C. Lindbladh et al., METABOLIC STUDIES ON SACCHAROMYCES-CEREVISIAE CONTAINING FUSED CITRATE SYNTHASE MALATE-DEHYDROGENASE, Biochemistry, 33(39), 1994, pp. 11684-11691
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 H
SA, respectively. The fusion proteins were expressed in mutants of Sac
charomyces cerevisiae in which CS1 and MDH1 had been deleted and the p
henotypes of the transformants characterized. The results show that th
e 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 n
ative 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 conjug
ate. Oxygen consumption studies on isolated mitochondria did not show
any significant differences between parental-type yeast and the strain
s expressing the different fusion proteins or native CSI. [3-C-13]Prop
ionate was used to study the Krebs TCA cycle metabolism of yeast cells
containing CS1/MDH1 fusion constructs. The C-13 NMR Study was perform
ed in respiratory-competent parental yeast cells and using the genetic
ally engineered yeast cells consisting of CS1(-) mutants expressing na
tive 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-C
oA 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 me
tabolites such as alanine. If the symmetrical intermediates randomly d
iffuse between the enzymes in the mitochondria, the C-13 label should
be equally distributed on the C2 and C3 positions of malate and alanin
e. However, if succinate and fumarate are directly transferred with co
nserved 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 asymm
etrical. 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 am
ounts of malonate resulted in a concentration-dependent decrease in th
e asymmetric labeling of alanine. When [3-C-13]propionate oxidation wa
s performed in the CS1(-) yeast cells containing CS1, CS1/MDH1, and CS
1/HSA, the CS/HSA transformant displayed significantly decreased asymm
etry 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 ti
me. These data show that, in yeast cells containing the CS1/MDH1 fusio
n 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 gen
etically engineered CS1/HSA fusion protein. We also discuss these data
in the context of the metabolon hypothesis for the Krebs TCA cycle.