The carboxyl terminus of isocitrate lyase is not essential for import into glyoxysomes in an in vitro system.
A procedure has been established for the isolation of intact and import-competent glyoxysomes from the cotyledons of sunflower (Helianthus annuus) seedlings. Radiolabeled isocitrate lyase (a glyoxysomal matrix protein) was produced by in vitro transcription of the castor bean cDNA and translation of the RNA in a wheat germ lysate. The heterologous isocitrate lyase was imported into isolated sunflower glyoxysomes and protease-protected. Dihydrofolate reductase, a cytoplasmic protein, neither bound to nor imported into the glyoxysomes. Import of isocitrate lyase was not observed when the glyoxysome fraction was replaced by a mitochondrial fraction. The import of isocitrate lyase into glyoxysomes was temperature- and ATP-dependent. Progressive carboxyl-terminal truncations of the isocitrate lyase gene were transcribed and translated to yield polypeptides with the same amino terminus but lacking varying amounts of the carboxyl terminus. All these polypeptides imported with the same characteristics as the full-length protein, suggesting that targeting information must be present within the first 168 amino acids, and, unlike some other peroxisomal proteins, the carboxyl terminus is dispensable for targeting and import.
Behari
R
R
Department of Biochemistry, University of Cambridge, United Kingdom.
Baker
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001693:Biological Transport, Active; D015153:Blotting, Western; D006368:Helianthus / Q000201:enzymology* / Q000648:ultrastructure; D066298:In Vitro Techniques; D007522:Isocitrate Lyase / Q000378:metabolism*; D008830:Microbodies / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014176:Protein Biosynthesis
D000255:Adenosine Triphosphate; D007522:Isocitrate Lyase