AbstractWe have previously shown that citrate synthase binds to an intrinsic protein of the mitochondrial inner membrane (D'Souza and Srere, 1983). In this paper we present evidence that this citrate synthase binding protein is the citrate transporter. We have used citrate synthase 1 mutants of Saccharomyces cerevesiae and transformants containing citrate synthase inactivated by site‐directed mutagenesis to study the effect of the CS1 protein upon mitochondrial function (Kispal and Srere). In the present study citrate uptake and oxidation were measured during state 3 conditions (presence of 200 μM ADP) in the mitochondria of several strains of Saccharomyces cerevesiae: a parental strain containing wild‐type mitochondrial citrate synthase (CS1) and strains derived from a CS1 deficient strain in which the CS1 gene was disrupted by insertation of the LEU2 gene. These strains were generated from the CS1− cells by transformation with vectors encoding site‐specific mutants of CS1 possessing very low levels of enzymatic activity. One such strain in this study was subsequently found to have undergone reversion to produce a strain which had activity very similar to wild type. Positive correlation between citrate uptake and the rate of citrate oxidation was found, suggesting coupling of the two processes. Both mitochondrial citrate uptake and oxidation were decreased in the mutant lacking any form of CS1 protein. Reintroduction of mutagenized CS1 into yeast causes an enhancement in the rate of state 3 oxygen consumption and of citrate uptake. The observed respiratory increase produces a state 3 oxygen consumption rate which is 1.5‐ to 17‐fold greater than the rate of reintroduced mutagenized CS1 activity; conversely, wild‐type CS1 enzyme levels are about 30‐fold greater than the respiratory rate in wild‐type cells. Further evidence for uncoupling of the process of citrate utilization from CS1 activity is also seen in the revertant strain which has lower respiratory rate than the wild‐type strain. This difference is not due to decreased concentration of the citrate carrier of those cells with lower respiratory rates, a change in equilibrium for citrate across the mitochondrial membrane, or to decreased citrate saturability of such mitochondria. These results, supported by the interaction observed during column chromatography between matrix‐immobilized pig citrate synthase and the protein from yeast mitochondrial membrane preparations which exhibits citrate transport activity, strongly suggest physical interaction between CS1 and the mitochondrial citrate transport protein.