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

Natalie Gibson,Lee Mcalister-Henn

doi:10.1074/jbc.m213231200

Abstract

A truncated form (deltanMDH2) 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 Deltamdh2 strain on minimal medium with ethanol or acetate as the carbon source. The DeltanMDH2 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 deltanMDH2 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 deltanMDH2, 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 deltanMDH2 with PCK1 and no interaction between deltanMDH2 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.

Highlights

Three differentially compartmentalized isozymes of malate dehydrogenase (MDH)1 in Saccharomyces cerevisiae catalyze the NAD(H)-specific interconversion of malate and oxaloacetate
This phenotype was attributed to the requirement for gluconeogenesis for growth in minimal medium that lacks carbon compounds sufficiently abundant in rich medium to allow for growth of these strains. (Note, no growth phenotypes are observed with glucose media because gluoneogenic enzymes are not expressed or functional under this growth condition.)
We have found that a truncated form of this enzyme (⌬nMDH2) lacking the first 12 amino acids fails to provide this function, despite retention of kinetic properties that are essentially indistinguishable from those of the authentic enzyme

Summary

EXPERIMENTAL PROCEDURES

Yeast Strains and Growth Conditions—Gene disruption mutants were constructed using the parental yeast strain S173-6B (MATa, leu112, his, ura, trp1-289) [20]. For subcloning into two-hybrid vectors (Clontech, Inc.), PCR was used to introduce BamHI restriction sites onto the 5Ј and 3Ј ends of the coding region of MDH2 for in-frame fusions with the 3Ј end of the GAL4 DNA-binding domain sequence in pAS2-1 (carrying GAL4(1–147) and TRP1 for selection) and with the 3Ј end of the GAL4 transcriptionactivation domain sequence in pACT2 (carrying GAL4(768–881) and LEU2 for selection). This generated plasmids designated pASMDH2 and pACTMDH2. After centrifugation to remove the beads, the supernatants were used for gel electrophoresis and immunoblot analysis

Interactions among Gluconeogenic Enzymes

RESULTS

Carbon sourcea

Negative controlsd

Negative controls

DISCUSSION