C9orf72 binds SMCR8, localizes to lysosomes, and regulates mTORC1 signaling.
C9orf72 binds SMCR8, localizes to lysosomes, and regulates mTORC1 signaling.
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DOI：
10.1091/mbc.e16-01-0003
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发表时间：
2016-10-15
影响因子：
3.3
通讯作者：
Ferguson SM
中科院分区：
文献类型：
--
作者：
Amick J;Roczniak-Ferguson A;Ferguson SM
C9 orf 72与SMCR 8强烈相互作用，并依赖于这种相互作用来维持其稳定性。溶酶体是C9 orf 72亚细胞定位的主要场所，并且在其缺失时观察到异常的溶酶体形态。在C9 orf 72 KO细胞中发现溶酶体定位的mTORC 1信号通路的调节缺陷。C9 orf 72基因内含子中的六核苷酸扩增导致肌萎缩侧索硬化和额颞叶痴呆然而，除了生物信息学预测表明与Birt-Hogg-Dubé综合征肿瘤抑制因子卵泡素结构相似之外，对C9 orf 72蛋白的正常功能知之甚少。为了解决这个问题，我们使用基因组编辑策略来研究C9 orf 72相互作用、亚细胞定位和敲除（KO）表型。我们发现C9 orf 72与SMCR 8（一种以前未知功能的蛋白质）强烈相互作用。我们还观察到C9 orf 72定位于溶酶体，并且这种定位受到氨基酸可用性的负调控。C9 orf 72 KO、SMCR 8 KO和双KO细胞系的分析揭示了与C9 orf 72在溶酶体的功能一致的表型。这些包括在不存在C9 orf 72的情况下溶酶体异常肿胀，以及在C9 orf 72或SMCR 8耗尽后mTORC 1信号传导对氨基酸可用性变化的反应受损（溶酶体依赖性过程）。总的来说，这些结果确定了C9 orf 72和SMCR 8之间强的物理和功能相互作用，并支持该蛋白质复合物的溶酶体作用位点。
C9orf72 interacts strongly with SMCR8 and depends on this interaction for its stability. Lysosomes are major sites of C9orf72 subcellular localization, and abnormal lysosome morphology is seen in its absence. Defects are found in the regulation of the lysosome-localized mTORC1 signaling pathway in C9orf72 KO cells. Hexanucleotide expansion in an intron of the C9orf72 gene causes amyotrophic lateral sclerosis and frontotemporal dementia. However, beyond bioinformatics predictions that suggested structural similarity to folliculin, the Birt-Hogg-Dubé syndrome tumor suppressor, little is known about the normal functions of the C9orf72 protein. To address this problem, we used genome-editing strategies to investigate C9orf72 interactions, subcellular localization, and knockout (KO) phenotypes. We found that C9orf72 robustly interacts with SMCR8 (a protein of previously unknown function). We also observed that C9orf72 localizes to lysosomes and that such localization is negatively regulated by amino acid availability. Analysis of C9orf72 KO, SMCR8 KO, and double-KO cell lines revealed phenotypes that are consistent with a function for C9orf72 at lysosomes. These include abnormally swollen lysosomes in the absence of C9orf72 and impaired responses of mTORC1 signaling to changes in amino acid availability (a lysosome-dependent process) after depletion of either C9orf72 or SMCR8. Collectively these results identify strong physical and functional interactions between C9orf72 and SMCR8 and support a lysosomal site of action for this protein complex.