Mitochondrial localization of a NADR-dependent isocitrate dehydrogenase isoenzyme by using the green fluorescent protein as a marker

In this work, we describe the isolation of a new cDNA encoding an NADP-dependent isocitrate dehydrogenase (ICDH). The nucleotide sequence in its 5* region gives a deduced amino acid sequence indicative of a targeting peptide. However, even if this cDNA clearly encodes a noncytosolic ICDH, it is not possible to say from the targeting peptide sequence to which subcellular compartment the protein is addressed. To respond to this question, we have transformed tobacco plants with a construct containing the entire targeting signal-encoding sequence in front of a modified green fluorescent protein (GFP) gene. This construct was placed under the control of the caulif lower mosaic virus 35S promoter, and transgenic tobacco plants were regenerated. At the same time, and as a control, we also have transformed tobacco plants with the same construct but lacking the nucleotide sequence corresponding to the ICDH-targeting peptide, in which the GFP is retained in the cytoplasm. By optical and confocal microscopy of leaf epiderm and Western blot analyses, we show that the putative-targeting sequence encoded by the cDNA addresses the GFP exclusively into the mitochondria of plant cells. Therefore, we conclude that this cDNA encodes a mitochondrial ICDH. A common feature within both plant and animal kingdoms is the presence of isoenzymes encoded by multi-gene families. Often they are located in different subcellular compartments within the same cell, each isoenzyme being exposed to different chemical environments and each participating in different metabolic pathways. With the development of molecular biology, an ever increasing number of cDNAs encoding different isoforms of a given protein have been described. The establishment of a correspondence between a cDNA and the isoenzyme it encodes is primordial in determining the physiological role(s) of the protein (1, 2). In general, the most used criterium is the suggestion of a putative-targeting peptide (as deduced from the nucleotide sequence) followed by a study of its composition; however, this kind of analysis is often inconclusive (3–5). The assimilation of ammonia is a good example of a plant metabolic pathway in which differently located isoforms are implicated (6–9). Ammonia produced from nitrogen fixation, nitrate reduction, or photorespiration is incorporated into an organic compound by the glutamine synthetaseyglutamate synthase (GSyGOGAT) pathway (10). To be active, this cycle requires a continuous supply of 2-oxoglutarate, which is synthesized by the action of isocitrate dehydrogenase. Two different enzymes catalyzing the oxidative decarboxylation of isocitrate to 2-oxoglutarate can be distinguished by their cofactor specificity: An NAD-dependent form (IDH; E.C.1.1.1.41) is located exclusively in the mitochondria where it plays a role in the Krebs cycle, and several NADP-dependent forms (ICDH; E.C.1.1.1.42) are located in cytosol, mitochondria, chloroplasts, and peroxisomes (see ref. 9 for a review); as yet, their physiological roles have not been elucidated. Although it has been proposed that the cytosolic ICDH isoform is responsible for producing the 2-oxoglutarate necessary for ammonia assimilation (11), proof is still lacking. Up until now, several plant ICDH-encoding cDNAs have been described in the literature (12–14), but they all resemble the tobacco cytosolic isoform (2). A recent and potentially powerful tool for studying the in vivo subcellular localization of a protein is the green fluorescent protein (GFP). An obvious advantage of this reporter protein is that the fluorescence emission does not require any added cofactor or substrate, and it is not necessary to destroy the tissue to visualize the fluorescence emission. Because the fluorescence emission of wild-type GFP is poorly detectable in a number of plant species including tobacco, several GFP mutants have been created (15–18) to make this protein a suitable marker in plant systems. Major modifications include altering the codon usage to be optimal for humans, maize, and Arabidopsis, and thus eliminating a cryptic intron site and changing the amino acid composition of the chromophore, e.g., 65-SYG–65-TYG. Such modifications have been carried out by J. Sheen and coworkers (15), and the resulting GFP [sGFP(S65T)] gives a 120-fold brighter signal than the original GFP in plant cells. This result has led to several examples of brightly f luorescent transgenic tobacco lines transformed with a modified GFP (16, 18). In this work, we report the isolation and characterization of the first plant cDNA encoding a noncytosolic ICDH. A modified GFP has been used to study the subcellular localization of this isoform. A chimeric gene encoding the putativetargeting sequence of the ICDH fused to the coding sequence of the sGFP(S65T) mutant has been used to transform tobacco plants. The analysis of the resulting transgenic plants shows that the modified GFP is exclusively located in the mitochondria of plant cells. Therefore, we conclude that the cDNA described in this work encodes a mitochondrial isoform of the plant enzyme. MATERIALS AND METHODS Isolation of cDNA Clones. A partial cDNA was isolated from a tobacco cell suspension library constructed in lZAPII as The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y957813-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: ICDH, NADP-dependent isocitrate dehydrogenase; GFP, green fluorescent protein; pSK1, pBluescript SK1. Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. X96728). ‡To whom reprint requests should be addressed. [email protected]. u-psud.fr.