Many mitochondrial and chloroplast proteins are encoded in the nucleus and subsequently imported into the organelles via active protein transport systems. While usually highly specific, some proteins are dual-targeted to both organelles. In tobacco (<i>Nicotiana tabacum L.</i>), the cDNA encoding the mitochondrial isoform of NADP+-dependent isocitrate dehydrogenase (NADP+-ICDH) contains two translational ATG start sites, indicating the possibility of two tandem targeting signals. In this work the putative mitochondrial and chloroplastic targeting signals from NADP+-ICDH were fused to a yellow fluorescent protein (YFP) to generate a series of constructs and introduced into tobacco leaves by <i>Agrobacterium</i>-mediated transient transfection. The subsequent sub-cellular locations of the ICDH:YFP fusion proteins were then examined under the confocal microscope. Constructs predicted to be targeted to the chlroplast all localized to the chloroplast. However, this was not the case for constructs that were predicted to be mitochondrial targeted. While some constructs localized to mitochondria, others appeared to be chloroplast localized. This was attributed to an additional 50 amino acid residues of the mature NADP+-ICDH protein which was present in those constructs. In addition, during the process of generating these constructs our sequence analysis indicated a stop codon present at amino acid position 161 of the mature NADP+-ICDH protein from both Xanthi and Petit Havana cultivars of tobacco. This was confirmed by multiple sequencing reactions and created discrepancies with the reported sequence present in the database. The results of this study raise interesting questions with regard to the targeting and processing of NADP+-ICDH.

Many mitochondrial and chloroplast proteins are encoded in the nucleus and subsequently imported into the organelles via active protein transport systems. While usually highly specific, some proteins are dual-targeted to both organelles. In tobacco (<i>Nicotiana tabacum L.</i>), the cDNA encoding the mitochondrial isoform of NADP+-dependent isocitrate dehydrogenase (NADP+-ICDH) contains two translational ATG start sites, indicating the possibility of two tandem targeting signals. In this work the putative mitochondrial and chloroplastic targeting signals from NADP+-ICDH were fused to a yellow fluorescent protein (YFP) to generate a series of constructs and introduced into tobacco leaves by <i>Agrobacterium</i>-mediated transient transfection. The subsequent sub-cellular locations of the ICDH:YFP fusion proteins were then examined under the confocal microscope. Constructs predicted to be targeted to the chlroplast all localized to the chloroplast. However, this was not the case for constructs that were predicted to be mitochondrial targeted. While some constructs localized to mitochondria, others appeared to be chloroplast localized. This was attributed to an additional 50 amino acid residues of the mature NADP+-ICDH protein which was present in those constructs. In addition, during the process of generating these constructs our sequence analysis indicated a stop codon present at amino acid position 161 of the mature NADP+-ICDH protein from both Xanthi and Petit Havana cultivars of tobacco. This was confirmed by multiple sequencing reactions and created discrepancies with the reported sequence present in the database. The results of this study raise interesting questions with regard to the targeting and processing of NADP+-ICDH.