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

Xiaojian Shi, Bryn Reinstadler, Hardik Shah, Tsz-Leung To, Katie Byrne, Luanna Summer, Sarah E. Calvo, Olga Goldberger, John G. Doench, Vamsi K. Mootha and Hongying Shen ()
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Xiaojian Shi: Yale School of Medicine
Bryn Reinstadler: Massachusetts General Hospital
Hardik Shah: Massachusetts General Hospital
Tsz-Leung To: Massachusetts General Hospital
Katie Byrne: Yale School of Medicine
Luanna Summer: Yale School of Medicine
Sarah E. Calvo: Massachusetts General Hospital
Olga Goldberger: Massachusetts General Hospital
John G. Doench: Broad Institute
Vamsi K. Mootha: Massachusetts General Hospital
Hongying Shen: Yale School of Medicine

Nature Communications, 2022, vol. 13, issue 1, 1-15

Abstract: Abstract 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.

Date: 2022
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DOI: 10.1038/s41467-022-30126-9

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