Combinatorial GxGxE CRISPR screen identifies SLC25A39 in mitochondrial glutathione transport linking iron homeostasis to OXPHOS.
Combinatorial GxGxE CRISPR screen identifies SLC25A39 in mitochondrial glutathione transport linking iron homeostasis to OXPHOS.
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DOI：
10.1038/s41467-022-30126-9
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发表时间：
2022-05-05
影响因子：
16.6
通讯作者：
Shen, Hongying
中科院分区：
文献类型：
--
作者：
Shi, Xiaojian;Reinstadler, Bryn;Shah, Hardik;To, Tsz-Leung;Byrne, Katie;Summer, Luanna;Calvo, Sarah E.;Goldberger, Olga;Doench, John G.;Mootha, Vamsi K.;Shen, Hongying
SLC 25载体家族由53种转运蛋白组成，它们将营养物质和辅助因子穿梭于线粒体膜之间。该家族是高度冗余的，它们的运输活动与代谢状态相耦合。在这里，我们使用了一种合并的双重CRISPR筛选策略，该策略敲除了四种代谢状态下的成对转运蛋白-葡萄糖，半乳糖，OXPHOS抑制和丙酮酸缺乏-旨在揭示这些基因的相互依赖性。总的来说，我们在四种代谢状态下筛选了63个基因，对应于2016年的单个和成对遗传扰动。我们恢复了19个基因与环境（GxE）的相互作用和9个基因与基因（GxG）的相互作用。一个GxE相互作用命中表明，线粒体叶酸载体（SLC 25 A32）KO细胞中的适应性缺陷由于从头嘌呤生物合成中缺乏底物而在半乳糖中得到遗传缓冲。GxG分析突出了铁转运蛋白SLC 25 A37（A37）和表征不佳的SLC 25 A39（A39）之间的缓冲相互作用。线粒体代谢物分析、细胞器转运测定和结构引导的诱变鉴定A39为线粒体谷胱甘肽（GSH）输入的关键。功能研究表明，A39介导的谷胱甘肽稳态和A37介导的线粒体铁摄取共同支持线粒体OXPHOS。我们的工作强调了研究不同代谢环境中全家族遗传相互作用的价值。靶向人SLC 25转运蛋白家族的组合基因×基因×环境CRISPR筛选使得能够在线粒体谷胱甘肽输入中鉴定SLC 25 A39及其与线粒体铁输入的协调以支持OXPHOS。
The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes. The family is highly redundant and their transport activities coupled to metabolic state. Here, we use a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states — glucose, galactose, OXPHOS inhibition, and absence of pyruvate — designed to unmask the inter-dependence of these genes. In total, we screen 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recover 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrates that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells is genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. GxG analysis highlights a buffering interaction between the iron transporter SLC25A37 (A37) and the poorly characterized SLC25A39 (A39). Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis identify A39 as critical for mitochondrial glutathione (GSH) import. Functional studies reveal that A39-mediated glutathione homeostasis and A37-mediated mitochondrial iron uptake operate jointly to support mitochondrial OXPHOS. Our work underscores the value of studying family-wide genetic interactions across different metabolic environments. Combinatorial Gene×Gene×Environment CRISPR screen targeting human SLC25 transporter family enables the identification of SLC25A39 in mitochondrial glutathione import and its coordination with mitochondrial iron import in supporting OXPHOS.
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影响因子：
16.2
作者：
Iossifov I;Ronemus M;Levy D;Wang Z;Hakker I;Rosenbaum J;Yamrom B;Lee YH;Narzisi G;Leotta A;Kendall J;Grabowska E;Ma B;Marks S;Rodgers L;Stepansky A;Troge J;Andrews P;Bekritsky M;Pradhan K;Ghiban E;Kramer M;Parla J;Demeter R;Fulton LL;Fulton RS;Magrini VJ;Ye K;Darnell JC;Darnell RB;Mardis ER;Wilson RK;Schatz MC;McCombie WR;Wigler M
通讯作者：
Wigler M
影响因子：
30.8
作者：
Krumm, Niklas;Turner, Tychele N.;Baker, Carl;Vives, Laura;Mohajeri, Kiana;Witherspoon, Kali;Raja, Archana;Coe, Bradley P.;Stessman, Holly A.;He, Zong-Xiao;Leal, Suzanne M.;Bernier, Raphael;Eichler, Evan E.
通讯作者：
Eichler, Evan E.
影响因子：
29
作者：
Arroyo JD;Jourdain AA;Calvo SE;Ballarano CA;Doench JG;Root DE;Mootha VK
通讯作者：
Mootha VK
影响因子：
20.8
作者：
Chen, Li;Zhang, Zhaoyue;Rabinowitz, Joshua D.
通讯作者：
Rabinowitz, Joshua D.
DOI：
10.1073/pnas.1632471100
发表时间：
2003-09-02
影响因子：
11.1
作者：
Luk, E;Carroll, M;Culotta, VC
通讯作者：
Culotta, VC