Preferential Transport of Glutathione versusGlutathione Disulfide in Rat Liver Microsomal Vesicles

Abstract

A bi-directional, saturable transport of glutathione (GSH) was found in rat liver microsomal vesicles. GSH transport could be inhibited by the anion transport blockers flufenamic acid and 4,4-diisothiocyanostilbene-2,2-disulfonic acid. A part of GSH taken up by the vesicles was metabolized to glutathione disulfide (GSSG) in the lumen. Microsomal membrane was virtually nonpermeable toward GSSG; accordingly, GSSG generated in the microsomal lumen could hardly exit. Therefore, GSH transport, contrary to previous assumptions, is preferred in the endoplasmic reticulum, and GSSG entrapped and accumulated in the lumen creates the oxidized state of its redox buffer. The endoplasmic reticulum (ER)1 of the cell is the site of the synthesis, posttranslational modification, and folding of proteins transported along the secretory pathway. The oxidizing environment in the lumen of the ER is necessary for the formation of disulfide bonds and for the proper folding of these proteins (1). The oxidative effects are reflected in and supported by the GSH redox buffer; the ratio of GSH and GSSG is around 2:1 within the lumen of ER and along the secretory pathway, whereas the cytosolic ratio ranges from 30:1 to 100:1 (2). However, the primary source(s) of the oxidative effect has not been demonstrated. Recent observations suggest two possible mechanisms. First, the preferential uptake of the oxidized member of a redox couple through the ER membrane and/or the efflux (or exocytosis) of its reduced form could ensure the oxidative environment. Alternatively, enzymes resident in the membrane or lumen of the ER could produce oxidizing compounds (e.g. reactive oxygen species) toward the lumen. Experimental evidence supports both mechanisms. Favoring the transport-based hypothesis, the preferential transport of dehydroascorbate (the oxidized form of ascorbate) has been described in rat liver microsomal vesicles (3). Similarly, the selective microsomal transport of GSSG was also reported (2, 4). On the other hand, several microsomal enzymes (cytochrome P-450s, NADPH cytochrome P-450 reductase, gulonolactone oxidase, microsomal iron protein, NADPH-dependent oxidase, sulfydryl oxidase, etc.) can produce reactive oxygen species (5-10). The recent exploration of the ER oxidase protein (ERO1) and its role in the protein folding also support the latter mechanism (11, 12). Because of the conflicting opinions, the microsomal transport of GSH and GSSG has not been unequivocally established. The presently available data are based on the detection of microsome-associated radioactivity by applying a rapid filtration method and radiolabeled compounds (2, 4); however, intraluminal GSH or GSSG contents upon transport have not been directly demonstrated. Therefore, experiments were undertaken to reinvestigate the transport of GSH and GSSG through the ER membrane.The main difficulties in the investigation of microsomal transport processes are deriving from the very small intraluminal space, the presence of (intraluminal) reactions affecting the transported c...

Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

“…In eukaryotes many proteins are synthesized and undergo posttranslational modification in the ER. These processes require an oxidized environment, which is re- flected in a relatively low GSH:GSSG ratio (approximately 2:1 compared with approximately 50:1 in the cytosol) and the presence of enzymes that produce oxidizing compounds (38)(39)(40). Paradoxically, in the ER and associated vesicular structures, millimolar levels of ascorbate have been reported (41,42).…”

Composition of the redox environment of the endoplasmic reticulum and sources of hydrogen peroxide

“…However, considering a gradient across the ER membrane, GSSG import would imply an active pumping activity. Systematic analysis of microsomal glutathione traffic revealed that GSH rather than GSSG is preferentially transported across the ER membrane by facilitated diffusion [18]. Although such a transport is theoretically bidirectional, the physiological GSH concentrations favor the uptake of GSH 6 into the ER lumen.…”

“…Due to the poor permeability of the ER membrane to GSSG [18], the intense local GSH oxidation in the aforementioned reactions results in the luminal accumulation of GSSG, which can still leave the compartment via the secretory pathway. GSSG can also be engaged in thiol-disulfide exchange reactions with substrate proteins of oxidative folding, although these reactions are kinetically unfavorable.…”

Radical-free biology of oxidative stress

“…The chloride channel, which is mutated in cystic fibrosis (CFTR), also transports GSH (113), and GSH is transported into mitochondria by the dicarboxylate carrier (DIC) and a monocarboxylate carrier (OGCP) (103). GSH is transported into the cisternae of the endoplasmic reticulum (13), but the molecular nature of the transporter is not known. 3) GSH is used by a number of GSH transferases (GST), which include microsomal and nonmicrosomal locations, to modify electrophilic chemicals (9).…”