Combinatorial G x G x E CRISPR screening and functional analysis highlights SLC25A39 in mitochondrial GSH transport

The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes1-3. The family is highly redundant and their transport activities coupled to metabolic state. Here, we introduce 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 screened 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recovered 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrated that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells was genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. Another GxE interaction hit revealed non-equivalence of the paralogous ATP/ADP exchangers (ANTs) with ANT2 specifically required during OXPHOS inhibition. GxG analysis highlighted a buffering interaction between the iron transporter SLC25A37 and the poorly characterized SLC25A39. Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis suggests SLC25A39 is critical for mitochondrial glutathione (GSH) transport. Our work underscores the importance of systemetically investigating family-wide genetic interactions between mitochondrial transporters across many metabolic environments.