Plant Molecular Biology 36: 691–698, 1998.
1998 Kluwer Academic Publishers. Printed in Belgium.
-dependent isocitrate dehydrogenase from Arabidopsis thaliana.
Characterization of two closely related subunits
Robert H. Behal
and David J. Oliver
Department of Botany, Iowa State University, Ames, IA, 50011–1020, USA (
author for correspondence)
Received 11 July 1997; accepted in revised form 20 October 1997
Key words: NAD
-dependent isocitrate dehydrogenase, Arabidopsis thaliana
Two cDNA clones which appear to encode different subunits of NAD
-dependent isocitrate dehydrogenase (IDH;
EC 220.127.116.11) were identiﬁed by homology searches from the Arabidopsis EST database. These cDNA clones were
obtained and sequenced; both encoded full-length messages and displayed 82.7% nucleotide sequence identity over
the coding region. The deduced amino acid sequences revealed preprotein lengths of 367 residues, with an amino
acid identity of 86.1%. Genomic Southern blot analysis showed distinct single-copy genes for both IDH subunits.
Both IDH subunits were expressed as recombinant proteins in Escherichia coli, and polyclonal antibodies were
raised to each subunit. The Arabidopsis cDNA clones were expressed in Saccharomyces cerevisiae mutants which
were deﬁcient in either one or both of the yeast NAD
-dependent IDH subunits. The Arabidopsis cDNA clones
failed to complement the yeast mutations; although both IDH-I and IDH-II were expressed at detectable levels,
neither protein was imported into the mitochondria.
The mitochondrial NAD
dehydrogenase (IDH) performs a critical role in the
metabolism of all organisms. Its position as the ﬁrst
decarboxylative enzyme of the TCA cycle leads to
IDH a unique regulatory opportunity. Under condi-
tions in which carbon ﬂux is directed towards biosyn-
thesis instead of energy production (such as the early
stages of oil-seed germination), other researchers have
indicated a decrease in activity of IDH [17, 24]. Incid-
ental to exploring this phenomenon, we identiﬁed and
characterized Arabidopsis thaliana cDNA clones rep-
resenting two subunits of IDH.
All known eukaryotic IDHs have a hetero-
oligomeric structure. The simplest of these systems,
Saccharomyces cerevisiae, has been intensively stud-
ied [3, 4, 5, 16, 37]. Yeast IDH exists as a hetero-
octamer, consisting of four copies of IDH1 and four
copies of IDH2. Kinetic analysis of yeast complexes
Thenucleotidesequencedatareportedwill appear intheEMBL,
GenBank and DDBJ Nucleotide Sequence Databases under the
accession numbers U81993, U81994, U82203 and AF015923.
that IDH1 serves a regulatory role, while IDH2 is
the catalytic moiety. Gel ﬁltration analysis of strains
expressing only IDH1 or IDH2 demonstrated that
IDH1 and IDH2 exist as monomers; subunit interac-
tion would seem to require both the IDH1 and IDH2
IDH from higher eukaryotes has been studied
primarily in mammals [9, 10, 28, 29]. These IDHs
consist of three differentsubunits (
the basic form
; the smallest oligomeric state has
a estimated molecular mass of 160 kDa. Heterogen-
eity of the bovine
subunit has been reported [29,
36]. Catalytic activity resides in the
this manuscript we present the ﬁrst molecular analysis
-dependent isocitrate dehydrogenase from a
plant, Arabidopsis thaliana.
Materials and methods
from the Arabidopsis Biological Research Center at