{"title":"线粒体外膜转运酶的质量控制途径分析。","authors":"Lara Calvo Santos, Fabian den Brave","doi":"10.1016/bs.mie.2024.07.050","DOIUrl":null,"url":null,"abstract":"<p><p>The functionality of mitochondria depends on the import of proteins synthesized on cytosolic ribosomes. Impaired import into mitochondria results in mitochondrial dysfunction and proteotoxic accumulation of precursor proteins in the cytosol. All proteins sorted to inner mitochondrial compartments are imported via the translocase of the outer membrane (TOM) complex. Premature protein folding, a reduction of the mitochondrial membrane potential or defects in translocases can result in precursor arrest during translocation, thereby clogging the TOM channel and blocking protein import. In recent years, different pathways have been identified, which employ the cytosolic ubiquitin-proteasome system in the extraction and turnover of precursor proteins from the TOM complex. Central events in this process are the modification of arrested precursor proteins with ubiquitin, their extraction by AAA-ATPases and subsequent degradation by the 26 S proteasome. Analysis of these processes is largely facilitated by the expression of model proteins that function as efficient \"cloggers\" of the import machinery. Here we describe the use of such clogger proteins and how their handling by the protein quality control machinery can be monitored. We provide protocols to study the extent of clogging, the ubiquitin-modification of arrested precursor proteins and their turnover by the 26 S proteasome.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"707 ","pages":"565-584"},"PeriodicalIF":0.0000,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Analysis of quality control pathways for the translocase of the outer mitochondrial membrane.\",\"authors\":\"Lara Calvo Santos, Fabian den Brave\",\"doi\":\"10.1016/bs.mie.2024.07.050\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The functionality of mitochondria depends on the import of proteins synthesized on cytosolic ribosomes. Impaired import into mitochondria results in mitochondrial dysfunction and proteotoxic accumulation of precursor proteins in the cytosol. All proteins sorted to inner mitochondrial compartments are imported via the translocase of the outer membrane (TOM) complex. Premature protein folding, a reduction of the mitochondrial membrane potential or defects in translocases can result in precursor arrest during translocation, thereby clogging the TOM channel and blocking protein import. In recent years, different pathways have been identified, which employ the cytosolic ubiquitin-proteasome system in the extraction and turnover of precursor proteins from the TOM complex. Central events in this process are the modification of arrested precursor proteins with ubiquitin, their extraction by AAA-ATPases and subsequent degradation by the 26 S proteasome. Analysis of these processes is largely facilitated by the expression of model proteins that function as efficient \\\"cloggers\\\" of the import machinery. Here we describe the use of such clogger proteins and how their handling by the protein quality control machinery can be monitored. We provide protocols to study the extent of clogging, the ubiquitin-modification of arrested precursor proteins and their turnover by the 26 S proteasome.</p>\",\"PeriodicalId\":18662,\"journal\":{\"name\":\"Methods in enzymology\",\"volume\":\"707 \",\"pages\":\"565-584\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2024-01-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Methods in enzymology\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1016/bs.mie.2024.07.050\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/8/15 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q3\",\"JCRName\":\"Biochemistry, Genetics and Molecular Biology\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods in enzymology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1016/bs.mie.2024.07.050","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/8/15 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 0
摘要
线粒体的功能取决于输入在细胞质核糖体上合成的蛋白质。导入线粒体的功能受损会导致线粒体功能障碍和前体蛋白在细胞质中的蛋白毒性积累。所有被分拣到线粒体内部的蛋白质都是通过外膜转运酶(TOM)复合体导入的。蛋白质折叠过早、线粒体膜电位降低或转运酶缺陷都可能导致前体在转运过程中停滞,从而堵塞 TOM 通道,阻碍蛋白质导入。近年来,人们发现了不同的途径,这些途径利用细胞质泛素-蛋白酶体系统从 TOM 复合物中提取和周转前体蛋白。这一过程的核心事件是用泛素修饰停滞的前体蛋白,由 AAA-ATP 酶提取它们,然后由 26 S 蛋白酶体降解。通过表达能有效 "堵塞 "导入机制的模型蛋白在很大程度上促进了对这些过程的分析。在此,我们介绍了此类堵塞蛋白的使用方法,以及如何监测蛋白质质量控制机制对它们的处理。我们提供了研究堵塞程度、被堵塞前体蛋白的泛素修饰及其在 26 S 蛋白酶体中的周转的方案。
Analysis of quality control pathways for the translocase of the outer mitochondrial membrane.
The functionality of mitochondria depends on the import of proteins synthesized on cytosolic ribosomes. Impaired import into mitochondria results in mitochondrial dysfunction and proteotoxic accumulation of precursor proteins in the cytosol. All proteins sorted to inner mitochondrial compartments are imported via the translocase of the outer membrane (TOM) complex. Premature protein folding, a reduction of the mitochondrial membrane potential or defects in translocases can result in precursor arrest during translocation, thereby clogging the TOM channel and blocking protein import. In recent years, different pathways have been identified, which employ the cytosolic ubiquitin-proteasome system in the extraction and turnover of precursor proteins from the TOM complex. Central events in this process are the modification of arrested precursor proteins with ubiquitin, their extraction by AAA-ATPases and subsequent degradation by the 26 S proteasome. Analysis of these processes is largely facilitated by the expression of model proteins that function as efficient "cloggers" of the import machinery. Here we describe the use of such clogger proteins and how their handling by the protein quality control machinery can be monitored. We provide protocols to study the extent of clogging, the ubiquitin-modification of arrested precursor proteins and their turnover by the 26 S proteasome.
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.