Combinatorial G x G x E CRISPR screening and functional analysis highlights SLC25A39 in mitochondrial GSH transport
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Abstract
The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes 1-3 . 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.
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Consolidated peer review report (23 November 2021)
GENERAL ASSESSMENT
Transport of metabolites across the mitochondrial inner membrane is a vital process which sustains the metabolic pathways connecting mitochondria and cytosol. Approximately one-third of the mitochondrial carriers of the SLC25 family, consisting of 53 members, are currently orphan transporters, limiting our understanding of mitochondrial transport and mitochondrial metabolism. This preprint reports a combinatorial CRISPR screening approach to probe interactions between mitochondrial carriers and their dependence on the metabolic environment, comparing different metabolic states of the cell and conditions under which mitochondrial respiration is or is not required. This is a potentially powerful approach to facilitate the identification of orphan transporters.
The …
Consolidated peer review report (23 November 2021)
GENERAL ASSESSMENT
Transport of metabolites across the mitochondrial inner membrane is a vital process which sustains the metabolic pathways connecting mitochondria and cytosol. Approximately one-third of the mitochondrial carriers of the SLC25 family, consisting of 53 members, are currently orphan transporters, limiting our understanding of mitochondrial transport and mitochondrial metabolism. This preprint reports a combinatorial CRISPR screening approach to probe interactions between mitochondrial carriers and their dependence on the metabolic environment, comparing different metabolic states of the cell and conditions under which mitochondrial respiration is or is not required. This is a potentially powerful approach to facilitate the identification of orphan transporters.
The authors identify a number of gene-by-gene, gene-by-environment and gene-by-gene-environment interactions. They confirm known functions of previously identified carriers, and they analyze gene-by-environment interactions of the proposed mitochondrial folate carrier, SLC25A32. A main highlight of this study is the conclusion that the orphan carrier SLC25A39 is involved in glutathione transport. Glutathione is a very important metabolite which plays a key role in protecting mitochondria against oxidative damage but the carrier responsible for this function remained undefined. Building on a strong observed interaction with a known iron transporter, the authors performed mitochondrial metabolite profiling where they observed depletion of both the reduced and the oxidized glutathione in mitochondria of the SLC25A39 knockout, while no changes were observed in the whole cell glutathione content. Additionally, they reported reduced glutathione transport in the mitochondria of the knockout compared to control and site directed mutagenesis in residues predicted via homology modeling to be part of the substrate binding site. Altogether, the study provides strong evidence on the importance of SLC25A39 in mitochondrial transport of glutathione and therefore it is an important contribution in the fields of mitochondrial transport, metabolism, physiology, and medicine.
The dual Cas9 screening approach employed here brings an important innovation to the study of mitochondrial carriers, which have traditionally been difficult to identify due to considerable functional redundancy. The screening in different media conditions has highlighted links that would have remained unidentified in a single medium, and the use of knockout pairs can overcome issues relevant to functional redundancy of the carriers. It is evident that this approach can provide a wealth of testable ideas on the physiological role of mitochondrial carriers and could provide a framework for exciting future studies on the identification of other orphan transporters in mitochondria. A strength of this study that this approach provides strong support for a role for SLC25A39 in glutathione transport, as also proposed by an independent study by Wang et al (2021) Nature 599, 136-140. The findings presented here provide strong motivation for undertaking in vitro reconstitution experiments to definitively demonstrate that SLC25A39 is a glutathione transporter.
Beyond the novel finding on the involvement of SLC25A39 in glutathione transport, the authors showcase the usefulness of their combinatorial CRISPR screen on the SLC25A19 and SLC25A32 carriers and the paralogous genes encoding the mitochondrial ADP/ATP carriers. In this part of the study, which covers half of the manuscript, they provide many interesting observations and raise a few questions. For example, they find no fitness defect for the SLC25A32 KO in galactose, which could imply that this carrier does not transport FAD as previously proposed or alternatively, there could be a redundant mechanism. Another example is that they propose there could be functional differences between the ADP/ATP carriers. These efforts are admirable, and we can appreciate it was challenging to combine the methodological validation of the CRISPR screen and a new functional identification in a single manuscript, but these unresolved issues do leave the reader wanting to know more.
RECOMMENDATIONS
Revisions essential for endorsement:
Experiments:
- If the authors could report the difference in the initial rates of glutathione transport between SLC25A39 KO and control in isolated mitochondria, it would strengthen the conclusion that SLC25A39 is a glutathione transporter. To calculate this accurately, the authors would need collect more time points in the first five minutes of the time course. It is also not clear, and should be indicated, against which protein they normalize their data.
Discussion:
A gene-by-gene buffering interaction has been identified between SLC25A39 and SLC25A37, an iron transporter, in all four media conditions, with the strongest in antimycin. As this is an important observation powered by the combinatorial screen approach, a further discussion seems warranted. Although the link between glutathione and iron metabolism is obvious and known, it is not clear why this would be a buffering interaction. Could the authors provide a specific metabolic explanation for these data?
The recent study by Wang et al., is associating SLC25A39, but also SLC25A40, with glutathione transport. It would be helpful to the reader if the authors could discuss this finding in the context of their results.
As the highlight of the manuscript relates to glutathione transport, more background information on glutathione, its role in metabolism and pathophysiology and previous attempts to identify this carrier would be helpful for the reader.
Please discuss the debate around SLC25A32 function in the text more deliberately as convincing molecular experimental evidence on its substrate is still lacking.
Additional suggestions for the authors to consider:
In vitro studies can prove unequivocally that SLC25A39 is the mitochondrial glutathione transporter. This manuscript will have an advantage if the authors can show in an in vitro system that SLC25A39 can transport glutathione.
Previous studies had proposed that mitochondrial dicarboxylate and 2-oxoglutarate carriers can transport glutathione, although this has been disputed. Could the authors discuss how the results of this screen relate to these carriers?
Why is there a growth defect for the SLC25A19 KO in the -pyruvate condition (Supplementary Figure 2)?
Given the significant number of still unidentified mitochondrial carriers, it is somewhat surprising that more interactions with uncharacterized carriers were not identified here. This is likely due to the limited number of media conditions used in the study and could be considered in design of future studies.
REVIEWING TEAM
Reviewed by:
Nora Kory, Assistant Professor, Harvard School of Public Health, USA: functional identification of mitochondrial transporters using metabolite profiling and functional genomics approaches
Sotiria Tavoulari, Research Associate, University of Cambridge, UK: molecular mechanisms of transport, mitochondrial transport, mitochondrial carriers (SLC25), mitochondrial pyruvate carrier (SLC54)
Curated by:
Merritt Maduke, Associate Professor, Stanford University School of Medicine, USA
(This consolidated report is a result of peer review conducted by Biophysics Colab on version 1 of this preprint. Minor corrections and presentational issues have been omitted for brevity.)
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