Behari R, Baker A
Department of Biochemistry, University of Cambridge, United Kingdom.
J Biol Chem. 1993 Apr 5;268(10):7315-22.
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.
已建立了一种从向日葵（Helianthus annuus）幼苗子叶中分离完整且具有导入能力的乙醛酸循环体的方法。通过蓖麻籽cDNA的体外转录以及在小麦胚芽裂解物中对RNA进行翻译，产生了放射性标记的异柠檬酸裂解酶（一种乙醛酸循环体基质蛋白）。异源异柠檬酸裂解酶被导入分离的向日葵乙醛酸循环体中，并受到蛋白酶保护。二氢叶酸还原酶，一种细胞质蛋白，既不与乙醛酸循环体结合也不被导入其中。当用线粒体部分替代乙醛酸循环体部分时，未观察到异柠檬酸裂解酶的导入。异柠檬酸裂解酶导入乙醛酸循环体是温度和ATP依赖性的。对异柠檬酸裂解酶基因进行逐步的羧基末端截短，转录并翻译以产生具有相同氨基末端但羧基末端缺失不同量的多肽。所有这些多肽都以与全长蛋白相同的特征进行导入，这表明靶向信息必定存在于前168个氨基酸内，并且与一些其他过氧化物酶体蛋白不同，羧基末端对于靶向和导入是可有可无的。
