Glutathione (GSH) functions as a major sulfur repository and hence occupies an important position in primary sulfur metabolism. GSH degradation results in sulfur reallocation and is believed to be carried out mainly by c-glutamyl cyclotransferases (GGCT2;1, GGCT2;2, and GGCT2;3), which, however, do not fully explain the rapid GSH turnover. Here, we discovered that c-glutamyl peptidase 1 (GGP1) contributes to GSH degradation through a yeast complementation assay. Recombinant proteins of GGP1, as well as GGP3, showed high degradation activity of GSH, but not of oxidized glutathione (GSSG), in vitro. Notably, the GGP1 transcripts were highly abundant in rosette leaves, in agreement with the ggp1 mutants constantly accumulating more GSH regardless of nutritional conditions. Given the lower energy requirements of the GGP-than the GGCT-mediated pathway, the GGP-mediated pathway could be a more efficient route for GSH degradation than the GGCT-mediated pathway. Therefore, we propose a model wherein cytosolic GSH is degraded chiefly by GGP1 and likely also by GGP3. Another noteworthy fact is that GGPs are known to process GSH conjugates in glucosinolate and camalexin synthesis; indeed, we confirmed that the ggp1 mutant contained higher levels of O-acetyl-L-Ser, a signaling molecule for sulfur starvation, and lower levels of glucosinolates and their degradation products. The predicted structure of GGP1 further provided a rationale for this hypothesis. In conclusion, we suggest that GGP1 and possibly GGP3 play vital roles in both primary and secondary sulfur metabolism.

SummaryOrganic sulfur is stored as glutathione (GSH) in plants. In Arabidopsis, γ-glutamyl cyclotransferases (GGCT2;1, GGCT2;2, and GGCT2;3) degrade cytosolic GSH, but they do not fully explain the rapid GSH turnover. Here, we demonstrate that γ-glutamyl peptidases, GGP1 and GGP3, play a substantial role in degrading GSH in the cytosol.We conducted yeast complementation assay and activity assay of recombinant proteins to identify the novel GSH degradation enzymes. The expression patterns were investigated by RT-qPCR. GSH concentrations in the mutants were also analyzed.GGP1 complemented the yeast phenotype. Recombinant GGP1 and GGP3 showed reasonable Km values considering cytosolic GSH concentration, and their activity was comparable to that of GGCTs. The GGP1 transcript was highly abundant in mature organs such as rosette leaves. The expression of GGCT2;1 was conspicuously enhanced under sulfur deficiency. GSH concentration was higher in ggp1 knockout mutants regardless of nutritional conditions; the concentration was higher in ggct2;1 knockout mutants under sulfur-deficient conditions.We propose a model wherein cytosolic GSH is degraded fundamentally by GGP1. The degradation is accelerated by GGCT2;1 under sulfur deficiency. Given the energy cost throughout the reactions, GGPs could render a more efficient route for GSH degradation than GGCTs.