Control of mitochondrial glutathione homeostasis by SLC25A39

Mitochondria are a class of organelles with mysterious overtones, both in their origin and central role in eukaryotic cells. As the energy and metabolic center of the cell, mitochondria have a double-layer membrane structure. Metabolite transporters on the inner membrane maintain the transport of substances between the mitochondria and the rest of the cell, and transport disorders can result in many diseases, such as neurodegenerative diseases and metabolic syndromes [ 1, 2] .

Glutathione (GSH) was discovered by De-Rey-Pailhade in 1888 and is present in most cells at high concentrations, ranging from 1‒10 mM, similar to glucose and cholesterol [3]. GSH plays important roles in the defense of antioxidant systems, homeostasis of iron-sulfur (Fe-S) clusters and other cellular functions. GSH is a tripeptide that is exclusively synthesized in the cytosol by two sequential reactions ( Figure 1A). As the powerhouses of cells, mitochondria are the strongest organelles involved in redox reactions, which produce energy primarily through electron transport chains, requiring large amounts of GSH for protective and biosynthetic functions. Generally, up to 10%–15% of cellular GSH is stored in the mitochondria. However, mitochondrion itself cannot synthesize negatively charged GSH, and how GSH enters the mitochondrial matrix remains a mystery [4].

Figure 1 .

Schematic diagram of cellular glutathione (GSH) synthesis in the cytosol and the regulatory mechanism of GSH transport to mitochondria

(A) GSH is synthesized from its constituent amino acids in the cytosol through the sequential action of γ-GCS and GS. Once synthesized in the cytosol, GSH can be transported to the mitochondrial matrix by the SLC25A39 transporter, which is located in the mitochondrial inner membrane. (B) The regulatory mechanism of the mitochondrial GSH transporter SLC25A39 by AFG3L2 and the Fe-S cluster. Under physiological conditions, SLC25A39 is degraded by AFG3L2 through matrix loop 1 to reduce mitochondrial GSH import (left). When the mitochondrial GSH concentration is low, SLC25A39 is protected from AFG3L2 proteolysis by Fe-S cluster binding loop 1, causing a compensatory increase in mitochondrial GSH uptake. Glu, glutamic acid; Cys, cysteine; γ-GCS, γ-glutamylcysteine synthase; ADP, adenosine diphosphate; ATP, adenosine triphosphate; Gly, glycine; GS, glutathione synthetase; Fe-S: Fe-S cluster; IMS: intermembrane space; IMM: inner membrane.

In 2021, Birsoy and colleagues [5] reported that mitochondrial GSH is transported by the transporter protein SLC25A39, which belongs to the SoLute Carrier 25 (SLC25) family, across the mitochondrial inner membrane ( Figure 1A). SLC25A39 accumulates upon GSH depletion, and cells lacking SLC25A39 exhibit reduced mitochondrial GSH import and abundance without affecting cellular GSH level. In addition, Shen’s team [6] reported that SLC25A39 is responsible for mitochondrial GSH import and its coordination with iron homeostasis in supporting mitochondrial OXPHOS in 2022. Therefore, a new question has arisen about how SLC25A39 senses and maintains mitochondrial GSH level.

In a recent paper, Birsoy’s team [7] reported that the protein level of SLC25A39 is maintained by an ingenious regulatory mechanism mediated by AFG3L2 (a zinc metalloprotease and an ATPase localized in the inner mitochondrial membrane) and Fe-S clusters to control mitochondrial GSH homeostasis. SLC25A39 serves as both a sensor and a transporter through its two completely independent domains: one region senses GSH, and the other transports GSH ( Figure 1B). By comparing the structure of SLC25A39 with that of SLC25A40, another member of the SLC25 family, they found that SLC25A39 has a protruding loop (loop 1) facing the matrix side that mediates its interaction with AFG3L2. Under physiological conditions, SLC25A39 is rapidly degraded by AFG3L2 (with a half-life of approximately 15 min) in a GSH-dependent manner to maintain mitochondrial GSH homeostasis ( Figure 1B). When the mitochondrial GSH concentration is low, Fe-S clusters competitively bind to loop1 to prevent the degradation of SLC25A39 by AFG3L2, thereby increasing the uptake of GSH ( Figure 1B). Depletion of GSH greatly extends the half-life of SLC25A39 (>300 min), and supplementation with GSH restores the rapid degradation of SLC25A39.

Coincidentally, Shen’s group [8] also independently reported a dual regulatory mechanism of SLC25A39 by AFG3L2 and Fe-S clusters. Based on amino acid sequence analysis of the SLC25A39 protein and AlphaFold structure prediction, Shen’s group identified an extramatochondrial matrix loop (loop1) between transmembrane domains 1 and 2 of SLC25A39 that contains four cysteine residues. Protein stability experiments revealed that the SLC25A39 protein is regulated by two mechanisms: (1) SLC25A39 is a protein with a very short half-life that does not depend on the four cysteine residues of loop1, and (2) SLC25A39 degradation is inhibited by low concentrations of GSH, which depends on the four cysteine residues of loop1. Further experiments revealed a dual regulatory mechanism of SLC25A39 involving the AFG3L2 and Fe-S clusters which directly control mitochondrial GSH homeostasis ( Figure 1B). The authors also explored the regulatory effect of SLC25A39 on mitochondrial protein quality control, GSH, and iron homeostasis in neuronal cells.

These mechanistic findings have a far-reaching impact on the understanding of mitochondrial GSH homeostasis at both the fundamental and translational levels. First, the autoregulatory control mechanism of GSH transport in mitochondria provides us with a classic example of negative feedback regulation of metabolic homeostasis at the organelle level. Similar to pH regulation in lysosomes [9], coupling GSH sensing in the mitochondrial matrix coupled with the degradation of its transporter SLC25A39 regulates mitochondrial GSH homeostasis, reflecting the subtle self-regulation of the intracellular compartment microenvironment. In addition, there have been many attempts to target GSH for the treatment of diseases such as cardiovascular and metabolic diseases, but these attempts have been limited due to the important physiological functions of GSH [ 3, 10] . We now know how SLC25A39 works in the mitochondrial inner membrane, which suggests that researchers can target SLC25A39 to design drugs without affecting the GSH levels of other cellular compartments, thereby reducing the side effects of altering GSH level. However, the exact mechanism by which Fe-S clusters and AFG3L2 collaboratively sense GSH to regulate SLC25A39 degradation needs to be further investigated. Additionally, further studies should focus on whether the regulation of GSH homeostasis by AFG3L2 is involved in AFG3L2 mutation-associated diseases such as neurological disorders [ 2, 8] .

Overall, a series of studies by two independent research groups, Birsoy and Shen, revealed the function and mechanism of SLC25A39 in mitochondria for GSH transport and homeostasis control. Understanding this control mechanism of GSH may provide important insights into the systemic role of metabolic regulation and may provide new ideas for the treatment of mitochondria/GSH-related diseases.

COMPETING INTERESTS

The authors declare that they have no conflict of interest.

Funding Statement

This work was supported by the grants from the National Key R&D Program of China (No. 2021YFF0702103) and the National Natural Science Foundation of China (Nos. 82171792 and 82200429).