Physical and genetic interactions of cytosolic malate dehydrogenase with other gluconeogenic enzymes

Natalie Gibson, Lee McAlister-Henn

Research output: Contribution to journalArticlepeer-review

20 Scopus citations

Abstract

A truncated form (ΔnMDH2) 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 Amdh2 strain on minimal medium with ethanol or acetate as the carbon source. The AnMDH2 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 AnMDH2 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 AnMDH2, 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 AnMDH2 with PCK1 and no interaction between AnMDH2 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.

Original languageEnglish (US)
Pages (from-to)25628-25636
Number of pages9
JournalJournal of Biological Chemistry
Volume278
Issue number28
DOIs
StatePublished - Jul 11 2003

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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