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Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae
Published on: February 16, 2015
Disulfide bond formation in yeast NAD+-specific isocitrate dehydrogenase
Joshua A Garcia1, Karyl I Minard, An-Ping Lin
1Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229, USA.
Biochemistry
|August 4, 2009
Summary
A specific cysteine residue (Cys-150) in yeast isocitrate dehydrogenase (IDH) forms a disulfide bond, inhibiting enzyme activity. This cysteine is crucial for IDH regulation during cellular stationary phase.
Area of Science:
- Biochemistry
- Molecular Biology
- Yeast Metabolism
Background:
- The NAD+-specific isocitrate dehydrogenase (IDH) in Saccharomyces cerevisiae is a key enzyme in the tricarboxylic acid cycle.
- Structural studies indicated that Cys-150 residues in adjacent IDH2 subunits are in close proximity, suggesting a role in enzyme regulation.
- Disulfide bond formation was hypothesized to stabilize an inactive enzyme conformation.
Purpose of the Study:
- To investigate the role of Cys-150 in the regulation of yeast IDH activity.
- To determine if disulfide bond formation involving Cys-150 inhibits IDH function both in vitro and in vivo.
- To assess the physiological relevance of Cys-150 mediated inhibition during the stationary phase.
Main Methods:
- Construction of IDH mutants: C150S (single cysteine mutant) and C56S/C242S (sole cysteine at 150).
- In vitro enzyme activity assays using affinity-purified wild-type and mutant IDH treated with diamide (disulfide bond inducer) and dithiothreitol (reductant).
- In vivo studies using yeast transformants expressing different IDH constructs, analyzing disulfide bond formation and enzyme activity under various conditions, including stationary phase.
Main Results:
- Diamide treatment induced disulfide bond formation and decreased activity in wild-type and C56S/C242S IDH, effects reversible by dithiothreitol.
- The C150S mutant enzyme showed no diamide-induced inhibition, confirming Cys-150's essential role in disulfide bond-mediated inactivation.
- Diamide-induced Cys-150 disulfide bond formation and natural stationary phase inhibition were observed in vivo for wild-type and C56S/C242S strains, but not for the C150S mutant.
- The C150S mutant exhibited reduced viability in stationary phase, suggesting IDH activity decrease is vital for metabolic adaptation.
Conclusions:
- Cys-150 is critical for the formation of an inhibitory disulfide bond in yeast IDH.
- This Cys-150 mediated inactivation mechanism is active both in vitro and in vivo, particularly during the stationary phase.
- The regulation of IDH activity via Cys-150 disulfide bonding plays a significant role in yeast metabolic adaptation and survival during stationary phase.
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