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Transport of Proteins into Mitochondria

将蛋白质转运至线粒体

基本信息

批准号：
02454147
负责人：
MORI Masataka
金额：
$ 4.42万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

Ornithine transcarbamylase (OTC), a mitochondrial matrix enzyme of the urea cycle, is initially synthesized on cytosolic free ribosomes as a larger precursor pOTC with an NH_2-terminal presequence of 32 amino acid residues. pOTC is released into a cytosolic pool and is imported into the mitochondrial matrix with a half life of 1-2 min. We established an in vitro import system in which the purified pOTC was imported into isolated mitochondria. Using this system, we showed that a cytosolic protein factor (s) in the reticulocyte lysate is required for the import of the purified pOTC. A protein factor that binds to pOTC but not to mature OTC and was named presequence binding factor (PBF), was purified from the lysate. The purified PBF migrated as a single polypeptide of about 50, 000 Da on SDS-PAGE. On sucrose gradients, pOTC and PBF cosedimented as a complex of 7S. The purified PBF markedly stimulated the import of the purified pOPC into the mitochondria. PBF-stimulated pOTC import was further enhanced by hsp70 purified from yeast ; the hsp70 alone had little effect. Thus, PBF binds to the presequence portion of the precursor and may hold it in a transport-competent form in cooperation with hsp70. We next asked whether PBF is also involved in transport of other mitochondrial proteins. The precursor for aspartate aminotransferase synthesized in the PBF-depleted lysate failed to be imported into the mitochondria. Readdition of the purified PBF to the depleted lysate fully restored the import. Therefore, PBF appears to be involved in transport of a set of mitochondrial proteins having presequences.

鸟氨酸转氨甲酰酶（OTC）是一种线粒体尿素循环的基质酶，最初在胞浆游离核糖体上合成为一个较大的前体pOTC，其NH_2端前序列为32个氨基酸残基。pOTC被释放到胞质池中，并被导入到线粒体基质中，半衰期为1-2分钟。我们建立了一个体外导入系统，其中纯化的pOTC被导入到分离的线粒体中。使用该系统，我们表明网织红细胞裂解物中的胞质蛋白因子是纯化的pOTC输入所需的。从裂解物中纯化出一种与pOTC结合但不与成熟OTC结合的蛋白因子，称为前序列结合因子（PBF）。纯化的PBF在SDS-PAGE上以约50，000 Da的单一多肽迁移。在蔗糖梯度上，pOTC和PBF共沉淀为7S的复合物。纯化的PBF显著刺激纯化的pOPC进入线粒体。PBF刺激的pOTC输入进一步增强从酵母中纯化的热休克蛋白70;单独的热休克蛋白70几乎没有影响。因此，PBF结合到前体的前序列部分，并可能与hsp 70合作将其保持在运输能力形式。我们接下来询问PBF是否也参与其他线粒体蛋白的转运。在PBF耗尽的裂解物中合成的天冬氨酸氨基转移酶的前体未能被输入线粒体。将纯化的PBF再加入到耗尽的裂解物中完全恢复了输入。因此，PBF似乎参与了一组具有前序列的线粒体蛋白的运输。