RNase H activity and was renamed RNH70 (Frank et al., 1999). a Calculated as the nucleotide position of each ORF in reference to the complete sequence of chromosome VII .

Research paper thumbnail of Regulation of the expression of theKluyveromyces lactis PDC1 gene: carbon source-responsive elements and autoregulation

Yeast, 1999

The yeast Kluyveromyces lactis has a single structural gene coding for pyruvate decarboxylase (Kl... more The yeast Kluyveromyces lactis has a single structural gene coding for pyruvate decarboxylase (KlPDC1). In order to study the regulation of the expression of KlPDC1, we have sequenced (EMBL Accession No. Y15435) its promoter and have fused the promoter to the reporter gene lacZ from E. coli. Transcription analysis in a Klpdc1 strain showed that KlPDC1 expression is subject to autoregulation. The PDC1 gene from Saccharomyces cerevisiae was able to complement the Rag phenotype of the Klpdc1 mutant strain and it could also repress transcription of the KlPDC1-lacZ fusion on glucose. A deletion analysis of the promoter region was performed to study carbon source-dependent regulation and revealed that at least two cis-acting regions are necessary for full induction of gene expression on glucose. Other cis-elements mediate repression on ethanol.

Research paper thumbnail of Molecular characterization of a plasmid from Pseudomonas fluorescens involved in styrene degradation

Plasmid, 1984

In this paper evidence is given that in a strain of Pseudomonas jluorescens able to grow on styre... more In this paper evidence is given that in a strain of Pseudomonas jluorescens able to grow on styrene as the sole carbon source, the degradation pathway of styrene is inducible and plasmid dependent. The plasmid, which we have called PEG is self-transmissible between Pseudomonas strains and has a size of 37 kb. A restriction map has been constructed and evidence for an inducible transcription of two separate regions of the plasmid has been obtained. io 1981 Academic Press. Inc.

Research paper thumbnail of Action of Rifamycin on Incorporation of Amino-acids into Protein in Cell-free Systems from B. subtilis

Research paper thumbnail of The complete DNA sequence of yeast chromosome III

Nature, 1992

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined... more The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map

Research paper thumbnail of Effect of Rifamycin on Protein Synthesis

Research paper thumbnail of Heterogeneity of mitochondrial DNA from Saccharomyces cerevisiae and genetic information for tRNA

Nucleic Acids Research, 1975

Mitochondrial DNA from wild-type Saccharomyces cerevisiae and from an "extreme" petite mutant wer... more Mitochondrial DNA from wild-type Saccharomyces cerevisiae and from an "extreme" petite mutant were analyzed by hybridization of several tRNAs on DNA fragments of different buoyant den sity, obtained by sonication and fractionation on a CsCl gradient. The hybridization patterns show that the genes for tRNAser, tRNAphe, tRNAhisv tRNAvalstRNAileu are present on wild-type mitochondrial DNA, while only genes for tRNAser and tRNAhis are present on petite mitochondrial DNA; moreover the hybridization patterns indicate that these genes are not clustered and suggest that more than one gene might exist for tRNAser and tRNAhis.

Research paper thumbnail of Dissection of the Carboxyl-Terminal Domain of the Proteasomal Subunit Rpn11 in Maintenance of Mitochondrial Structure and Function

Molecular Biology of the Cell, 2007

We have previously demonstrated that the C-terminal part of Rpn11, a deubiquitinating enzyme in t... more We have previously demonstrated that the C-terminal part of Rpn11, a deubiquitinating enzyme in the lid of the proteasome, is essential for maintaining a correct cell cycle and normal mitochondrial morphology and function. The two roles are apparently unlinked as the mitochondrial role is mapped to the Carboxy-terminus, whereas the catalytic deubiquitinating activity is found within the N-terminal region. The mitochondrial defects are observed in rpn11-m1 (originally termed mpr1-1), a mutation that generates Rpn11 lacking the last 31 amino acids. No mitochondrial phenotypes are recorded for mutations in the MPN؉/JAMM motif. In the present study, we investigated the participation of the last 31 amino acids of the Rpn11 protein by analysis of intragenic revertants and site-specific mutants. We identified a putative ␣-helix necessary for the maintenance of a correct cell cycle and determined that a very short region at the C-terminus of Rpn11 is essential for the maintenance of tubular mitochondrial morphology. Furthermore, we show that expression of the C-terminal part of Rpn11 is able to complement in trans all of the rpn11-m1 mitochondrial phenotypes. Finally, we investigate the mechanisms by which Rpn11 controls the mitochondrial shape and show that Rpn11 may regulate the mitochondrial fission and tubulation processes.

Research paper thumbnail of A Saccharomyces cerevisiae gene essential for viability has been conserved in evolution

Gene, 1995

To identify the gene coding for the endonuclease which processes the 3' end of mitochondrial (mt)... more To identify the gene coding for the endonuclease which processes the 3' end of mitochondrial (mt) tRNA transcripts in Saccharomyces cerevisiae, nuclear mutations able to complement a mt mutant (Ts932) defective for this process were isolated and analyzed. One of these mutants exhibited a growth defect both on respiratory and fermentable media. Complementation of this phenotype with a S. cerevisiae centromeric wild-type genomic library has allowed us to identify a new essential S. cerevisiae gene strongly conserved in various eukaryotic organisms.

