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

  • Nat Commun. 2022 May 5;13(1):2483. doi: 10.1038/s41467-022-30126-9.
Xiaojian Shi  #  1  2 ,  Bryn Reinstadler  #  3  4  5 ,  Hardik Shah  3  4  5 ,  Tsz-Leung To  3  4  5 ,  Katie Byrne  1  2 ,  Luanna Summer  1  2 ,  Sarah E Calvo  3  4  5 ,  Olga Goldberger  3  4 ,  John G Doench  5 ,  Vamsi K Mootha  3  4  5 ,  Hongying Shen  6  7
Affiliations
  • 1. Cellular and Molecular Physiology Department, Yale School of Medicine, New Haven, CT, USA.
  • 2. Systems Biology Institute, Yale West Campus, West Haven, CT, USA.
  • 3. Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA.
  • 4. Department of Systems Biology, Harvard Medical School, Boston, MA, USA.
  • 5. Broad Institute, Cambridge, MA, USA.
  • 6. Cellular and Molecular Physiology Department, Yale School of Medicine, New Haven, CT, USA. [email protected].
  • 7. Systems Biology Institute, Yale West Campus, West Haven, CT, USA. [email protected].
  • # Contributed equally.
PMID: 35513392 DOI: 10.1038/s41467-022-30126-9
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.