Mukhopadhyay Abhijit, Hammen Philip, Waltner-Law Mary, Weiner Henry
Department of Biochemistry, Purdue University, West Lafayette, IN 47907-1153, USA.
Protein Sci. 2002 May;11(5):1026-35. doi: 10.1110/ps.3760102.
Most mitochondrial matrix space proteins are synthesized as a precursor protein, and the N-terminal extension of amino acids that served as the leader sequence is removed after import by the action of a metalloprotease called mitochondrial processing peptidase (MPP). The crystal structure of MPP has been solved very recently, and it has been shown that synthetic leader peptides bind with MPP in an extended conformation. However, it is not known how MPP recognizes hundreds of leader peptides with different primary and secondary structures or when during import the leader is removed. Here we took advantage of the fact that the structure of the leader from rat liver aldehyde dehydrogenase has been determined by 2D-NMR to possess two helical portions separated by a three amino acid (RGP) linker. When the linker was deleted, the leader formed one long continuous helix that can target a protein to the matrix space but is not removed by the action of MPP. Repeats of two and three leaders were fused to the precursor protein to determine the stage of import at which processing occurs, if MPP could function as an endo peptidase, and if it would process if the cleavage site was part of a helix. Native or linker deleted constructs were used. Import into isolated yeast mitochondria or processing with recombinantly expressed MPP was performed. It was concluded that processing did not occur as the precursor was just entering the matrix space, but most likely coincided with the folding of the protein. Further, finding that hydrolysis could not take place if the processing site was part of a stable helix is consistent with the crystal structure of MPP. Lastly, it was found that MPP could function at sites as far as 108 residues from the N terminus of the precursor protein, but its ability to process decreases exponentially as the distance increases.
大多数线粒体基质空间蛋白最初都是以前体蛋白的形式合成的，在导入线粒体后，由一种名为线粒体加工肽酶（MPP）的金属蛋白酶作用去除作为前导序列的N端氨基酸延伸部分。MPP的晶体结构最近已被解析，结果表明合成的前导肽以伸展构象与MPP结合。然而，尚不清楚MPP如何识别数百种具有不同一级和二级结构的前导肽，以及在前导肽导入过程中的何时被去除。在此，我们利用了大鼠肝脏醛脱氢酶前导序列的结构已通过二维核磁共振确定这一事实，该前导序列具有两个由三个氨基酸（RGP）连接子分隔的螺旋部分。当连接子缺失时，前导序列形成一个长的连续螺旋，该螺旋可将蛋白质靶向基质空间，但不会被MPP作用去除。将两个或三个前导序列重复片段与前体蛋白融合，以确定加工发生的导入阶段、MPP是否可作为内切肽酶发挥作用，以及如果切割位点是螺旋的一部分时MPP是否会进行加工。使用了天然或缺失连接子的构建体。进行了导入分离的酵母线粒体或用重组表达的MPP进行加工的实验。得出的结论是，加工并非在前体刚进入基质空间时发生，而是很可能与蛋白质的折叠同时发生。此外，发现如果加工位点是稳定螺旋的一部分则无法发生水解，这与MPP的晶体结构一致。最后，发现MPP可在前体蛋白N端起作用的位点距离可达108个残基，但随着距离增加其加工能力呈指数下降。
