Sjöling S, Waltner M, Kalousek F, Glaser E, Weiner H
Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, Sweden.
Eur J Biochem. 1996 Nov 15;242(1):114-21. doi: 10.1111/j.1432-1033.1996.0114r.x.
The plant mitochondrial processing peptidase (MPP) that catalyses the cleavage of the presequences from precursor proteins during or after protein import is a membrane-bound enzyme that constitutes an integral part of the bc1 complex of the respiratory chain. In contrast, MPP from mammals is soluble in the matrix space and does not form part of the respiratory chain. In the present study, we have compared the substrate specificity of the isolated spinach leaf bc1/MPP with rat liver MPP using synthetic signal peptides and different mitochondrial precursor proteins. Inhibition studies of processing with synthetic peptides showed a similar inhibition pattern for plant and rat MPP activity. A peptide derived from the presequence of rat liver mitochondrial aldehyde dehydrogenase (ALDH) was a potent inhibitor of the spinach and rat MPP. Two nonprocessed signal peptides, rhodanese and linker-deleted ALDH (a form of ALDH that lacks the RGP linker connecting two helices in the presequence) had lower inhibitory effects towards each protease. The signal peptide from thiolase, another nonprocessed protein, had little inhibitory effect on MPP. Peptides derived from presequence of the plant Nicotiana plumbaginifolia F1 beta also showed a similar inhibitory pattern with rat MPP as with spinach MPP processing. In-vitro synthesised precursors of plant N. plumbaginifolia F1 beta and rat liver ALDH were cleaved to mature form by both spinach and rat MPP. However, the efficiency of processing was higher with the homologous precursor. Linker-deleted ALDH, rhodanese, and thiolase were not processed by the mammalian or plant MPP. However, both forms of MPP cleaved a mutated form of rhodanese that possesses a typical MPP cleavage motif, RXY S. Addition of the same cleavage motif to thiolase did not result in processing by either MPP. These results show that similar higher-order structural elements upstream from the cleavage site are important for processing by both the membrane-bound plant and the soluble mammalian MPP.
植物线粒体加工肽酶（MPP）在蛋白质导入期间或之后催化从前体蛋白中切除前导序列，它是一种膜结合酶，是呼吸链bc1复合体的一个组成部分。相比之下，哺乳动物的MPP可溶于线粒体基质空间，并非呼吸链的组成部分。在本研究中，我们使用合成信号肽和不同的线粒体前体蛋白，比较了分离的菠菜叶bc1/MPP与大鼠肝脏MPP的底物特异性。用合成肽进行的加工抑制研究表明，植物和大鼠MPP活性具有相似的抑制模式。源自大鼠肝脏线粒体醛脱氢酶（ALDH）前导序列的一种肽是菠菜和大鼠MPP的有效抑制剂。两种未加工的信号肽，硫氰酸酶和缺失连接子的ALDH（一种ALDH形式，其前导序列中缺少连接两个螺旋的RGP连接子）对每种蛋白酶的抑制作用较低。来自硫解酶（另一种未加工蛋白）的信号肽对MPP几乎没有抑制作用。源自植物蓝烟草F1β前导序列的肽对大鼠MPP也表现出与菠菜MPP加工相似的抑制模式。体外合成的植物蓝烟草F1β和大鼠肝脏ALDH的前体均被菠菜和大鼠MPP切割成成熟形式。然而，同源前体的加工效率更高。缺失连接子的ALDH、硫氰酸酶和硫解酶均未被哺乳动物或植物MPP加工。然而，两种形式的MPP都切割了具有典型MPP切割基序RXY S的硫氰酸酶突变体形式。在硫解酶中添加相同的切割基序不会导致两种MPP对其进行加工。这些结果表明，切割位点上游类似的高阶结构元件对于膜结合的植物MPP和可溶性哺乳动物MPP的加工都很重要。
