Szigyarto C, Dessi P, Smith M K, Knorpp C, Harmey M A, Day D A, Glaser E, Whelan J
Department of Biochemistry, Stockholm University, Sweden.
Plant Mol Biol. 1998 Jan;36(1):171-81. doi: 10.1023/a:1005977716814.
Nuclear-encoded mitochondrial precursor proteins are proteolytically processed inside the mitochondrion after import. The general mitochondrial processing activity in plant mitochondria has been shown to be integrated into the cytochrome bc1 complex of the respiratory chain. Here we investigate the occurrence of an additional, matrix-located processing activity by incubation of the precursors of the soybean mitochondrial proteins, alternative oxidase, the FAd subunit of the ATP synthetase and the tobacco F1 beta subunit of the ATP synthase, with the membrane and soluble components of mitochondria isolated from soybean cotyledons and spinach leaves. A matrix-located peptidase specifically processed the precursors to the predicted mature form in a reaction which was sensitive to orthophenanthroline, a characteristic inhibitor of mitochondrial processing peptidase (MPP). The specificity of the matrix peptidase was illustrated by the inhibition of processing of the alternative oxidase precursor in both soybean and spinach matrix extracts upon altering a single amino acid residue in the targeting presequence (-2 Arg to Gly). Additionally, there was no evidence for general proteolysis of precursor proteins incubated with the matrix. The purity of the matrix fractions was ascertained by spectrophotometric and immunological analyses. The results demonstrate that there is a specific processing activity in the matrix of soybean and spinach in addition to the previously well characterized membrane-bound MPP integrated into the cytochrome bcl complex of the respiratory chain.
核编码的线粒体前体蛋白在导入后在线粒体内进行蛋白水解加工。植物线粒体中的一般线粒体加工活性已被证明整合到呼吸链的细胞色素bc1复合物中。在这里，我们通过将大豆线粒体蛋白、交替氧化酶、ATP合酶的FAd亚基和烟草ATP合酶的F1β亚基的前体与从大豆子叶和菠菜叶中分离的线粒体的膜和可溶性组分一起孵育，来研究另一种位于基质中的加工活性的存在。一种位于基质中的肽酶在对邻菲罗啉敏感的反应中将前体特异性加工成预测的成熟形式，邻菲罗啉是线粒体加工肽酶（MPP）的一种特征性抑制剂。当在靶向前导序列中改变单个氨基酸残基（-2 Arg变为Gly）时，大豆和菠菜基质提取物中交替氧化酶前体加工的抑制说明了基质肽酶的特异性。此外，没有证据表明与基质一起孵育的前体蛋白会发生一般的蛋白水解。通过分光光度法和免疫学分析确定了基质组分的纯度。结果表明，除了先前已充分表征的整合到呼吸链细胞色素bcl复合物中的膜结合MPP外，大豆和菠菜的基质中还存在一种特异性加工活性。
