Instituto de Investigação e Inovação em Saúde (I3S), Universidade do Porto, Porto, Portugal.
Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal.
Methods Mol Biol. 2023;2643:333-343. doi: 10.1007/978-1-0716-3048-8_23.
Cell-free in vitro systems are invaluable tools to study the molecular mechanisms of protein translocation across biological membranes. We have been using such a strategy to dissect the mechanism of the mammalian peroxisomal matrix protein import machinery. Here, we provide a detailed protocol to import proteins containing a peroxisomal targeting signal type 2 (PTS2) into the organelle. The in vitro system consists of incubating a S-labeled reporter protein with a post-nuclear supernatant from rat/mouse liver. At the end of the incubation, the organelle suspensions are generally treated with an aggressive protease to degrade reporter proteins that did not enter peroxisomes, and the organelles are isolated by centrifugation and analyzed by SDS-PAGE and autoradiography. This in vitro system is particularly suited to characterize the functional consequences of PEX5 and PEX7 mutations found in patients affected with a peroxisomal biogenesis disorder.
无细胞体外系统是研究蛋白质跨生物膜转运的分子机制的宝贵工具。我们一直使用这种策略来剖析哺乳动物过氧化物酶体基质蛋白导入机制。在此，我们提供了一个详细的方案，用于将含有过氧化物酶体靶向信号类型 2（PTS2）的蛋白质导入细胞器。该体外系统包括用放射性标记的报告蛋白与来自大鼠/小鼠肝脏的核后上清液孵育。孵育结束时，通常用强烈的蛋白酶处理细胞器悬浮液，以降解未进入过氧化物酶体的报告蛋白，然后通过离心分离细胞器，并通过 SDS-PAGE 和放射自显影进行分析。该体外系统特别适合于表征在患有过氧化物酶体生物发生障碍的患者中发现的 PEX5 和 PEX7 突变的功能后果。
