We have constructed two different fusion proteins consisting of the C-terminal end of CS1 fused in-frame to the N-terminal end of MDH1 and HSA, respectively. The fusion proteins were expressed in mutants of Saccharomyces cerevisiae in which CS1 and MDH1 had been deleted and the phenotypes of the transformants characterized. The results show that the fusion proteins are transported into the mitochondria and that they restore the ability for the yeast mutants CS1-, MDH1-, and CS1-/MDH1- to grow on acetate. Determination of CS1 activity in isolated mitochondria showed a 10-fold increase for the strain that expressed native CS1, relative to the parental. In the transformant with CS1/MDH1 fusion protein, parental levels of CS1 were observed, while one-fifth this amount was observed for the strain expressing the CS1/HSA conjugate. Oxygen consumption studies on isolated mitochondria did not show any significant differences between parental-type yeast and the strains expressing the different fusion proteins or native CS1. [3(-13)C]Propionate was used to study the Krebs TCA cycle metabolism of yeast cells containing CS1/MDH1 fusion constructs. The 13C NMR study was performed in respiratory-competent parental yeast cells and using the genetically engineered yeast cells consisting of CS1- mutants expressing native CS1 and the fusion proteins CS1/MDH1 and CS1/HSA, respectively. [3(-13)C]Propionate is believed to be metabolized to [2(-13)C]succinyl-CoA before it enters the TCA cycle in the mitochondria. This metabolite is then oxidized through two symmetrical intermediates, succinate and fumarate, followed by conversion to malate, oxalacetate, and other metabolites such as alanine.(ABSTRACT TRUNCATED AT 250 WORDS)