Faculty of Biology and Signalling Research Centres BIOSS and CIBSS, University of Freiburg, 79104 Freiburg, Germany.
Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, 79104 Freiburg, Germany.
Cells. 2024 Oct 9;13(19):1671. doi: 10.3390/cells13191671.
Mitochondria provide cells with energy and regulate the cellular metabolism. Almost all mitochondrial proteins are nuclear-encoded, translated on ribosomes in the cytoplasm, and subsequently transferred to the different subcellular compartments of mitochondria. Here, we developed OptoMitoImport, an optogenetic tool to control the import of proteins into the mitochondrial matrix via the presequence pathway on demand. OptoMitoImport is based on a two-step process: first, light-induced cleavage by a TEV protease cuts off a plasma membrane-anchored fusion construct in close proximity to a mitochondrial targeting sequence; second, the mitochondrial targeting sequence preceding the protein of interest recruits to the outer mitochondrial membrane and imports the protein fused to it into mitochondria. Upon reaching the mitochondrial matrix, the matrix processing peptidase cuts off the mitochondrial targeting sequence and releases the protein of interest. OptoMitoImport is available as a two-plasmid system as well as a P2A peptide or IRES sequence-based bicistronic system. Fluorescence studies demonstrate the release of the plasma membrane-anchored protein of interest through light-induced TEV protease cleavage and its localization to mitochondria. Cell fractionation experiments confirm the presence of the peptidase-cleaved protein of interest in the mitochondrial fraction. The processed product is protected from proteinase K treatment. Depletion of the membrane potential across the inner mitochondria membrane prevents the mitochondrial protein import, indicating an import of the protein of interest by the presequence pathway. These data demonstrate the functionality of OptoMitoImport as a generic system with which to control the post-translational mitochondrial import of proteins via the presequence pathway.
线粒体为细胞提供能量并调节细胞代谢。几乎所有的线粒体蛋白都是核编码的，在细胞质核糖体上翻译，然后被转运到线粒体的不同亚细胞区室。在这里，我们开发了 OptoMitoImport，这是一种光遗传学工具，可以按需通过前导序列途径控制蛋白质进入线粒体基质的导入。OptoMitoImport 基于两步过程：首先，TEV 蛋白酶的光诱导切割将靠近线粒体靶向序列的质膜锚定融合构建体切断；其次，感兴趣的蛋白质之前的线粒体靶向序列募集到外线粒体膜，并将与其融合的蛋白质导入线粒体。到达线粒体基质后，基质加工肽将切断线粒体靶向序列并释放感兴趣的蛋白质。OptoMitoImport 有两种质粒系统以及 P2A 肽或 IRES 序列双顺反子系统。荧光研究表明，通过光诱导的 TEV 蛋白酶切割释放质膜锚定的感兴趣的蛋白质，并将其定位到线粒体。细胞分级实验证实了肽切割的感兴趣的蛋白质存在于线粒体部分中。处理产物可防止蛋白酶 K 处理。跨内膜的膜电位耗竭可防止线粒体蛋白导入，表明通过前导序列途径导入感兴趣的蛋白质。这些数据表明 OptoMitoImport 作为一种通用系统的功能，可通过前导序列途径控制蛋白质的翻译后线粒体导入。
