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John M Kelly - One of the best experts on this subject based on the ideXlab platform.
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erratum to the amino terminal domain of a novel wd repeat protein from trypanosoma cruzi contains a non canonical Mitochondrial Targeting Signal int j parasitol 34 1 2004 63 71
2004Co-Authors: Elizabeth Bromley, Martin C Taylor, Shane R Wilkinson, John M KellyAbstract:Fig. 5. Subcellular location of N-GFP in relation to an endoplasmic reticulum-specific marker. Panel 1, a metacyclic trypomastigote stained with TOTO-3 to identify DNA (blue). The intense staining identifies the kinetoplast. Panel 2, the same parasite visualised to identify the endoplasmic reticulum-localised protein BiP. Panel 3, localisation of N-GFP (green). Panel 4, merged images of panels 1–3. Panel 5, phase image. In panel 4, the yellow coloration at the posterior end of the cell indicates where the mitochondrion spirals over and under the endoplasmic reticulum. Bar, 5 mm.
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the amino terminal domain of a novel wd repeat protein from trypanosoma cruzi contains a non canonical Mitochondrial Targeting Signal
2004Co-Authors: Elizabeth Bromley, Martin C Taylor, Shane R Wilkinson, John M KellyAbstract:WD (tryptophan/aspartic acid) repeat proteins perform a wide variety of functions in eukaryotic cells. They are characterised by the presence of a number of conserved repeat motifs that contribute to the beta-propeller structures which are the common feature of this large group of proteins. We report here the properties of the first characterised member of this family in the American trypanosome, Trypanosoma cruzi (TcBPP1). In the CL Brener clone the protein is 482 amino acids long and is predicted to contain four WD repeat motifs, flanked by amino and carboxyl terminal extensions. TcBPP1 is a single copy gene present on a 1.0/1.6 Mb pair of homologous chromosomes in a locus that is syntenic with the corresponding regions of Trypanosoma brucei and Leishmania major chromosomes. Consistent with the proposed hybrid nature of the CL Brener clone, the proteins encoded by the two different alleles share only 97% identity at the amino acid level. To determine subcellular location, we examined transfected parasites for the distribution of green fluorescent protein (GFP) fused with different regions of TcBPP1. These studies demonstrated that a 115 amino acid peptide derived from the amino terminal domain of TcBPP1 is able to target GFP to the mitochondrion. Interestingly this region lacks a typical amino terminal presequence suggesting that Mitochondrial import is mediated by an alternative Targeting Signal.
Joanna Tripp - One of the best experts on this subject based on the ideXlab platform.
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requirement of a functional flavin mononucleotide prenyltransferase for the activity of a bacterial decarboxylase in a heterologous muconic acid pathway in saccharomyces cerevisiae
2017Co-Authors: Heike Weber, Christine Brückner, Eckhard Boles, Manuela Gottardi, Mislav Oreb, Joanna TrippAbstract:Biotechnological production of cis, cis -muconic acid from renewable feedstocks is an environmentally sustainable alternative to conventional, petroleum-based methods. Even though a heterologous production pathway for cis, cis -muconic acid has already been established in the host organism Saccharomyces cerevisiae , the generation of industrially relevant amounts of cis, cis -muconic acid is hampered by the low activity of the bacterial protocatechuic acid (PCA) decarboxylase AroY subunit C iso (AroY-C iso ), leading to secretion of high amounts of the intermediate PCA to the medium. In the current study, we show that activity of AroY-C iso in S. cerevisiae strongly depends on the strain background. We could demonstrate that the strain dependency is caused by the presence or absence of an intact genomic copy of PAD1, which encodes a Mitochondrial enzyme responsible for the biosynthesis of a prenylated form of the cofactor flavin mononucleotide (prFMN). The inactivity of AroY-C iso in strain CEN.PK2-1 could be overcome by plasmid borne expression of Pad1 or its bacterial homologue aroY subunit B (AroY-B). Our data reveal that the two enzymes perform the same function in decarboxylation of PCA by AroY-C iso , although coexpression of Pad1 led to higher decarboxylase activity. Conversely, AroY-B can replace Pad1 in its function in decarboxylation of phenylacrylic acids by ferulic acid decarboxylase Fdc1. Targeting of the majority of AroY-B to mitochondria by fusion to a heterologous Mitochondrial Targeting Signal did not improve decarboxylase activity of AroY-C iso , suggesting that Mitochondrial localization has no major impact on cofactor biosynthesis. IMPORTANCE In Saccharomyces cerevisiae , decarboxylation of protocatechuic acid (PCA) to catechol is the bottleneck reaction in the heterologous biosynthetic pathway for production of cis, cis -muconic acid, a valuable precursor for the production of bulk chemicals. In our work, we demonstrate the importance of the strain background for the activity of a bacterial PCA decarboxylase in S. cerevisiae. Inactivity of the decarboxylase is due to a nonsense mutation in a gene encoding a Mitochondrial enzyme involved in the biosynthesis of a cofactor required for decarboxylase function. Our study reveals functional interchangeability of Pad1 and a bacterial homologue, irrespective of their intracellular localization. Our results open up new possibilities to improve muconic acid production by engineering cofactor supply. Furthermore, the results have important implications for the choice of the production strain.
