Molecular characterization of higher plant NAD-dependent isocitrate dehydrogenase: evidence for a heteromeric structure by the complementation of yeast mutants
M. Lancien et al., Molecular characterization of higher plant NAD-dependent isocitrate dehydrogenase: evidence for a heteromeric structure by the complementation of yeast mutants, PLANT J, 16(3), 1998, pp. 325-333
NAD-dependent isocitrate dehydrogenase (IDH) is a key enzyme controlling th
e activity of the citric acid cycle. Despite more than 30 years of work, th
e plant enzyme remains poorly characterized. In this paper, a molecular cha
racterization of the plant IDH is presented. Starting from probes defined a
ccording to sequence comparisons, three full-length cDNAs named Ntidha Ntid
hb and Ntidhc encoding different IDH subunits have been isolated from a Nic
otiana tabacum cell suspension library. Sequence comparisons of the tobacco
IDH subunits with the E. coli NADP-dependent enzyme, and the yeast IDH1 an
d IDH2 subunits suggested that only IDHa had the capacity to be catalytic a
s IDHb and IDHc were lacking certain residues implied in catalysis. The abi
lity of antibodies raised against the recombinant IDHa protein to preferent
ially cross-react with IDH2 indicated that IDHa was more closely related to
IDH2 than to IDH1. Complementation of yeast single IDH mutants showed that
IDHb and IDHc could replace the function of the yeast regulatory IDH1 subu
nit. Although IDHa was unable to complement the IDH2 mutant, its catalytic
function was revealed by the ability of two heteromeric enzymes, composed o
f either IDHa with IDHb or IDHa with IDHc, to replace IDH function in a yea
st double mutant lacking both subunits. Expression studies at the protein a
nd mRNA levels show that each subunit is present in both root and leaf tiss
ues and that the three IDH genes respond in the same way to nitrate additio
n. Taken together, such observations suggest that the physiologically activ
e enzyme is composed of the three different subunits. These results show fo
r the first time that the plant IDH is heteromeric and that IDH subunit com
position appears to be conserved between plant and animal kingdoms.