Function and expression of yeast mitochondrial NAD- and NADP-specific isocitrate dehydrogenases

R.J Haselbeck,L Mcalister-Henn

doi:10.1016/s0021-9258(19)50315-5

Abstract

The three isozymes of isocitrate dehydrogenase in Saccharomyces cerevisiae differ in subunit structure, subcellular location, and cofactor specificity. The two mitochondrial isozymes, IDH and IDP1, are NAD- and NADP-specific, respectively. Several lines of evidence presented here confirm the importance of IDH to respiratory processes. Expression of IDH RNA and protein is low with growth on glucose and is elevated with growth on non-fermentable carbon sources, a pattern of expression similar to that seen for other tricarboxylic acid cycle enzymes. In addition, a disruption mutant lacking IDH activity exhibits reduced growth rates on non-fermentable carbon sources, and mitochondria isolated from this mutant are incapable of respiration with added citrate. In contrast, IDP1 expression levels appear to be unresponsive to carbon source, and an IDP1 disruption mutant is not significantly impaired for growth or mitochondrial respiration. These results strongly suggest that IDP1 is incapable of participating in tricarboxylic acid cycle-based respiration despite its mitochondrial location. Analysis of the IDP1 and IDH disruption mutants for glutamate auxotrophy showed that either enzyme can contribute alpha-ketoglutarate for endogenous glutamate synthesis. IDH expression levels were found to be repressed in response to added glutamate during growth on glucose, while IDP1 expression levels remained unchanged. A double mutant lacking both IDP1 and IDH activities proved to be auxotrophic for glutamate during growth on glucose, but was capable of growth independent of added glutamate on non-fermentable carbon sources. These results suggest that the cytosolic NADP-specific IDP2 isozyme may provide alpha-ketoglutarate both for tricarboxylic acid cycle carbon flux and for cytosolic glutamate synthesis during growth on non-fermentable carbon sources in the absence of mitochondrial isocitrate dehydrogenase activity.

Highlights

The two tinct isozymes of isocitrate dehydrogenase, which differ in mitochondrial isozymes, IDHand IDP1, are NAD- and subunit structure, regulation, cofactor specificity, and subcel
Chondria isolated from this mutant are incapable of specific mitochondrial isozyme (IDP1) has been shown to be respirationwith added citrate.Incontrast, IDPl a dimer of identical subunits and is not known to exhibit any expression levels appear tobe unresponsive to carbon allosteric control, it is subject to end-product inhisource, and an IDPl disruption mutant is not signifi- bition by NADPH or a-ketoglutarate [7]
Analysis hydrogenase enzymes in response to yeast growth on ferof the IDPl and IDHdisruption mutants for glutamatmeentable and non-fermentable carbon sources has been reauxotrophy showed that eitherenzyme can contribute ported [1,4].IDH mitochondrial protein levels are low during a-ketoglutarate for endogenous glutamate synthesis. growth on glucose and increase with growth on 3-carbon or IDH expression levels were found to be repressed in 2-carbon non-fermentable metabolites

Summary

In the currenrteport we describe biochemical and molecular

NADP- and NAD-isocitrate dehydrogenase activity was measured as previously described [1, 4]. Protein concentrations were measured by the Bradford method [25]. Mitochondrial and cytosolic cellular fractions were prepared as described by Daum et al [26], except that bovine serum albumin was EXPERIMENTAL PROCEDURES. State 111respiratory rateswere measured in the presence peptone) or YNB medium (0.17% yeast nitrogen base, 0.5% ammo- of 167 g M ADP with 13.3 mMof one of the following substrates: anium sulfate, pH 6.0) with carbon sources including glucose,glycerol, ketoglutarate, isocitrate, citrate, or pyruvate (12.0mM) plus malate lactate, ethanol, or acetate added to 2%. Uracil were added to YNB medium a t concentrations of 0.1 mg/ml to satisfy auxotrophic requirements. Cell growth was measured spectrophotometricaly at Amnm and logarithmic growth rates were deter-

RESULTS

Yeast MIitsoocchitornadterial

DISCUSSION

Findings

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