Mitochondrial localization of a NADP-dependent [corrected] 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 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 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.
Gálvez
S
S
Institut de Biotechnologie des Plantes (Centre National de la Recherche Scientifique ERS569), Bât 630, Université de Paris-Sud, 91405 Orsay Cédex, France.
Roche
O
O
Bismuth
E
E
Brown
S
S
Gadal
P
P
Hodges
M
M
D000595:Amino Acid Sequence; D015415:Biomarkers; D005434:Flow Cytometry; D015723:Gene Library; D005819:Genetic Markers; D049452:Green Fluorescent Proteins; D007521:Isocitrate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D014026:Nicotiana
D015415:Biomarkers; D005819:Genetic Markers; D007527:Isoenzymes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)