The MitoLuc assay for the analysis of the mechanism of mitochondrial protein import.

4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-22 DOI:10.1016/bs.mie.2024.07.033
Hope I Needs, Youmian Yan, Natalie M Niemi, Ian Collinson
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引用次数: 0

Abstract

The NanoLuc split luciferase assay has proven to be a powerful tool for the analysis of protein translocation. Its flexibility has enabled in vivo, ex vivo, and in vitro studies-including systems reconstituting protein transport from pure components. The assay has been particularly useful in the characterization of bacterial secretion and mitochondrial protein import. In the latter case, MitoLuc has been developed for the investigation of the TIM23-pathway via import into the matrix of isolated yeast mitochondria. Subsequent analysis identified three distinct phases of import, rather than in a single continuous step. The assay has also been developed to monitor import into the mitochondrial matrix of intact cultured cells. This latter innovation has laid the foundations for further analysis of the import process in humans, including the consequences of interactions with cytosolic factors and neighboring organelles. The versatility of the MitoLuc assay is conducive for its adaptation to also monitor import into the inter-membrane space (MIA-pathway), and into the inner-membrane via the TIM22- and TIM23-complexes. Here, we present detailed protocols for the application of MitoLuc to mitochondria isolated from yeast and to those within cultured human cells.

用于分析线粒体蛋白质导入机制的 MitoLuc 分析法。
事实证明,NanoLuc 分体荧光素酶测定法是分析蛋白质转运的有力工具。它的灵活性使得体内、体外和离体研究成为可能--包括从纯成分中重建蛋白质转运的系统。这种检测方法在鉴定细菌分泌和线粒体蛋白质导入方面尤其有用。在后一种情况下,MitoLuc 被开发用于研究通过导入分离的酵母线粒体基质的 TIM23 途径。随后的分析确定了导入的三个不同阶段,而不是单一的连续步骤。该检测方法还被开发用于监测向完整培养细胞线粒体基质的导入。后一项创新为进一步分析人类的导入过程奠定了基础,包括与细胞膜因子和邻近细胞器相互作用的后果。MitoLuc 检测方法的多功能性使其也能监测向膜间隙(MIA 途径)以及通过 TIM22 和 TIM23 复合物向内膜的导入。在此,我们介绍了将 MitoLuc 应用于从酵母分离的线粒体和培养的人体细胞线粒体的详细方案。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Methods in enzymology
Methods in enzymology 生物-生化研究方法
CiteScore
2.90
自引率
0.00%
发文量
308
审稿时长
3-6 weeks
期刊介绍: The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.
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