Reversible transdominant inhibition of a metabolic pathway - In vivo evidence of interaction between two sequential, tricarboxylic acid cycle enzymesin yeast

The enzymes of the Krebs tricarboxylic acid cycle in mitochondria are propo sed to form a supramolecular complex, in which there is channeling of inter mediates between enzyme active sites. While interactions have been demonstr ated in vitro between most of the sequential tricarboxylic acid cycle enzym es, no direct evidence has been obtained in vivo for such interactions. We have isolated, in the Saccharomyces cerevisiae gene encoding the tricarboxy lic acid cycle enzyme citrate synthase Cit1p, an "assembly mutation," i.e. a mutation that causes a tricarboxylic acid cycle deficiency without affect ing the citrate synthase activity. We have shown that a 15-amino acid pepti de from wild type Cit1p encompassing the mutation point inhibits the tricar boxylic acid cycle in a dominant manner, and that the inhibitory phenotype is overcome by a co-overexpression of Mdh1p, the mitochondrial malate dehyd rogenase. These data provide the first direct in vivo evidence of interacti on between two sequential tricarboxylic acid cycle enzymes, Cit1p and Mdh1p , and indicate that the characterization of assembly mutations by the rever sible transdominant inhibition method may be a powerful way to study multie nzyme complexes in their physiological context.