Protein import into mitochondria and its disorders

鸟氨酸转氨基甲酸酶(OTC)是尿素循环中的一种线粒体基质酶，最初在胞质游离核糖体上合成，是一种较大的POTC前体，其NH_2末端的前置序列为32个氨基酸残基，POTC被释放到细胞质池中，并被输入到线粒体基质中，半衰期为1-2分钟。我们建立了将纯化的重组POTC导入分离的线粒体的体外导入系统。利用该系统，我们证明了网织红细胞裂解液中的胞浆蛋白因子(S)是纯化的pOTC输入所必需的。从裂解液中纯化出一种与POTC结合而不与成熟OTC结合的蛋白因子，称为序列前结合因子(PbF)。纯化的PBF在SDS-PAGE上以约50000 Da的单一多肽迁移。在蔗糖梯度上，POTC和PBF以7S的复合物形式存在。纯化的PBF显著刺激POTC进入线粒体。带…的受PBF刺激的POTC进口从酵母中纯化的HSP70进一步增强；单独的HSP70几乎没有作用。因此，PBF与前体的前序列部分结合，并可能与HSP70合作以运输能力的形式持有它。PBF耗竭的裂解液中合成的天冬氨酸氨基转移酶和苹果酸脱氢酶前体未能进入线粒体。将纯化的PBF重新提取到耗尽的裂解物中，完全恢复了进口。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，从裂解液中去除PBF对没有可切割前序列的3-氧酰基-CoA硫解酶的输入几乎没有影响。这些结果表明，线粒体蛋白输入确实存在PBF依赖和非依赖的途径。后一种途径的蛋白质包括3-氧乙酰-辅酶A硫解酶，可以跳过依赖PBF的步骤，直接与线粒体表面的“前序列受体”结合。根据纯化的PBF的部分氨基酸序列，分离出小鼠和人PBF的cDNAs。小鼠PBF在大肠杆菌中高效表达，纯化到接近均一的水平。重组PBF的鉴定正在进行中。较少

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