Pavlov Pavel F, Rudhe Charlotta, Bhushan Shashi, Glaser Elzbieta
Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Science, Stockholm University, Sweden.
Methods Mol Biol. 2007;372:297-314. doi: 10.1007/978-1-59745-365-3_22.
In plants, the majority of mitochondrial and chloroplast proteins are nuclear encoded, synthesized on cytosolic polyribosomes, and then imported into the organelle. Most of the nuclear encoded precursor proteins contain an N-terminal extension called signal or targeting peptide that directs the protein to the correct organelle. Here, we describe in vitro and in vivo methods to study mitochondrial protein import. In a common single-organelle in vitro import procedure, transcribed/translated precursor proteins are imported into isolated mitochondria. A novel semi-in vivo system for simultaneous import of precursor proteins into isolated mitochondria and chloroplasts, called a dual-import system, is superior to the single-import system as it abolishes mistargeting of chloroplast precursors into mitochondria as observed in a single-organelle import system. Precursor proteins can also be imported into the organelles in vivo using an intact cellular system. In vivo approaches include import of transiently expressed fusion constructs containing a targeting peptide or a precursor protein fused to a reporter gene, most commonly the green fluorescence protein in protoplasts or in an Agrobacterium-mediated system in intact tobacco leaves.
在植物中，大多数线粒体和叶绿体蛋白是由核基因编码的，在胞质多聚核糖体上合成，然后导入细胞器。大多数核基因编码的前体蛋白含有一个N端延伸序列，称为信号肽或靶向肽，它将蛋白质导向正确的细胞器。在这里，我们描述了研究线粒体蛋白导入的体外和体内方法。在一个常见的单细胞器体外导入程序中，转录/翻译的前体蛋白被导入分离的线粒体。一种用于将前体蛋白同时导入分离的线粒体和叶绿体的新型半体内系统，称为双导入系统，优于单导入系统，因为它消除了在单细胞器导入系统中观察到的叶绿体前体蛋白错误靶向到线粒体的情况。前体蛋白也可以使用完整的细胞系统在体内导入细胞器。体内方法包括导入含有靶向肽或与报告基因融合的前体蛋白的瞬时表达融合构建体，最常见的是原生质体中的绿色荧光蛋白或完整烟草叶片中农杆菌介导系统中的绿色荧光蛋白。
