Combinatorial GxGxE CRISPR screen identifies SLC25A39 in mitochondrial glutathione transport linking iron homeostasis to OXPHOS

• Published in: Nature Communications Vol. 13; no. 1; pp. 2483 - 15
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: London Springer Science and Business Media LLC 05.05.2022; Nature Publishing Group UK; Nature Publishing Group; Nature Portfolio
• Subjects: 13/106; 13/109; 49/40; 631/80; 631/80/642/333/1465; 631/92/577; Biosynthesis; Buffers; Clustered Regularly Interspaced Short Palindromic Repeats; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; Combinatorial analysis; CRISPR; Folic acid; Galactose; Genes; Glutathione; Homeostasis; Humanities and Social Sciences; Imports; Iron; Membrane Transport Proteins; Membrane Transport Proteins - genetics; Metabolism; Metabolites; Mitochondria; multidisciplinary; Mutagenesis; Nutrients; Perturbation; Pyruvic acid; Q; Science; Science (multidisciplinary); Substrates
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-30126-9

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.