The mitoCPR unclogs mitochondria The import of proteins into mitochondria is essential for cell viability. How cells respond when mitochondrial protein import is impaired is poorly understood. Weidberg and Amon showed that upon mitochondrial import stress, yeast cells mounted a response known as the mitoCPR. mitoCPR was activated when mitochondrial protein import was impaired and unimported precursors accumulated on the organelle's surface. mitoCPR restored mitochondrial functions by clearing stalled proteins from the import channels. It did this by inducing expression of Cis1, which recruited the adenosine triphosphatase Msp1 to import channels to remove unimported precursors and target them for degradation by the proteasome. Science, this issue p. eaan4146 MitoCPR triggers the removal of unimported mitochondrial precursors and restores function during protein import stress in yeast. INTRODUCTION Mitochondria provide cells with energy and numerous essential metabolites such as lipids, amino acids, iron sulfur clusters, and heme. All mitochondrial functions rely on import of proteins into the organelle because the mitochondrial proteome is almost exclusively encoded by nuclear genes. Given the central importance of mitochondria for cell viability, it is not surprising that cells mount a nuclear response when mitochondrial functions are compromised. These mitochondria-to-nucleus signaling pathways include the mtUPR (mitochondrial unfolded protein response), which triggers expression of mitochondrial chaperones when mitochondrial protein folding is defective, and the UPRam (unfolded protein response activated by mistargeting of proteins) and mPOS (mitochondrial precursor over-accumulation stress) pathways, which reduce translation and induce degradation of unimported proteins in the cytosol when mitochondrial import is impaired. Even though mitochondrial import is central to all mitochondrial functions, no response to protein import defects had been described that protects mitochondria during this stress. RATIONALE To determine how cells respond to defects in mitochondrial protein import, we first developed a system in budding yeast with which to specifically inhibit this process. We found that overexpression of proteins that rely on a bipartite signal sequence for their mitochondrial localization inhibited mitochondrial import and led to the accumulation of mitochondrial precursors. Protease protection and carbonate extraction assays that were performed on isolated mitochondria revealed that these unimported proteins accumulated on the mitochondrial surface and in the import channel known as the translocase. RESULTS Having developed a system that allowed us to specifically inhibit mitochondrial protein import, we examined the cellular response to this defect. Transcriptome analysis of cells overexpressing bipartite signal–containing proteins identified a gene expression pattern related to the multi-drug resistance response. We termed this response mitochondrial compromised protein import response (mitoCPR). mitoCPR was triggered by protein import defects but not other mitochondrial deficiencies, such as respiratory failure, and was mediated by the transcription factor Pdr3. Our analyses further showed that mitoCPR was critical for the protection of mitochondria during import stress. Cells lacking PDR3 did not mount a mitoCPR during import stress and accumulated higher levels of unimported proteins on the organelle surface as compared with those of wild-type cells. Consequently, pdr3Δ cells exhibited decreased respiratory function and loss of mitochondrial DNA when mitochondrial import was restored. Our results also shed light on the mechanism by which mitoCPR protected mitochondria. Upon mitochondrial import stress, Pdr3 induced expression of Cis1. Coimmunoprecipitation analyses showed that Cis1 recruited the AAA+ adenosine triphosphatase Msp1 to the translocase by binding to the translocase receptor Tom70. There, the two proteins mediated the clearance and proteasomal degradation of proteins that failed to be imported into mitochondria. CONCLUSION We discovered a mitochondrial import surveillance mechanism in budding yeast. This surveillance mechanism, mitoCPR, is activated when mitochondrial import is stalled in order to induce the removal of mitochondrial proteins accumulating on the mitochondrial surface. Clearance of precursors is critical for maintaining mitochondrial functions during import stress. We propose that mitoCPR could be especially important when the import machinery is overwhelmed, as may occur in situations that require the rapid expansion of the mitochondrial compartment. MitoCPR protects mitochondria during import stress. (Left) Mitochondrial protein import deficiency leads to the accumulation of mitochondrial proteins on the organelle’s surface and in the translocases. (Right) Pdr3 induces CIS1 expression. Cis1 binds to the mitochondrial import receptor Tom70 and recruits Msp1 to mediate clearance of unimported precursors from the mitochondrial surface and their proteasomal degradation. This protects mitochondrial functions during import stress. ILLUSTRATION: ELLA MARU STUDIO Mitochondrial functions are essential for cell viability and rely on protein import into the organelle. Various disease and stress conditions can lead to mitochondrial import defects. We found that inhibition of mitochondrial import in budding yeast activated a surveillance mechanism, mitoCPR, that improved mitochondrial import and protected mitochondria during import stress. mitoCPR induced expression of Cis1, which associated with the mitochondrial translocase to reduce the accumulation of mitochondrial precursor proteins at the mitochondrial translocase. Clearance of precursor proteins depended on the Cis1-interacting AAA+ adenosine triphosphatase Msp1 and the proteasome, suggesting that Cis1 facilitates degradation of unimported proteins. mitoCPR was required for maintaining mitochondrial functions when protein import was compromised, demonstrating the importance of mitoCPR in protecting the mitochondrial compartment.