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Requirement of a Functional Flavin Mononucleotide Prenyltransferase for the Activity of a Bacterial Decarboxylase in a Heterologous Muconic Acid Pathway in Saccharomyces cerevisiae
2017Co-Authors: Heike Weber, Christine Brückner, Eckhard Boles, Manuela Gottardi, Mislav Oreb, Joanna TrippAbstract:Biotechnological production of cis,cis-muconic acid from renewable feedstocks is an environmentally sustainable alternative to conventional, petroleum-based methods. Even though a heterologous production pathway for cis,cis-muconic acid has already been established in the host organism Saccharomyces cerevisiae, the generation of industrially relevant amounts of cis,cis-muconic acid is hampered by the low activity of the bacterial protocatechuic acid (PCA) decarboxylase AroY isomeric subunit Ciso (AroY-Ciso), leading to secretion of large amounts of the intermediate PCA into the medium. In the present study, we show that the activity of AroY-Ciso in S. cerevisiae strongly depends on the strain background. We could demonstrate that the strain dependency is caused by the presence or absence of an intact genomic copy of PAD1, which encodes a Mitochondrial enzyme responsible for the biosynthesis of a prenylated form of the cofactor flavin mononucleotide (prFMN). The inactivity of AroY-Ciso in strain CEN.PK2-1 could be overcome by plasmid-borne expression of Pad1 or its bacterial homologue AroY subunit B (AroY-B). Our data reveal that the two enzymes perform the same function in decarboxylation of PCA by AroY-Ciso, although coexpression of Pad1 led to higher decarboxylase activity. Conversely, AroY-B can replace Pad1 in its function in decarboxylation of phenylacrylic acids by ferulic acid decarboxylase Fdc1. Targeting of the majority of AroY-B to mitochondria by fusion to a heterologous Mitochondrial Targeting Signal did not improve decarboxylase activity of AroY-Ciso, suggesting that Mitochondrial localization has no major impact on cofactor biosynthesis.IMPORTANCE In Saccharomyces cerevisiae, the decarboxylation of protocatechuic acid (PCA) to catechol is the bottleneck reaction in the heterologous biosynthetic pathway for production of cis,cis-muconic acid, a valuable precursor for the production of bulk chemicals. In our work, we demonstrate the importance of the strain background for the activity of a bacterial PCA decarboxylase in S. cerevisiae Inactivity of the decarboxylase is due to a nonsense mutation in a gene encoding a Mitochondrial enzyme involved in the biosynthesis of a cofactor required for decarboxylase function. Our study reveals functional interchangeability of Pad1 and a bacterial homologue, irrespective of their intracellular localization. Our results open up new possibilities to improve muconic acid production by engineering cofactor supply. Furthermore, the results have important implications for the choice of the production strain.
Ervin Fodor - One of the best experts on this subject based on the ideXlab platform.
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the pb2 subunit of the influenza virus rna polymerase affects virulence by interacting with the Mitochondrial antiviral Signaling protein and inhibiting expression of beta interferon
2010Co-Authors: Katy M Graef, Amber W Mccall, Kanta Subbarao, Simon M. Carr, Frank T. Vreede, Ervin FodorAbstract:The PB2 subunit of the influenza virus RNA polymerase is a major virulence determinant of influenza viruses. However, the molecular mechanisms involved remain unknown. It was previously shown that the PB2 protein, in addition to its nuclear localization, also accumulates in the mitochondria. Here, we demonstrate that the PB2 protein interacts with the Mitochondrial antiviral Signaling protein, MAVS (also known as IPS-1, VISA, or Cardif), and inhibits MAVS-mediated beta interferon (IFN-β) expression. In addition, we show that PB2 proteins of influenza viruses differ in their abilities to associate with the mitochondria. In particular, the PB2 proteins of seasonal human influenza viruses localize to the mitochondria while PB2 proteins of avian influenza viruses are nonMitochondrial. This difference in localization is caused by a single amino acid polymorphism in the PB2 Mitochondrial Targeting Signal. In order to address the functional significance of the Mitochondrial localization of the PB2 protein in vivo, we have generated two recombinant human influenza viruses encoding either Mitochondrial or nonMitochondrial PB2 proteins. We found that the difference in the Mitochondrial localization of the PB2 proteins does not affect the growth of these viruses in cell culture. However, the virus encoding the nonMitochondrial PB2 protein induces higher levels of IFN-β and, in an animal model, is attenuated compared to the isogenic virus encoding a Mitochondrial PB2. Overall this study implicates the PB2 protein in the regulation of host antiviral innate immune pathways and suggests an important role for the Mitochondrial association of the PB2 protein in determining virulence.
