Targeting of castor bean glyoxysomal isocitrate lyase to tobacco leaf peroxisomes.
The cDNA encoding castor bean endosperm isocitrate lyase (ICL) was expressed under the control of the promoter of the small subunit of pea ribulose bisphosphate carboxylase in transformed tobacco. ICL protein was detected using anti-ICL antibodies on immunoblots of total leaf protein extracts. Nycodenz density gradient separation of the extracts from the transgenic tobacco leaves showed ICL co-fractionated with hydroxypyruvate reductase, a peroxisomal matrix marker protein, and away from lactate dehydrogenase, a cytosolic marker protein. Immunoelectron microscopy of ultrathin leaf sections demonstrated the location of ICL within the matrix of the leaf peroxisomes of the transgenic plants. In vitro transcribed and translated ICL was also imported into leaf peroxisomes isolated from germinating sunflower seeds. The in vivo and in vitro import of this protein into leaf peroxisomes provides strong support for the notion that the import machinery of glyoxysomes and peroxisomes is very similar.
Onyeocha
I
I
Department of Biochemistry, University of Cambridge, UK.
Behari
R
R
Hill
D
D
Baker
A
A
D000429:Alcohol Oxidoreductases / Q000032:analysis; D001692:Biological Transport; D002499:Centrifugation, Density Gradient; D007887:Fabaceae / Q000235:genetics; D050537:Hydroxypyruvate Reductase; D007472:Iohexol; D007522:Isocitrate Lyase / Q000235:genetics*; D008830:Microbodies / Q000201:enzymology*; D016253:Microscopy, Immunoelectron; D030821:Plants, Genetically Modified; D010946:Plants, Medicinal; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics; D013347:Subcellular Fractions / Q000201:enzymology; D014026:Nicotiana / Q000235:genetics; D014162:Transfection
D011993:Recombinant Fusion Proteins; D007472:Iohexol; D000429:Alcohol Oxidoreductases; D050537:Hydroxypyruvate Reductase; D012273:Ribulose-Bisphosphate Carboxylase; D007522:Isocitrate Lyase