Protein import into mitochondria and its disorders

鸟氨酸转氨甲酰酶（Ornithine transcarbamylase，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被释放到细胞溶质池中，并以1- 100%的半衰期输入到线粒体基质中。我们建立了一个体外导入系统，其中纯化的重组pOTC导入分离的线粒体。利用该系统，我们发现网织红细胞裂解液中的一种蛋白因子是pOTC导入所必需的，并从裂解液中纯化了一种与pOTC结合但不与成熟OTC结合的蛋白因子，命名为前序列结合因子（PBF）。纯化的PBF在SDS-PAGE上以约50，000 Da的单一多肽形式迁移，在蔗糖梯度上，pOTC和PBF以7S的复合物形式共沉淀。PBF刺激的pOTC输入w 关于我们 从酵母中纯化的HSP 70进一步增强;单独的HSP 70几乎没有效果。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。天冬氨酸转氨酶和苹果酸脱氢酶的前体合成的PBF耗尽裂解液未能被输入到线粒体。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。另一方面，从裂解物中消耗PBF对没有可裂解前序列的3-氧代酰基-CoA硫解酶的输入几乎没有影响。这些结果表明，PBF依赖性和非依赖性的线粒体蛋白输入途径确实存在。后一途径的蛋白质包括3-氧代酰基-CoA硫解酶可以跳过PBF依赖性步骤并直接结合线粒体表面上的“前序列受体”。根据纯化的PBF的部分氨基酸序列，分离了小鼠和人PBF的cDNA，小鼠PBF在大肠杆菌中得到高效表达，并纯化至接近均一。重组PBF的表征正在进行中。少

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 recombinant 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 about50,000 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 pOTC into the mitochondria. PBF-stimulated pOTC import w … More as 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. The precursor for aspartate aminotransferase and malate dehydrogenase 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. On the other hand, depletion of PBF from the lysate had little effect on the import of 3-oxoacyl-CoA thiolase that has no cleavable presequence. These results indicate that PBF-dependent and -independent pathways of mitochondrial protein import do exist. Proteins of the latter pathway including 3-oxoacyl-CoA thiolase may skip a PBF-dependent step and bind directly to the "presequence receptor" on the mitochondrial surface. cDNAs for mouse and human PBF were isolated based on partial amino acid sequences of the purified PBF.Mouse PBF was highly expressed in Escherichia coli and was purified to near homogeneity. Characterization of the recombinant PBF is underway. Less