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

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.

MTCH2 is a mitochondrial outer membrane protein insertase

“…S13B) in a MTCH2 knockout background had an additive effect on biogenesis of many mitochondrial TAs. This result is consistent with recent pair-wise genetic interaction maps that have found a synthetic lethal relationship between MTCH1 and 2 (28), and the fact that MTCH1 was also a statistically significant hit in our genome-wide screen (fig. S12; S13C).…”

Metabolic determinants of tumour initiation

“…Likewise, simply changing the composition of the medium can induce large-scale metabolic alterations in cultured cells, implying that nutrient availability is a major factor dictating metabolic phenotypes 65,66 . These context-specific preferences can translate into altered metabolic demands: a subset of metabolic genes exhibit conditional essentiality depending on exogenous nutrient availability 67,68 . Somewhat surprisingly, therefore, genetic screens revealed that pancreatic cancer cells grown in 2D in vitro or as tumours in mice exhibited overall strikingly similar requirements for metabolic genes 69,70 .…”

MTCH2 is a mitochondrial outer membrane protein insertase

“…This result is consistent with our genome-wide screen (fig. S17C) and the synthetic lethal relationship between MTCH1 and 2 ( 21 ). We therefore propose that MTCH1 and 2 are the founding members of a distinctive class of membrane protein insertases that exploit the SLC25 transporter fold (fig.…”