SLC25A39 is necessary for mitochondrial glutathione import in mammalian cells

Abstract

Glutathione (GSH) is a small molecule thiol abundantly present in all eukaryotes with key roles in oxidative metabolism. Mitochondria, as the major site of oxidative reactions, must maintain sufficient levels of GSH to perform protective and biosynthetic functions. GSH is exclusively synthesized in the cytosol, yet the molecular machinery involved in mitochondrial GSH import remain elusive. Here, using organellar proteomics and metabolomics approaches, we identify SLC25A39, a mitochondrial membrane carrier of unknown function, to regulate GSH transport into mitochondria. SLC25A39 loss reduces mitochondrial GSH import and abundance without impacting whole cell GSH levels. Cells lacking both SLC25A39 and its paralog SLC25A40 exhibit defects in the activity and stability of iron-sulfur cluster containing proteins. Moreover, mitochondrial GSH import is necessary for cell proliferation in vitro and red blood cell development in mice. Remarkably, the heterologous expression of an engineered bifunctional bacterial GSH biosynthetic enzyme (GshF) in mitochondria enabled mitochondrial GSH production and ameliorated the metabolic and proliferative defects caused by its depletion. Finally, GSH availability negatively regulates SLC25A39 protein abundance, coupling redox homeostasis to mitochondrial GSH import in mammalian cells. Our work identifies SLC25A39 as an essential and regulated component of the mitochondrial GSH import machinery.

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

“…Our work shows that A39 is required for GSH transport, and the screen further suggests that the impact of impaired mitochondrial GSH transport is sensitive to genetic and environmental states, notably iron metabolism. While this manuscript was in revision, Wang et al manuscript in Biorxiv 66 independently reported the characterization of SLC25A39 as a mitochondrial GSH transporter and a secondary defect of iron-sulfur cluster deficiency in cells depleted of mitochondrial glutathione. This observation might explain the decreased mitochondrial respiration in the A39 KO cells we have observed and the buffering interaction with the iron transporter SLC25A37 in our screen.…”

Transsulfuration, minor player or crucial for cysteine homeostasis in cancer

“…Cysteine is rate-limiting for production of glutathione (GSH), a prominent antioxidant in mammalian cells [1,6,7]. Mitochondrial GSH import is essential for the activity of FeS cluster proteins and mitochondrial mRNA translation [8]. GSH dysregulation contributes to many pathological conditions including neurodegenerative disorders, anemia, aberrant aging, and cancer initiation and progression [7,8].…”

Nonalcoholic steatohepatitis and mechanisms by which it is ameliorated by activation of the CNC-bZIP transcription factor Nrf2

“…Therefore, ROS and lipid peroxidation products will probably perturb glutathionylation of these proteins, leading to loss of mitochondrial regulation. Furthermore, GSH in mitochondria is derived from the cytoplasmic pool by the actions of solute carrier (SLC) 25A39 and SLC25A40 [124], which indicates it is vulnerable to oxidative stress in other subcellular compartments. Interestingly, depletion of GSH is necessary for the biogenesis of iron-sulfur cluster proteins within the mitochondrion, such as succinate dehydrogenase, cytochrome c oxidase subunit II, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8 and mitochondrial rRNA methyltransferase 1 [124].…”

“…Furthermore, GSH in mitochondria is derived from the cytoplasmic pool by the actions of solute carrier (SLC) 25A39 and SLC25A40 [124], which indicates it is vulnerable to oxidative stress in other subcellular compartments. Interestingly, depletion of GSH is necessary for the biogenesis of iron-sulfur cluster proteins within the mitochondrion, such as succinate dehydrogenase, cytochrome c oxidase subunit II, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8 and mitochondrial rRNA methyltransferase 1 [124]. In this context it is notable that cholesterol enriched mitochondria exhibit diminished membrane fluidity and GSH transport (presumably via SLC25A39 and SLC25A40) that is likely to impair their function [125].…”

“…For example, it is not known whether the MRC functions efficiently in mitochondria from Nrf2-ko mice or if OXPHOS is inhibited when the knockout animals are placed on a NASH-provoking diet. Given that mitochondria obtain their GSH from the cytoplasmic pool through the actions of SLC25A39 [124], it is probable that the ability of mitochondria to inactivate ROS through GPX isoenzymes will be impaired in Nrf2-ko mice because the cytoplasmic GSH pool will be depleted.…”