Research paper thumbnail of Cesium chloride sensing and signaling in Saccharomyces cerevisiae : an interplay among the HOG and CWI MAPK pathways and the transcription factor Yaf9

FEMS Yeast Research, 2009

In yeast, many environmental stimuli are sensed and signaled by the MAP kinases pathways. In a pr... more In yeast, many environmental stimuli are sensed and signaled by the MAP kinases pathways. In a previous work, we showed that cesium chloride activates the HOG pathway and modulates the transcription of several genes, especially those involved in cell wall biosynthesis and organization. The response to cesium was largely overlapping with the response to salt and osmotic stress. However, when low cesium chloride concentrations were used, a specific response was eventually elicited. The cesium-specific response involved the Yaf9 protein and its activity of chromatin remodeling and transcription regulation. In this paper we show that the osmotic activity of cesium salt is detected and signaled by the two branches downstream of the Sln1 and Sho1 sensors of the HOG pathway, that seem to possess different but exchangeables functions in cesium signaling. However, the cesium-specific response mediated by Yaf9, that counteracts the efficiency of the HOG pathway, is not routed by these sensors. In addition, the cesium response also involves the cell wall integrity (CWI) pathway, which is activated by low concentration of cesium chloride. Mutations blocking the CWI pathway show sensitivity to this salt.

Research paper thumbnail of Regulation of primary carbon metabolism in Kluyveromyces lactis

Enzyme and Microbial Technology, 2000

In the recent past, through advances in development of genetic tools, the budding yeast Kluyverom... more In the recent past, through advances in development of genetic tools, the budding yeast Kluyveromyces lactis has become a model system for studies on molecular physiology of so-called "Nonconventional Yeasts." The regulation of primary carbon metabolism in K. lactis differs markedly from Saccharomyces cerevisiae and reflects the dominance of respiration over fermentation typical for the majority of yeasts. The absence of aerobic ethanol formation in this class of yeasts represents a major advantage for the "cell factory" concept and large-scale production of heterologous proteins in K. lactis cells is being applied successfully. First insight into the molecular basis for the different regulatory strategies is beginning to emerge from comparative studies on S. cerevisiae and K. lactis. The absence of glucose repression of respiration, a high capacity of respiratory enzymes and a tight regulation of glucose uptake in K. lactis are key factors determining physiological differences to S. cerevisiae. A striking discrepancy exists between the conservation of regulatory factors and the lack of evidence for their functional significance in K. lactis. On the other hand, structurally conserved factors were identified in K. lactis in a new regulatory context. It seems that different physiological responses result from modified interactions of similar molecular modules.

Research paper thumbnail of Participation of the proteasomal lid subunit Rpn11 in mitochondrial morphology and function is mapped to a distinct C-terminal domain

Biochemical Journal, 2004

Substrates destined for degradation by the 26 S proteasome are labelled with polyubiquitin chains... more Substrates destined for degradation by the 26 S proteasome are labelled with polyubiquitin chains. Rpn11/Mpr1, situated in the lid subcomplex, partakes in the processing of these chains or in their removal from substrates bound to the proteasome. Rpn11 also plays a role in maintaining mitochondrial integrity, tubular structure and proper function. The recent finding that Rpn11 participates in proteasome-associated deubiquitination focuses interest on the MPN+ (Mpr1, Pad1, N-terminal)/JAMM (JAB1/MPN/Mov34) metalloprotease site in its N-terminal domain. However, Rpn11 damaged at its C-terminus (the mpr1-1 mutant) causes pleiotropic effects, including proteasome instability and mitochondrial morphology defects, resulting in both proteolysis and respiratory malfunctions. We find that overexpression of WT (wild-type) RPN8, encoding a paralogous subunit that does not contain the catalytic MPN+ motif, corrects proteasome conformations and rescues cell cycle phenotypes, but is unable to correct defects in the mitochondrial tubular system or respiratory malfunctions associated with the mpr1-1 mutation. Transforming mpr1-1 with various RPN8-RPN11 chimaeras or with other rpn11 mutants reveals that a WT C-terminal region of Rpn11 is necessary, and more surprisingly sufficient, to rescue the mpr1-1 mitochondrial phenotype. Interestingly, single-site mutants in the catalytic MPN+ motif at the N-terminus of Rpn11 lead to reduced proteasome-dependent deubiquitination connected with proteolysis defects. Nevertheless, these rpn11 mutants suppress the mitochondrial phenotypes associated with mpr1-1 by intragene complementation. Together, these results point to a unique role for the C-terminal region of Rpn11 in mitochondrial maintenance that may be independent of its role in proteasome-associated deubiquitination.

Research paper thumbnail of How to bring orphan genes into functional families

Yeast, 1999

In the framework of the B1 Consortium of the EUROFAN-1 project, we set up a series of simple phen... more In the framework of the B1 Consortium of the EUROFAN-1 project, we set up a series of simple phenotypic tests that can be performed on a large number of strains at a time. This methodological approach was intended to help assign functions of putative genes coding for unknown proteins to several specific aspects of cell biology. The tests were chosen to study phenotypes which should be affected by numerous genes. In this report, we examined the sensitivity/resistance or the adaptation of the cell to physical or chemical stresses (thermotolerance, osmotolerance and ethanol sensitivity), the effects of the alteration of the level of protein phosphorylation (sensitivity or resistance to compounds affecting the activity of protein kinases or phosphatases) and the effects of compounds interfering with synthesis of nucleic acids or proteins. Deletions in 66 genes of unknown function have been tested in 21 different conditions. In many deletant strains, phenotypes were observed and, for the most promising candidates, tetrad analysis was performed in order to verify co-segregation of the deletion marker with the phenotype.