Institute of Biochemistry and Molecular Biology (IBMB), Faculty of Medicine, University of Bonn, Bonn, Germany.
Core Facility Mass Spectrometry, Institute of Biochemistry and Molecular Biology (IBMB), University of Bonn, Faculty of Medicine, Bonn, Germany.
J Biol Chem. 2022 Oct;298(10):102494. doi: 10.1016/j.jbc.2022.102494. Epub 2022 Sep 15.
Chaperones of the Hsp100/Clp family represent major components of protein homeostasis, conferring maintenance of protein activity under stress. The ClpB-type members of the family, present in bacteria, fungi, and plants, are able to resolubilize aggregated proteins. The mitochondrial member of the ClpB family in Saccharomyces cerevisiae is Hsp78. Although Hsp78 has been shown to contribute to proteostasis in elevated temperatures, the biochemical mechanisms underlying this mitochondria-specific thermotolerance are still largely unclear. To identify endogenous chaperone substrate proteins, here, we generated an Hsp78-ATPase mutant with stabilized substrate-binding behavior. We used two stable isotope labeling-based quantitative mass spectrometry approaches to analyze the role of Hsp78 during heat stress-induced mitochondrial protein aggregation and disaggregation on a proteomic level. We first identified the endogenous substrate spectrum of the Hsp78 chaperone, comprising a wide variety of proteins related to metabolic functions including energy production and protein synthesis, as well as other chaperones, indicating its crucial functions in mitochondrial stress resistance. We then compared these interaction data with aggregation and disaggregation processes in mitochondria under heat stress, which revealed specific aggregation-prone protein populations and demonstrated the direct quantitative impact of Hsp78 on stress-dependent protein solubility under different conditions. We conclude that Hsp78, together with its cofactors, represents a recovery system that protects major mitochondrial metabolic functions during heat stress as well as restores protein biogenesis capacity after the return to normal conditions.
Hsp100/Clp 家族的伴侣蛋白是蛋白质动态平衡的主要组成部分，在应激条件下赋予蛋白质活性的维持。该家族中的 ClpB 型成员存在于细菌、真菌和植物中，能够重新溶解聚集的蛋白质。酿酒酵母中 ClpB 家族的线粒体成员是 Hsp78。尽管已经表明 Hsp78 有助于高温下的蛋白质动态平衡，但这种线粒体特有的耐热性的生化机制在很大程度上仍不清楚。为了鉴定内源性伴侣蛋白底物，在这里，我们生成了一种具有稳定的底物结合行为的 Hsp78-ATP 酶突变体。我们使用两种基于稳定同位素标记的定量质谱方法来分析 Hsp78 在热应激诱导的线粒体蛋白聚集和去聚集过程中的作用，从蛋白质组水平上进行分析。我们首先鉴定了 Hsp78 伴侣蛋白的内源性底物谱，其中包括与代谢功能相关的广泛的各种蛋白质，包括能量产生和蛋白质合成，以及其他伴侣蛋白，表明其在线粒体应激抵抗中的关键功能。然后，我们将这些相互作用数据与热应激下线粒体中的聚集和去聚集过程进行了比较，这揭示了特定的聚集倾向蛋白群体，并证明了 Hsp78 在不同条件下对依赖于应激的蛋白质溶解度的直接定量影响。我们得出结论，Hsp78 与其辅助因子一起代表了一种恢复系统，该系统在热应激期间保护主要的线粒体代谢功能，并在恢复到正常条件后恢复蛋白质生物发生能力。
