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 tar geting peptide. However, even if this cDNA clearly encodes a noncytoso lic ICDH, it is not possible to say from the targeting peptide sequenc e to which subcellular compartment the protein is addressed. To respon d to this question, we have transformed tobacco plants with a construc t containing the entire targeting signal-encoding sequence in front of a modified green fluorescent protein (GFP) gene. This construct was p laced under the control of the cauliflower 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 co nstruct but lacking the nucleotide sequence corresponding to the ICDH- targeting peptide, in which the GFP is retained in the cytoplasm, By o ptical and confocal microscopy of leaf epiderm and Western blot analys es, we show that the putative-targeting sequence encoded by the cDNA a ddresses the GFP exclusively into the mitochondria of plant cells. The refore, we conclude that this cDNA encodes a mitochondrial ICDH.