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characterization of a Mitochondrial Targeting Signal in the pb2 protein of influenza viruses
2006Co-Authors: Simon M. Carr, Elena Carnero, Adolfo Garciasastre, George G Brownlee, Ervin FodorAbstract:Influenza virus RNA polymerase is a heterotrimeric complex consisting of PB1, PB2, and PA subunits. These polymerase subunits accumulate in the nucleus of infected cells. We report here that PB2, from both human and avian influenza viruses, could also localize to mitochondria in transfected cells. Importantly, cells infected with influenza A virus also displayed Mitochondrial PB2. We show that an N-terminal motif composed of 120 amino acids is sufficient for localization of PB2 to mitochondria. In particular, leucine residues at positions 7 and 10 were essential for Mitochondrial Targeting. Recombinant influenza A/WSN/33 viruses expressing PB2 proteins with L7A and/or L10A mutations showed reduced viral titers, but unaffected levels of transcription, replication, and protein expression. The introduction of L7A and/or L10A mutations into recombinant viruses correlated with reduced Mitochondrial membrane potential in infected cells, suggesting that Mitochondrial localization of PB2 contributes to the preservation of Mitochondrial function during influenza virus infection.
Elizabeth Bromley - One of the best experts on this subject based on the ideXlab platform.
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erratum to the amino terminal domain of a novel wd repeat protein from trypanosoma cruzi contains a non canonical Mitochondrial Targeting Signal int j parasitol 34 1 2004 63 71
2004Co-Authors: Elizabeth Bromley, Martin C Taylor, Shane R Wilkinson, John M KellyAbstract:Fig. 5. Subcellular location of N-GFP in relation to an endoplasmic reticulum-specific marker. Panel 1, a metacyclic trypomastigote stained with TOTO-3 to identify DNA (blue). The intense staining identifies the kinetoplast. Panel 2, the same parasite visualised to identify the endoplasmic reticulum-localised protein BiP. Panel 3, localisation of N-GFP (green). Panel 4, merged images of panels 1–3. Panel 5, phase image. In panel 4, the yellow coloration at the posterior end of the cell indicates where the mitochondrion spirals over and under the endoplasmic reticulum. Bar, 5 mm.
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the amino terminal domain of a novel wd repeat protein from trypanosoma cruzi contains a non canonical Mitochondrial Targeting Signal
2004Co-Authors: Elizabeth Bromley, Martin C Taylor, Shane R Wilkinson, John M KellyAbstract:WD (tryptophan/aspartic acid) repeat proteins perform a wide variety of functions in eukaryotic cells. They are characterised by the presence of a number of conserved repeat motifs that contribute to the beta-propeller structures which are the common feature of this large group of proteins. We report here the properties of the first characterised member of this family in the American trypanosome, Trypanosoma cruzi (TcBPP1). In the CL Brener clone the protein is 482 amino acids long and is predicted to contain four WD repeat motifs, flanked by amino and carboxyl terminal extensions. TcBPP1 is a single copy gene present on a 1.0/1.6 Mb pair of homologous chromosomes in a locus that is syntenic with the corresponding regions of Trypanosoma brucei and Leishmania major chromosomes. Consistent with the proposed hybrid nature of the CL Brener clone, the proteins encoded by the two different alleles share only 97% identity at the amino acid level. To determine subcellular location, we examined transfected parasites for the distribution of green fluorescent protein (GFP) fused with different regions of TcBPP1. These studies demonstrated that a 115 amino acid peptide derived from the amino terminal domain of TcBPP1 is able to target GFP to the mitochondrion. Interestingly this region lacks a typical amino terminal presequence suggesting that Mitochondrial import is mediated by an alternative Targeting Signal.
Heike Weber - One of the best experts on this subject based on the ideXlab platform.
