RT Journal Article
SR Electronic
T1 Combinatorial <em>G</em> x <em>G</em> x <em>E</em> CRISPR screening and functional analysis highlights SLC25A39 in mitochondrial GSH transport
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.09.22.461361
DO 10.1101/2021.09.22.461361
A1 Xiaojian Shi
A1 Bryn Reinstadler
A1 Hardik Shah
A1 Tsz-Leung To
A1 Katie Byrne
A1 Luanna Summer
A1 Sarah E. Calvo
A1 Olga Goldberger
A1 John G. Doench
A1 Vamsi K. Mootha
A1 Hongying Shen
YR 2021
UL http://biorxiv.org/content/early/2021/09/22/2021.09.22.461361.abstract
AB 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.Competing Interest StatementVKM serves on the SAB and receives equity from Janssen Pharmaceutics and 5AM Ventures. JGD consults for Foghorn Therapeutics, Maze Therapeutics, Merck, Agios, and Pfizer; JGD consults for and has equity in Tango Therapeutics.