METABOLIC STUDIES ON SACCHAROMYCES-CEREVISIAE CONTAINING FUSED CITRATE SYNTHASE MALATE-DEHYDROGENASE

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 CSI. [3-C-13]Propionate was used to study the Krebs TCA cycle metabolism of yeast cells containing CS1/MDH1 fusion constructs. The C-13 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-C-13] Propionate is believed to be metabolized to [2-C-13]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. If the symmetrical intermediates randomly diffuse between the enzymes in the mitochondria, the C-13 label should be equally distributed on the C2 and C3 positions of malate and alanine. However, if succinate and fumarate are directly transferred with conserved orientation between the active sites of the enzymes succinate thiokinase, succinate dehydrogenase, and fumarase, the labeling of the C2 and C3 positions of malate, oxalacetate, and alanine will be asymmetrical. During oxidation of [3-C-13]propionate in parental cells, we observed an asymmetric labeling of the C2 and C3 positions of alanine where the C-13 enrichment was significantly higher in the C3 position (C3/C2 14.3). Inhibition of succinate dehydrogenase with increasing amounts of malonate resulted in a concentration-dependent decrease in the asymmetric labeling of alanine. When [3-C-13]propionate oxidation was performed in the CS1(-) yeast cells containing CS1, CS1/MDH1, and CS1/HSA, the CS/HSA transformant displayed significantly decreased asymmetry in the labeling of the C2 and C3 positions of alanine (C3/C2 = 2.9). No significant difference was found between parental cells and the CS1 and CS1/MDH1 transformants. Growth experiments on rich medium did not show any differences between the transformants. On minimal medium, however, the CS1/HSA transformant displayed an increased doubling time. These data show that, in yeast cells containing the CS1/MDH1 fusion protein, symmetrical intermediates are transferred directly from TCA cycle enzyme to TCA cycle enzyme under in vivo conditions just as is observed in the parental cell. The data also show that it is possible to alter this effect in the TCA cycle pathway by introduction of a genetically engineered CS1/HSA fusion protein. We also discuss these data in the context of the metabolon hypothesis for the Krebs TCA cycle.