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requirement of a functional flavin mononucleotide prenyltransferase for the activity of a bacterial decarboxylase in a heterologous muconic acid pathway in saccharomyces cerevisiae
2017Co-Authors: Heike Weber, Christine Brückner, Eckhard Boles, Manuela Gottardi, Mislav Oreb, Joanna TrippAbstract:Biotechnological production of cis, cis -muconic acid from renewable feedstocks is an environmentally sustainable alternative to conventional, petroleum-based methods. Even though a heterologous production pathway for cis, cis -muconic acid has already been established in the host organism Saccharomyces cerevisiae , the generation of industrially relevant amounts of cis, cis -muconic acid is hampered by the low activity of the bacterial protocatechuic acid (PCA) decarboxylase AroY subunit C iso (AroY-C iso ), leading to secretion of high amounts of the intermediate PCA to the medium. In the current study, we show that activity of AroY-C iso in S. cerevisiae strongly depends on the strain background. We could demonstrate that the strain dependency is caused by the presence or absence of an intact genomic copy of PAD1, which encodes a Mitochondrial enzyme responsible for the biosynthesis of a prenylated form of the cofactor flavin mononucleotide (prFMN). The inactivity of AroY-C iso in strain CEN.PK2-1 could be overcome by plasmid borne expression of Pad1 or its bacterial homologue aroY subunit B (AroY-B). Our data reveal that the two enzymes perform the same function in decarboxylation of PCA by AroY-C iso , although coexpression of Pad1 led to higher decarboxylase activity. Conversely, AroY-B can replace Pad1 in its function in decarboxylation of phenylacrylic acids by ferulic acid decarboxylase Fdc1. Targeting of the majority of AroY-B to mitochondria by fusion to a heterologous Mitochondrial Targeting Signal did not improve decarboxylase activity of AroY-C iso , suggesting that Mitochondrial localization has no major impact on cofactor biosynthesis. IMPORTANCE In Saccharomyces cerevisiae , decarboxylation of protocatechuic acid (PCA) to catechol is the bottleneck reaction in the heterologous biosynthetic pathway for production of cis, cis -muconic acid, a valuable precursor for the production of bulk chemicals. In our work, we demonstrate the importance of the strain background for the activity of a bacterial PCA decarboxylase in S. cerevisiae. Inactivity of the decarboxylase is due to a nonsense mutation in a gene encoding a Mitochondrial enzyme involved in the biosynthesis of a cofactor required for decarboxylase function. Our study reveals functional interchangeability of Pad1 and a bacterial homologue, irrespective of their intracellular localization. Our results open up new possibilities to improve muconic acid production by engineering cofactor supply. Furthermore, the results have important implications for the choice of the production strain.
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Requirement of a Functional Flavin Mononucleotide Prenyltransferase for the Activity of a Bacterial Decarboxylase in a Heterologous Muconic Acid Pathway in Saccharomyces cerevisiae
2017Co-Authors: Heike Weber, Christine Brückner, Eckhard Boles, Manuela Gottardi, Mislav Oreb, Joanna TrippAbstract:Biotechnological production of cis,cis-muconic acid from renewable feedstocks is an environmentally sustainable alternative to conventional, petroleum-based methods. Even though a heterologous production pathway for cis,cis-muconic acid has already been established in the host organism Saccharomyces cerevisiae, the generation of industrially relevant amounts of cis,cis-muconic acid is hampered by the low activity of the bacterial protocatechuic acid (PCA) decarboxylase AroY isomeric subunit Ciso (AroY-Ciso), leading to secretion of large amounts of the intermediate PCA into the medium. In the present study, we show that the activity of AroY-Ciso in S. cerevisiae strongly depends on the strain background. We could demonstrate that the strain dependency is caused by the presence or absence of an intact genomic copy of PAD1, which encodes a Mitochondrial enzyme responsible for the biosynthesis of a prenylated form of the cofactor flavin mononucleotide (prFMN). The inactivity of AroY-Ciso in strain CEN.PK2-1 could be overcome by plasmid-borne expression of Pad1 or its bacterial homologue AroY subunit B (AroY-B). Our data reveal that the two enzymes perform the same function in decarboxylation of PCA by AroY-Ciso, although coexpression of Pad1 led to higher decarboxylase activity. Conversely, AroY-B can replace Pad1 in its function in decarboxylation of phenylacrylic acids by ferulic acid decarboxylase Fdc1. Targeting of the majority of AroY-B to mitochondria by fusion to a heterologous Mitochondrial Targeting Signal did not improve decarboxylase activity of AroY-Ciso, suggesting that Mitochondrial localization has no major impact on cofactor biosynthesis.IMPORTANCE In Saccharomyces cerevisiae, the decarboxylation of protocatechuic acid (PCA) to catechol is the bottleneck reaction in the heterologous biosynthetic pathway for production of cis,cis-muconic acid, a valuable precursor for the production of bulk chemicals. In our work, we demonstrate the importance of the strain background for the activity of a bacterial PCA decarboxylase in S. cerevisiae Inactivity of the decarboxylase is due to a nonsense mutation in a gene encoding a Mitochondrial enzyme involved in the biosynthesis of a cofactor required for decarboxylase function. Our study reveals functional interchangeability of Pad1 and a bacterial homologue, irrespective of their intracellular localization. Our results open up new possibilities to improve muconic acid production by engineering cofactor supply. Furthermore, the results have important implications for the choice of the production strain.