中文翻译：

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MitoCPR——一种保护线粒体以应对蛋白质输入应激的监测途径


mitoCPR 疏通线粒体蛋白质进入线粒体对于细胞活力至关重要。当线粒体蛋白质输入受损时细胞如何反应尚不清楚。 Weidberg 和 Amon 表明，在线粒体输入应激下，酵母细胞会产生一种称为 mitoCPR 的反应。当线粒体蛋白质输入受损且未输入的前体在细胞器表面积累时，mitoCPR 就会被激活。 mitoCPR 通过清除输入通道中停滞的蛋白质来恢复线粒体功能。它通过诱导 Cis1 的表达来做到这一点，Cis1 招募三磷酸腺苷酶 Msp1 进入输入通道，以去除未输入的前体并靶向它们被蛋白酶体降解。科学，本期第 14 页。 eaan4146 MitoCPR 触发未输入的线粒体前体的去除，并在酵母中蛋白质输入应激期间恢复功能。简介线粒体为细胞提供能量和许多必需代谢物，如脂质、氨基酸、铁硫簇和血红素。所有线粒体功能都依赖于蛋白质输入细胞器，因为线粒体蛋白质组几乎完全由核基因编码。鉴于线粒体对细胞活力的核心重要性，当线粒体功能受损时细胞产生核反应也就不足为奇了。 这些线粒体到细胞核的信号通路包括 mtUPR（线粒体未折叠蛋白反应），当线粒体蛋白折叠有缺陷时，它会触发线粒体伴侣的表达，以及 UPRam（由蛋白质定位错误激活的未折叠蛋白反应）和 mPOS（线粒体前体未折叠蛋白反应）。 -积累应激）途径，当线粒体输入受损时，该途径会减少翻译并诱导细胞质中未输入的蛋白质降解。尽管线粒体输入是所有线粒体功能的核心，但尚未描述在这种应激期间保护线粒体的对蛋白质输入缺陷的反应。基本原理为了确定细胞如何应对线粒体蛋白质输入缺陷，我们首先在芽殖酵母中开发了一种系统，用于特异性抑制这一过程。我们发现，依赖二分信号序列进行线粒体定位的蛋白质的过度表达会抑制线粒体输入并导致线粒体前体的积累。对分离的线粒体进行的蛋白酶保护和碳酸盐提取测定表明，这些未输入的蛋白质积聚在线粒体表面和称为转位酶的输入通道中。结果开发了一种能够特异性抑制线粒体蛋白质输入的系统后，我们检查了细胞对这种缺陷的反应。对过度表达含有二分信号的蛋白质的细胞进行转录组分析，确定了与多药耐药反应相关的基因表达模式。我们将这种反应称为线粒体受损蛋白输入反应（mitoCPR）。 mitoCPR 是由蛋白质输入缺陷触发的，但不是由其他线粒体缺陷（例如呼吸衰竭）触发，并且由转录因子 Pdr3 介导。我们的分析进一步表明，mitoCPR 对于输入应激期间保护线粒体至关重要。与野生型细胞相比，缺乏 PDR3 的细胞在输入应激期间不会安装 mitoCPR，并且在细胞器表面积累更高水平的未输入蛋白质。因此，当线粒体输入恢复时，pdr3Δ细胞表现出呼吸功能下降和线粒体DNA丢失。我们的结果还揭示了 mitoCPR 保护线粒体的机制。在线粒体输入应激下，Pdr3 诱导 Cis1 的表达。免疫共沉淀分析表明，Cis1 通过与转位酶受体 Tom70 结合，将 AAA+ 三磷酸腺苷酶 Msp1 招募到转位酶中。在那里，这两种蛋白质介导未能输入线粒体的蛋白质的清除和蛋白酶体降解。结论我们在芽殖酵母中发现了线粒体输入监视机制。当线粒体输入停滞时，这种监视机制 mitoCPR 就会被激活，以诱导去除积累在线粒体表面的线粒体蛋白。前体的清除对于输入应激期间维持线粒体功能至关重要。我们认为，当输入机制不堪重负时，mitoCPR 可能尤其重要，这可能发生在需要线粒体室快速扩张的情况下。 MitoCPR 在输入应激期间保护线粒体。 （左）线粒体蛋白输入缺陷导致线粒体蛋白在细胞器表面和易位酶中积累。 （右）Pdr3 诱导 CIS1 表达。 Cis1 与线粒体输入受体 Tom70 结合并招募 Msp1 介导线粒体表面未输入前体的清除及其蛋白酶体降解。这可以在输入应激期间保护线粒体功能。插图：ELLA MARU STUDIO 线粒体功能对于细胞活力至关重要，并依赖于细胞器中的蛋白质输入。各种疾病和应激条件都可能导致线粒体输入缺陷。我们发现，抑制芽殖酵母中的线粒体输入会激活一种监视机制 mitoCPR，该机制可改善线粒体输入并在输入应激期间保护线粒体。 mitoCPR 诱导 Cis1 的表达，Cis1 与线粒体转位酶相关，以减少线粒体转位酶处线粒体前体蛋白的积累。前体蛋白的清除取决于 Cis1 相互作用的 AAA+ 三磷酸腺苷酶 Msp1 和蛋白酶体，表明 Cis1 促进未输入蛋白的降解。当蛋白质输入受到损害时，mitoCPR 是维持线粒体功能所必需的，这证明了 mitoCPR 在保护线粒体区室中的重要性。