High-affinity transport of glutathione is part of a multicomponent system essential for mitochondrial function.

Abstract

Glutathione, an essential cellular antioxidant required for mitochondrial function, is not synthesized by mitochondria but is imported from the cytosol. Rat liver mitochondria have a multicomponent system that underlies the remarkable ability of mitochondria to take up and retain glutathione. At external glutathione levels of <1 mM, glutathione is transported into the mitochondrial anatrix by a high-affinity component (K., -'60 FM-; V ,K, 0.5 nmol/min per mg of protein), which is saturated at levels of 1-2 mM and stimulated by ATP. Another component has lower affinity (Km, -5.4 mM; V,,,,, -5.9 nmol/min per mg of protein) and is stimulated by ATP and ADP. Both components are inhibited by carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), glutamate, and ophthalmic acid. Increase of extramitochondrial glutathione promotes uptake and exchange; the intermembranous space seems to function as a recovery zone that promotes efficient recycling of matrix glutathione. The findings are in accord with in vivo data showing that (i) rapid exchange occurs between mitochondrial and cytosolic glutathione, (ii) lowering of cytosolic glutathione levels (produced by administration of buthionine siilfoximine) decreases export of glutathione from mitochondria to cytosol, and (iii) administration of glutathione esters increases glutathione levels in mitochondria more than those in the cytosol.A small but significant fraction of the oxygen utilized by mitochondria is converted to hydrogen peroxide (1). Much has been written about the toxicity of hydrogen peroxide, superoxide anion, and other reactive oxygen compounds and also about the antioxidant defenses that seem to protect cells against oxygen toxicity (see, for example, refs. 2-5). The suggested "primary defenses" (5) include the activities of such enzymes as superoxide dismutase, catalase, and glutathione (GSH) peroxidases and also smaller molecules such as ascorbate, a-tocopherol, GSH, B-carotene, and uric acid. Superoxide dismutase converts superoxide anion to hydrogen peroxide, which is destroyed in mitochondria (which lack catalase) by GSH peroxidase. GSH is involved in the reduction of dehydroascorbate to ascorbate and also in the maintenance of a-tocopherol in the reduced state. Thus, it appears that GSH plays a crucial role as a cellular antioxidant.In the course of studies in this laboratory on the functions of GSH, we induced GSH deficiency in vivo in mice and rats by administration of buthionine sulfoximine (BSO), a selective and irreversible transition-state inhibitor of y-glutamylcysteine synthetase (4, 6-9), the enzyme that catalyzes the first step of GSH synthesis. GSH deficiency leads to marked structural damage in several tissues, including skeletal muscle (10), lung (11), lens epithelia of newborns (12), and epithelia of the jejunum and colon (13). Cellular damage in each instance is characterized by severe mitochondrial degeneration and very low levels of mitochondrial GSH. Administration of GSH monoester (14-17) eliminated the BSO-induced GSH de...

Oxidative Stress Induced S-glutathionylation and Proteolytic Degradation of Mitochondrial Thymidine Kinase 2

“…Mitochondrial GSH/GSSG plays an important role in redox buffering and redox signaling. In humans, glutathione depletion has been associated with renal, hepatic, and brain tissue damage along with degradation of mitochondria (22,23). Severe impairment of glutathione homeostasis and changes in glutathione-dependent antioxidant defense have been found to be associated with Friedreich's ataxia (24).…”

Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line

“…Mitochondria do not possess the enzymes required for de novo GSH synthesis, 40 but utilize cytosolic GSH derived from a multicomponent, ATP dependent, mitochondrial transporter that translocates GSH from the cytosol into the mitochondrial matrix. 41,42 The transporter has a high affinity component which functions at low cytosolic GSH levels, to maintain mitochondrial GSH levels during periods of cytosolic GSH depletion. 41 Evidence for this transporter function in PW cells is provided by the observation that after cytosolic GSH depletion, the enriched mitochondrial GSH fraction still contains approximately 25% of the pretreatment GSH content compared with untreated control samples.…”

“…41,42 The transporter has a high affinity component which functions at low cytosolic GSH levels, to maintain mitochondrial GSH levels during periods of cytosolic GSH depletion. 41 Evidence for this transporter function in PW cells is provided by the observation that after cytosolic GSH depletion, the enriched mitochondrial GSH fraction still contains approximately 25% of the pretreatment GSH content compared with untreated control samples. After 48 h of treatment with BSO, GSH was undetectable in mitochondrial fractions.…”

The Human Hydroxyacylglutathione Hydrolase (HAGH) Gene Encodes Both Cytosolic and Mitochondrial Forms of Glyoxalase II

“…It is possible that the mitochondrial enzyme hydrolyzes S-D-lactoylglutathione that has diffused or been transported into the mitochondrial matrix from the cytosol (12). It has also been suggested that uptake of S-D-lactoylglutathione is a possible pathway for the mitochondrial import of GSH (12), although direct import of GSH also occurs (32). Alternatively mitochondrial glyoxalase II activity may be required to hydrolyze thiol esters of glutathione formed by non-enzymatic or enzymatic acyl transfer from mitochondrial thiol esters of coenzyme A (9).…”