Characterization of γ-Glutamyl Peptidases and γ-Glutamyl Cyclotransferases for Glutathione Degradation in Arabidopsis

Ito, T.; Kitaiwa, T.; Nishizono, K.; Umahashi, M.; Miyaji, S.; Agake, S.-i.; Kuwahara, K.; Yokoyama, T.; Maruyama-Nakashita, A.; Sugiyama, R.; Yokota Hirai, M.; Ohkama-Ohtsu, N.

doi:10.1101/2021.10.29.466391

bioRxiv · 10.1101/2021.10.29.466391

Characterization of γ-Glutamyl Peptidases and γ-Glutamyl Cyclotransferases for Glutathione Degradation in Arabidopsis

Ito, T.·Kitaiwa, T.·Nishizono, K.·Umahashi, M.·Miyaji, S.·Agake, S.-i.·Kuwahara, K.·Yokoyama, T.·Maruyama-Nakashita, A.·Sugiyama, R.·Yokota Hirai, M.·Ohkama-Ohtsu, N.

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Abstract

O_LIOrganic sulfur is stored as glutathione (GSH) in plants. In Arabidopsis, {gamma}-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 {gamma}-glutamyl peptidases, GGP1 and GGP3, play a substantial role in degrading GSH in the cytosol. C_LIO_LIWe 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. C_LIO_LIGGP1 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. C_LIO_LIWe 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. C_LI

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Ito, T., Kitaiwa, T., Nishizono, K., Umahashi, M., Miyaji, S., Agake, S.-i., Kuwahara, K., Yokoyama, T., Maruyama-Nakashita, A., Sugiyama, R., Yokota Hirai, M., Ohkama-Ohtsu, N.. 2021-10-31. Characterization of γ-Glutamyl Peptidases and γ-Glutamyl Cyclotransferases for Glutathione Degradation in Arabidopsis. https://doi.org/10.1101/2021.10.29.466391

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