Reversible Transdominant Inhibition of a Metabolic Pathway: IN VIVO EVIDENCE OF INTERACTION BETWEEN TWO SEQUENTIAL TRICARBOXYLIC ACID CYCLE ENZYMES IN YEAST

Christian Vélot,Paul A Srere

doi:10.1074/jbc.275.17.12926

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.

Highlights

From the Research Service of the Department of Veterans Affairs Medical Center, Dallas, Texas 75216 and the Department of Biochemistry, the University of Texas Southwestern Medical Center, Dallas, Texas 75235
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
Isolation of a cit1 Assembly Mutation—Genes CIT1 and CIT2 from S. cerevisiae encode mitochondrial and peroxisomal forms of citrate synthase (CS) involved in the Krebs tricarboxylic acid cycle and glyoxylate pathway, respectively

Summary

EXPERIMENTAL PROCEDURES

Growth Media, and Transformation Procedures—The S. cerevisiae null mutant cit (cit1::LEU2) and cit1cit The yeast media were synthetic complete (SC) media prepared as described by Sherman et al [15] with either 2% glucose (SCD) or 2% sodium acetate (SCAc) as the carbon source. All enzymatic reactions were performed as recommended by the manufacturers (Roche Molecular Biochemicals, Life Technologies, Inc., Qiagen, and Stratagene), and all recombinant manipulations were done according to Maniatis et al [21]. Cloned Pfu polymerase (Stratagene) was used in all polymerase chain reaction amplifications, except in those of the construction pRS-C-hcit2/1* that were performed with Taq DNA polymerase (Roche Molecular Biochemicals). The nitrocellulose membrane was blocked by 5% nonfat dry milk in PBS (1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, pH 7.0), incubated with a 1:1000 dilution of a mixture of two mouse monoclonal anti-GFP antibodies (Roche Molecular Biochemicals) in PBS containing a final concentration of 2.5% nonfat dry milk (PBS-M), and washed in PBS containing 0.1% Tween 20 (PBS-T). Protein concentrations were determined spectrophotometrically at 562 nm using the BCA protein assay as described by Smith et al [26], with bovine serum albumin as the standard

RESULTS

48 Ϯ 18 4Ϯ3

DISCUSSION

96 Ϯ 23 99 Ϯ 18 136 Ϯ 28 137 Ϯ 25 130 Ϯ 27 122 Ϯ 24 133 Ϯ 31 120 Ϯ 19