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Protein import into mitochondria and its disorders

蛋白质进入线粒体及其疾病

基本信息

批准号：
04454174
负责人：
MORI Masataka
金额：
$ 4.54万
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：
1992
资助国家：
日本
起止时间：
1992 至 1993
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 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

鸟氨酸转氨基甲酰基酶（OTC）是一种尿素循环的线粒体基质酶，最初是在胞质游离核糖体上合成的一个较大的前体poc，其NH_2末端有32个氨基酸残基，poc被释放到胞质池中并进入线粒体基质，其半衰期为1-2 min。我们建立了一个体外导入系统，将纯化的重组pOTC导入离体线粒体。使用该系统，我们发现网状细胞裂解液中的细胞质蛋白因子(s)是纯化的pOTC进口所必需的。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，命名为前置结合因子（PBF）。纯化后的PBF在SDS-PAGE上以单个约50,000 Da的多肽形式迁移。在蔗糖梯度上，pOTC和PBF以7S络合物的形式共同沉积。纯化的PBF显著刺激纯化的pOTC进入线粒体。从酵母中纯化的hsp70进一步增强了pbf刺激的poc进口；单独使用hsp70几乎没有效果。因此，PBF结合前体的前体部分，并可能与hsp70合作将其保持在运输能力的形式。在pbf缺失的裂解物中合成的天冬氨酸转氨酶和苹果酸脱氢酶的前体不能输入线粒体。将纯化的PBF重新读取到耗尽的裂解物完全恢复了进口。因此，PBF似乎参与了一组具有先兆序列的线粒体蛋白的运输。另一方面，从裂解物中消耗PBF对没有可切割前体的3-氧酰基辅酶a硫醇酶的输入影响不大。这些结果表明，线粒体蛋白输入的pbf依赖性和非依赖性途径确实存在。后一途径的蛋白质，包括3-氧酰基辅酶a硫酶，可能跳过pbf依赖的步骤，直接与线粒体表面的“前序受体”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cdna。小鼠PBF在大肠杆菌中高度表达，纯化后接近均匀性。重组PBF的表征正在进行中。少