19 Jan 2023
by Richa

Summary- paper 8: SLC25A39 is necessary for mitochondrial glutathione import in mammalian cells

Ying Wang, Frederick S. Yen, Xiphias Ge Zhu, Rebecca C. Timson, Ross Weber, Changrui Xing, Yuyang Liu, Benjamin Allwein, Hanzhi Luo, Hsi-Wen Yeh, Søren Heissel, Gokhan Unlu, Eric R. Gamazon, Michael G. Kharas, Richard Hite & Kıvanç Birsoy

Nature, 2021

Questions/gaps addressed:

  • Mitochondria (highly redox-active), contain 10–15% of total cellular Glutathione (GSH), but lack GSH biosynthetic machinery. How does GSH get inside the mitochondria?

Major hypotheses:

  • Since GSH is negatively charged under physiological conditions, there must be a dedicated GSH transporter in the mitochondrial membrane.

Key methods:

  • Mito-IP from HeLa cells expressing a mitochondrial tag (3×HA–OMP25–mCherry) grown in standard medium or treated with a GSH synthesis inhibitor, buthionine sulfoximine (BSO), followed by proteomics to identify proteins with altered abundance.

  • Metabolomics on mito-IPs to profile changes in mitochondrial metabolites in WT vs SLC25A39-KO cell lines.

  • in vitro GSH transport assay using isotope-labeled GSH (GSH-(glycine-13C2,15N))-uptake assays using mitochondria isolated from WT or SLC25A39-KO cells.

  • Genetic interaction analysis: CRISPR–Cas9-based genetic screens in both Jurkat and HEK 293T cells using a metabolism-focused sgRNA library to screen for genes essential for cellular proliferation in the absence of SLC25A39.

  • Mitochondrially targeted GshF (Streptococcus thermophilus bifunctional enzyme with both glutamate–cysteine ligase and GSH synthetase) to complement mitochondrial GSH loss.

Major takeaways:

  • Identified SLC25A39 (uncharacterized mitochondrial membrane small molecule transporter) as highly upregulated proteins upon GSH depletion with BSO, or upon loss of GCLC (rate-limiting enzyme of GSH synthesis).

  • Observed largest reductions in the levels of GSH or GSH disulfide in mitochondria (Mito-IP) of SLC25A39-knockout cells by metabolomics.

  • SLC25A39 dependent mitochondrial uptake specific for GSH (observed no uptake of GSSG or GSH disulfide) in in-vitro uptake assay. Mutating K329A or D226A (conserved residues as potential substrate-binding residues, modeled based on the structure of the bovine ADP/ATP transporter) abolished GSH uptake. Expression of mito-GshF complemented mito GSH levels in SLC25A39-KO cells.

  • Redundant role of SLC25A40 and SLC25A39 in mitochondrial GSH uptake. Identified SLC25A40 (mitochondrial SLC25A family transporter) with the highest sequence homology to SLC25A39, in the genetic interaction screen. Double mutants unable to proliferate under standard growth conditions. (But did not identify SLC25A40 levels upregulated upon GSH inhibition?)

  • SLC25A40 and SLC25A39 are highly conserved: yeast Mtm1, Drosophila Shawn.

  • Downregulation of mitochondrial translation and iron–sulfur cluster-containing proteins in unbiased proteomics on SLC25A39/40 double-ko Jurkat cells. Slc25a39-KO mice embryonically lethal E13.5, and embryos appeared pale, severely anaemic. Importance of GSH import during erythropoiesis? Effect on iron–sulfur cluster biogenesis due to GSH’s role as a cofactor for mitochondrial glutaredoxins?