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

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 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.

Dual regulation of SLC25A39 by AFG3L2 and iron controls mitochondrial glutathione homeostasis

“…Second, we immunoisolated mitochondria from BSO-treated A39 CRISPR KO cells expressing either A39-FLAG or A39 4CA -FLAG where only A39-FLAG can be upregulated but not A39 4CA -FLAG (Fig. 3F); and then performed in vitro mitochondria-based uptake assay using stable isotope labeled GSH [M+3] (GSH-[glycine- 13 C2, 15 N]) for 15 min. The uptake assay revealed significantly higher GSH uptake in the wild type A39-FLAG expressing cells (Fig.…”

“…Glutathione 9 critically supports redox signaling and biosynthesis of iron-sulfur (Fe-S) cluster cofactors inside the mitochondrial matrix 10-12 . Because glutathione is exclusively synthesized in the cytosol, how glutathione enters the mitochondrial matrix remained a decades-old mystery until the recent identification that a previously uncharacterized mitochondrial metabolite transporter, SLC25A39 (A39), is critical for mitochondrial glutathione transport 13-15 . A39 belongs to the S o L ute C arrier 25 (SLC25) family, the largest transporter family that is responsible for translocating diverse metabolite ligands across the mitochondrial inner membrane 1-3 .…”

Molecular evolution of IRG1 shapes itaconate production in metazoans and alleviates the “double-edged dilemma” of innate immune defense

“…Aliquots of 50 µl were taken at 0, 10, 30, and 60 minutes and were immediately quenched with 2 % formic acid, vortexed, and centrifuged at 14,000 rpm for 20 minutes at 4 ℃. The supernatant was then transferred to a new centrifuge tube and mixed 1:9 with a 3:1 acetonitrile:methanol solvent for LC-MS analysis 20 following the established polar metabolite profiling protocol in the lab 86 . Itaconate production was confirmed using chemical standard itaconic acid (Sigma Aldrich #I29204).…”