Reversible transdominant inhibition of a metabolic pathway. In vivo evidence of interaction between the sequential tricarboxylic acid cycle enzymes in yeast

Christian Vélot, P. Srere

Research output: Article

40 Citations (Scopus)

Abstract

The enzymes of the Krebs tricarboxylic acid cycle in mitochondria are proposed to form a supramolecular complex, in which there is channeling of intermediates between enzyme active sites. While interactions have been demonstrated in vitro between most of the sequential tricarboxylic acid cycle enzymes, no direct evidence has been obtained in vivo for such interactions. We have isolated, in the Saccharomyces cerevisiae gene encoding the tricarboxylic acid cycle enzyme citrate synthase Cit1p, an 'assembly mutation,' i.e. a mutation that causes a tricarboxylic acid cycle deficiency without affecting the citrate synthase activity. We have shown that a 15- amino acid peptide from wild type Cit1p encompassing the mutation point inhibits the tricarboxylic acid cycle in a dominant manner, and that the inhibitory phenotype is overcome by a co-overexpression of Mdh1p, the mitochondrial malate dehydrogenase. These data provide the first direct in vivo evidence of interaction between two sequential tricarboxylic acid cycle enzymes, Cit1p and Mdh1p, and indicate that the characterization of assembly mutations by the reversible transdominant inhibition method may be a powerful way to study multienzyme complexes in their physiological context.

Original languageEnglish
Pages (from-to)12926-12933
Number of pages8
JournalJournal of Biological Chemistry
Volume275
Issue number17
DOIs
Publication statusPublished - ápr. 28 2000

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Citric Acid Cycle
Metabolic Networks and Pathways
Yeast
Yeasts
Enzymes
Citrate (si)-Synthase
Mutation
Multienzyme Complexes
Malate Dehydrogenase
Mitochondria
Gene encoding
Point Mutation
Saccharomyces cerevisiae
Catalytic Domain
Phenotype
Amino Acids
Peptides
Genes

ASJC Scopus subject areas

  • Biochemistry

Cite this

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abstract = "The enzymes of the Krebs tricarboxylic acid cycle in mitochondria are proposed to form a supramolecular complex, in which there is channeling of intermediates between enzyme active sites. While interactions have been demonstrated in vitro between most of the sequential tricarboxylic acid cycle enzymes, no direct evidence has been obtained in vivo for such interactions. We have isolated, in the Saccharomyces cerevisiae gene encoding the tricarboxylic acid cycle enzyme citrate synthase Cit1p, an 'assembly mutation,' i.e. a mutation that causes a tricarboxylic acid cycle deficiency without affecting the citrate synthase activity. We have shown that a 15- amino acid peptide from wild type Cit1p encompassing the mutation point inhibits the tricarboxylic acid cycle in a dominant manner, and that the inhibitory phenotype is overcome by a co-overexpression of Mdh1p, the mitochondrial malate dehydrogenase. These data provide the first direct in vivo evidence of interaction between two sequential tricarboxylic acid cycle enzymes, Cit1p and Mdh1p, and indicate that the characterization of assembly mutations by the reversible transdominant inhibition method may be a powerful way to study multienzyme complexes in their physiological context.",
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T1 - Reversible transdominant inhibition of a metabolic pathway. In vivo evidence of interaction between the sequential tricarboxylic acid cycle enzymes in yeast

AU - Vélot, Christian

AU - Srere, P.

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Y1 - 2000/4/28

N2 - The enzymes of the Krebs tricarboxylic acid cycle in mitochondria are proposed to form a supramolecular complex, in which there is channeling of intermediates between enzyme active sites. While interactions have been demonstrated in vitro between most of the sequential tricarboxylic acid cycle enzymes, no direct evidence has been obtained in vivo for such interactions. We have isolated, in the Saccharomyces cerevisiae gene encoding the tricarboxylic acid cycle enzyme citrate synthase Cit1p, an 'assembly mutation,' i.e. a mutation that causes a tricarboxylic acid cycle deficiency without affecting the citrate synthase activity. We have shown that a 15- amino acid peptide from wild type Cit1p encompassing the mutation point inhibits the tricarboxylic acid cycle in a dominant manner, and that the inhibitory phenotype is overcome by a co-overexpression of Mdh1p, the mitochondrial malate dehydrogenase. These data provide the first direct in vivo evidence of interaction between two sequential tricarboxylic acid cycle enzymes, Cit1p and Mdh1p, and indicate that the characterization of assembly mutations by the reversible transdominant inhibition method may be a powerful way to study multienzyme complexes in their physiological context.

AB - The enzymes of the Krebs tricarboxylic acid cycle in mitochondria are proposed to form a supramolecular complex, in which there is channeling of intermediates between enzyme active sites. While interactions have been demonstrated in vitro between most of the sequential tricarboxylic acid cycle enzymes, no direct evidence has been obtained in vivo for such interactions. We have isolated, in the Saccharomyces cerevisiae gene encoding the tricarboxylic acid cycle enzyme citrate synthase Cit1p, an 'assembly mutation,' i.e. a mutation that causes a tricarboxylic acid cycle deficiency without affecting the citrate synthase activity. We have shown that a 15- amino acid peptide from wild type Cit1p encompassing the mutation point inhibits the tricarboxylic acid cycle in a dominant manner, and that the inhibitory phenotype is overcome by a co-overexpression of Mdh1p, the mitochondrial malate dehydrogenase. These data provide the first direct in vivo evidence of interaction between two sequential tricarboxylic acid cycle enzymes, Cit1p and Mdh1p, and indicate that the characterization of assembly mutations by the reversible transdominant inhibition method may be a powerful way to study multienzyme complexes in their physiological context.

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