Physical and Genetic Interactions of Cytosolic Malate Dehydrogenase with Other Gluconeogenic Enzymes

Abstract

A truncated form (⌬nMDH2) of yeast cytosolic malate dehydrogenase (MDH2) lacking 12 residues on the amino terminus was found to be inadequate for gluconeogenic function in vivo because the mutant enzyme fails to restore growth of a ⌬mdh2 strain on minimal medium with ethanol or acetate as the carbon source. The ⌬nMDH2 enzyme was also previously found to be refractory to the rapid glucose-induced inactivation and degradation observed for authentic MDH2. In contrast, kinetic properties measured for purified forms of MDH2 and ⌬nMDH2 enzymes are very similar. Yeast two-hybrid assays indicate weak interactions between MDH2 and yeast phosphoenolpyruvate carboxykinase (PCK1) and between MDH2 and fructose-1,6-bisphosphatase (FBP1). These interactions are not observed for ⌬nMDH2, suggesting that differences in cellular function between authentic and truncated forms of MDH2 may be related to their ability to interact with other gluconeogenic enzymes. Additional evidence was obtained for interaction of MDH2 with PCK1 using Hummel-Dreyer gel filtration chromatography, and for interactions of MDH2 with PCK1 and with FBP1 using surface plasmon resonance. Experiments with the latter technique demonstrated a much lower affinity for interaction of ⌬nMDH2 with PCK1 and no interaction between ⌬nMDH2 and FBP1. These results suggest that the interactions of MDH2 with other gluconeogenic enzymes are dependent on the amino terminus of the enzyme, and that these interactions are important for gluconeogenic function in vivo.Three differentially compartmentalized isozymes of malate dehydrogenase (MDH) 1 in Saccharomyces cerevisiae catalyze the NAD(H)-specific interconversion of malate and oxaloacetate. Mitochondrial MDH1 catalyzes a reaction of the tricarboxylic acid cycle, and disruption of the corresponding gene results in an inability to grow with acetate as a carbon source (1, 2), a phenotype shared with yeast mutants containing disruptions in genes encoding other tricarboxylic acid cycle enzymes (3-5). Cytosolic MDH2 is a gluconeogenic enzyme and is required for growth on minimal medium with ethanol or acetate as the carbon source (6), as are the other yeast gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PCK1) (7) and fructose-1,6-bisphosphatase (FBP1) (8). Peroxisomal MDH3 is proposed to catalyze a step in the glyoxylate pathway, which permits formation of C 4 metabolites from C 2 precursors (9, 10), and has also been shown to be necessary for growth with oleate as the carbon source (11), suggesting an additional role in providing NADH for peroxisomal ␤-oxidation.The yeast MDH isozymes are all homodimers with similar subunit M r values (33,500 for MDH1; 40,700 for MDH2; and 37,200 for MDH3), and they share primary sequence identities of 43 to 50%. Distinct differences among the enzymes include a 17-residue mitochondrial targeting sequence for MDH1 that is removed upon import (2), and a carboxyl-terminal tripeptide targeting sequence (Ser-Lys-Leu) required for peroxisomal import of MDH3 (11). In addition, MDH2 has a 12-r...

Degradation of the Gluconeogenic Enzymes Fructose-1,6-bisphosphatase and Malate Dehydrogenase Is Mediated by Distinct Proteolytic Pathways and Signaling Events

“…However, the vacuolar degradation pathway is unlikely to utilize this system, because FBPase ubiquitination was not observed under the vacuolar degradation conditions (38). FBPase is known to interact with MDH2 under glucose starvation conditions (44). Therefore, we determined whether the degradation defect seen in the P1S-FBPase and P1S-MDH2 mutants resulted from a reduction in their interactions.…”

“…Mu-tagenesis studies have shown that the first 12 amino acids at the N terminus in MDH2 (⌬nMDH2) are essential for degradation. More recently, an interaction between MDH2 and FBPase has been demonstrated using the yeast two-hybrid assay and surface plasmon resonance analysis (44). Again, the Nterminal 12 amino acids are important for MDH2-FBPase interaction, because FBPase interacted with ⌬nMDH2 with a much lower affinity.…”

Mitochondrial involvement to methylglyoxal detoxification: d-Lactate/Malate antiporter in Saccharomyces cerevisiae

“…In virtue of the close physical associations between MDH2 and PCK1 (phosphoenolpyruvate carboxykinase; see Gibson and McAlister-Henn, 2003) and since the close proximity with the next enzyme within a metabolic pathway may be important to ensure direct delivery of oxaloacetate because an interaction between sequential enzymes in a given metabolic pathway is frequently cited as evidence for the possibility of ''channeling'' of intermediates within the pathway (Estojak et al 1995) this article proposes that D-lactate translocators and D-lactate dehydrogenases work together for the removal of MG from cytosol as well as for D-lactate-dependent gluconeogenesis.…”

“…Our data show that as a result of D-lactate uptake and metabolism by S. cerevisiae mitochondria, reducing equivalents were exported from the mitochondrial matrix to the cytosol in the form of malate via a novel putative D-lactate/malate carrier. Malate is converted to oxaloacetate via yeast cytosolic gluconeogenic isozyme of malate dehydrogenase (MDH2) and could become a substrate for gluconeogenesis by virtue of the close physical associations between MDH2 and PCK1 (phosphoenolpyruvate carboxykinase; see Gibson and McAlister-Henn 2003).…”

Molecular analysis of cytosolic and mitochondrial malate dehydrogenases isolated from domestic cats (Felis catus)

“…MDH1 is the key enzyme in the malate/aspartate shuttle, which is the dominant pathway in the liver and heart (Scholz et al, 1998), while MDH2 is known to be a key enzyme in the TCA cycle. Cytosolic MDH, mentioned as MDH2 in yeast, has been shown to work as a gluconeogenic enzyme, because it is required for the growth of yeasts on a minimal medium in the presence of gluconeogenic precursors (Gibson and McAlister-Henn, 2003).…”