Identification of a tobacco cDNA encoding a cytosolic NADP-isocitrate dehydrogenase

Abstract

A cDNA which encodes a specific member of the NADP-dependent isocitrate dehydrogenase (ICDH) multi-isoenzyme family has been isolated from a tobacco cell suspension library, and the expression pattern of ICDH transcripts examined in various plant tissues. To assign this cDNA to a specific ICDH isoenzyme, the major, cytosolic ICDH isoenzyme of tobacco leaves (ICDH1) was purified to homogeneity and its N-terminus as well as several tryptic peptides, representing 30% of the protein, were sequenced. The comparison of these amino acid sequences with the deduced protein sequence of the cDNA confirmed that this clone encodes for ICDH1. The total ICDH specific activity and protein content were higher in vascular-enriched tobacco leaf tissue than in deveined (depleted in midrib and first-order veins) leaves. Taking advantage of antibodies raised against either ICDH1 or the chloroplastic ICDH2 isoenzyme from tobacco cell suspensions, an immuno-cytochemical approach indicated that the ICDH1 isoenzyme, located in the cytosolic compartment of tobacco leaf cells, is responsible for this expression pattern. This observation was confirmed by northern blot analyses, using a specific probe obtained from the 3' non-coding region of the ICDH1 cDNA. A comparison of ICDH protein sequences shows a large degree of similarity between eukaryotes (> 60%) but a poor homology is observed when compared to Escherichia coli ICDH (< 20%). However, it was found that the amino acids implicated in substrate binding, deduced from the 3-dimensional structure of the E. coli NADP-ICDH, appear to be conserved in all the deduced eukaryotic ICDH proteins reported until now.