Genes of succinyl‐CoA ligase from Saccharomyces cerevisiae

Abstract

Succinyl-CoA ligase (succinyl-CoA synthetase) catalyzes the nucleotide-dependent conversion of succinyl-CoA to succinate. This enzyme functions in the tricarboxylic acid (TCA) cycle and is also involved in ketone-body breakdown in animals. The enzyme is composed of A and β subunits that are required for catalytic activity. Two genes, LSC1 (YOR142W) and LSC2 (YGR244C), with high similarity to succinylCoA ligase subunits from other species were isolated from Saccharomyces cerevisiae. The expression of these genes was repressed by growth on glucose and was induced threefold to sixfold during growth on nonfermentable carbon sources. The LSC genes were deleted singly and in combination. Unlike other yeast strains with defects in TCA cycle genes, strains lacking either or both LSC genes were able to grow with acetate as a carbon source. However, growth on glycerol or pyruvate was impaired. An antiserum against both subunits of the Escherichia coli enzyme was capable of recognizing the yeast succinyl-CoA ligase A subunit, and this band was absent in ∆lsc1 deletion strains. Succinyl-CoA ligase activity was absent in mitochondria isolated from strains deleted for one or both LSC genes, but activity was restored by the presence of the appropriate LSC gene on a plasmid. The yeast succinyl-CoA ligase was shown to utilize ATP but not GTP for succinyl-CoA synthesis.Keywords : succinyl-CoA ligase ; tricarboxylic acid cycle; mitochondria; Saccharomyces cerevisiae; yeast.Succinyl-CoA ligase (succinyl-CoA synthetase ; succinate thiokinase) catalyzes the nucleotide-dependent conversion of succinyl-CoA to succinate [1]. This is the only enzyme of the tricarboxylic acid (TCA) cycle that directly produces a highenergy phosphate. Succinyl-CoA ligase is composed of A and β subunits that function either as dimers or tetramers. The Escherichia coli enzyme is an A 2 β 2 tetramer, in which two independently active heterodimers are capable of synergistic interaction with the substrate [1Ϫ3]. The porcine heart mitochondrial enzyme has also been well characterized and, like other eukaryotic isoforms, it functions as an Aβ heterodimer [4]. In both prokaryotes and eukaryotes, the A subunit is phosphorylated on a conserved histidine residue, and the phosphate is subsequently transferred to form ATP or GTP [1]. Alteration of this residue by mutagenesis results in an inactive enzyme [5].Isoforms of succinyl-CoA ligase have been distinguished on the basis of their nucleotide preference for either GTP or ATP. The E. coli and Pseudomonas aeruginosa enzymes can utilize either ATP or GTP [1,6,7]. Eukaryotic succinyl-CoA ligases can be either ATP or GTP dependent [1]. While the ATP-dependent form is involved in TCA-cycle metabolism, the GTP-dependent form has been associated with ketone-body breakdown and heme biosynthesis [8,9]. The purified enzyme from Dichtyostelium discoideum shows specificity for GTP, while the enzyme Correspondence to M. T. McCammon,

TCA cycle‐independent acetate metabolism via the glyoxylate cycle in Saccharomyces cerevisiae

“…In addition, the mutant strains lacking any of the enzymes involved in both the TCA and glyoxylate cycles (∆aco1 and ∆fum1 ) or solely in the glyoxylate cycle (∆icl1, ∆mls1 and ∆mdh2 strains) also showed growth impairment on YPA as well as on YNBA. However, in accordance with a previous report (Przybyla-Zawislak et al, 1998), the ∆lsc1 strain did not show any growth defect on YPA. Together, these results indicate that most of the TCA and glyoxylate cycle enzymes are indispensable for the growth of yeast cells on YPA.…”

“…However, on YNBA, growth was classified into two phenotypic categories; the members of the first group ( cit1, ∆idh1, ∆kgd1, ∆lsc1 and ∆mdh1 ) grew on acetate as the sole carbon source, while those of the second group (∆aco1, ∆fum1, ∆sdh1, ∆mls1, ∆icl1 and ∆mdh2 ) failed to grow on this carbon source. These results suggest that most of the TCA cycle-specific mitochondrial enzymes, such as Cit1, NAD + -dependent isocitrate dehydrogenase (an octamer composed of four Idh1 and four Idh2 subunits; McAlister-Henn, 1991, 1992), KGDC (a heterotrimer of Kgd1p, Kgd2p and Lpd1p) and succinyl-CoA ligase (LSC; a heterodimer of Lsc1 and Lsc2; Przybyla-Zawislak et al, 1998), are dispensable for acetate utilization on YNBA plates. It thus appears that the succinate dehydrogenase complex (SDHC), a tetramer of Sdh1, Sdh2, Sdh3 and Sdh4 (Lombardo et al, 1990;Chapman et al, 1992;Bullis and Lemire, 1994;Daignan-Fornier et al, 1994), is the only TCA cycle-specific enzyme required for acetate utilization on YNBA.…”

Suppression of metabolic defects of yeast isocitrate dehydrogenase and aconitase mutants by loss of citrate synthase

“…Growth phenotypes and protein expression in idhΔcit1 Δ and aco1 Δcit1 Δ mutants Yeast mutants lacking IDH or ACO1 have been reported to share some growth phenotypes, including an inability to grow with acetate as a carbon source [14] and a propensity for generation of petite segregants [10]. To better understand the phenotypes exhibited by IDH mutants and their suppression by CIT1 defects, we wished to first compare the properties of idhΔ and idhΔ cit1 Δ mutant strains with those of aco1 Δ and aco1 Δcit1 Δ mutant strains.…”

“…To better understand the phenotypes exhibited by IDH mutants and their suppression by CIT1 defects, we wished to first compare the properties of idhΔ and idhΔ cit1 Δ mutant strains with those of aco1 Δ and aco1 Δcit1 Δ mutant strains. In initial experiments with previously constructed aco1 Δ gene disruption strains [10,14], we could not retrieve any respiratory-competent colonies capable of growth with non-fermentable carbon sources, i.e. these strains had become entirely petite during storage.…”

“…Construction of an idhΔ mutant strain with LEU2 and HIS3 deletion/ disruptions in IDH1 and IDH2 loci, respectively, and of the idhΔcit1 Δ mutant strain were previously described [9,14]. Mutant aco1 Δ and aco1 Δcit1 Δ strains were newly constructed by deletion/disruption mutagenesis using the kanMX4 cassette [15] to replace the ACO1 coding regions in parental and cit1 Δ strains, respectively.…”