Frederick S. Yen

Mitochondria must maintain adequate amounts of metabolites for protective and biosynthetic functions. However, how mitochondria sense the abundance of metabolites and regulate metabolic homeostasis is not well understood. In this work, we focused on glutathione (GSH), a critical redox metabolite in mitochondria, and identified a feedback mechanism that controls its abundance through the mitochondrial GSH transporter, SLC25A39. Under physiological conditions, SLC25A39 is rapidly degraded by mitochondrial protease AFG3L2. Depletion of GSH dissociates AFG3L2 from SLC25A39, causing a compensatory increase in mitochondrial GSH uptake. Genetic and proteomic analyses identified a putative iron-sulfur cluster in the matrix-facing loop of SLC25A39 as essential for this regulation, coupling mitochondrial iron homeostasis to GSH import. Altogether, our work revealed a paradigm for the autoregulatory control of metabolic homeostasis in organelles.

SUMMARY Glutathione (GSH) is a small molecule thiol abundantly present in all eukaryotes with key roles in oxidative metabolism 1 . Mitochondria, as the major site of oxidative reactions, must maintain sufficient levels of GSH to perform protective and biosynthetic functions 2 . 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 ironsulfur 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.