Cellular oxidation is essential for many physiological processes but poses a constant threat to redoxvulnerable proteins.Eukaryotic cells maintain millimolar concentrations of reduced glutathione(GSH)as a primary anti...Cellular oxidation is essential for many physiological processes but poses a constant threat to redoxvulnerable proteins.Eukaryotic cells maintain millimolar concentrations of reduced glutathione(GSH)as a primary antioxidative defense;however,how this highly reduced pool is locally reorganized to permit necessary oxidation without triggering widespread proteolysis remains poorly understood.Here,we demonstrate that protein S-glutathionylation promotes stress granule(SG)assembly,thereby creating reductive microenvironments within an otherwise oxidized cytosol to protect redox-sensitive proteins and modulate GSH biosynthesis during salicylic acid(SA)-induced oxidative stress in Arabidopsis thaliana.SA enhances GSH oxidation and protein glutathionylation both in vitro and in vivo.To visualize glutathionylated proteins under native conditions,we developed CamLog,a transgene-free,click-activated metabolic labeling method.Using CamLog method,we revealed that SA induces glutathionylated protein condensates that strongly colocalize with SG markers but not with processing bodies.These condensates share more than 77% of their components with canonical SGs,with translation-related proteins being particularly enriched,and pharmacological analyses confirmed their identity as oxidative stressinduced SGs.Glutathionylation of the SG marker RBP47B regulates its mobility and SA responsiveness,and global inhibition of glutathionylation abolishes SG formation.Conversely,SGs sequester glutathionylated proteins,including components of the translation machinery,forming a reductive niche that prevents oxidation-induced proteolysis.SGs also sequester GSH1,the rate-limiting enzyme in GSH biosynthesis,suggesting a mechanism for fine-tuning GSH metabolism rather than fully counteracting oxidation.Taken together,these findings uncover an organelle-level role for SGs in shaping cytosolic redox heterogeneity and reveal a spatial antioxidative strategy critical for maintaining proteostasis in oxidation-vulnerable systems.展开更多
基金supported by the National Natural Science Foundation of China(32470734 to W.W.)the State Key Laboratory for Gene Function and Modulation Research,School of Life Sciences,Peking University(to W.W.)+5 种基金the Center for Life Sciences(to W.W.)the National Natural Science Foundation of China(31970283 and 32370288 to M.Z.)the Beijing Nova Program of Science and Technology(Z191100001119027 to M.Z.)the Support Project of High-Level Teachers in Beijing Municipal Universities during the 14th Five-Year Plan(BPHR20220114 to M.Z.)Capital Normal University(to M.Z.),the Beijing Natural Science Foundation(5254024 to S.Z.)the Postdoctoral Fellowship of the Center for Life Sciences(to S.Z.,Z.X.,Y.L.,and C.C.).
文摘Cellular oxidation is essential for many physiological processes but poses a constant threat to redoxvulnerable proteins.Eukaryotic cells maintain millimolar concentrations of reduced glutathione(GSH)as a primary antioxidative defense;however,how this highly reduced pool is locally reorganized to permit necessary oxidation without triggering widespread proteolysis remains poorly understood.Here,we demonstrate that protein S-glutathionylation promotes stress granule(SG)assembly,thereby creating reductive microenvironments within an otherwise oxidized cytosol to protect redox-sensitive proteins and modulate GSH biosynthesis during salicylic acid(SA)-induced oxidative stress in Arabidopsis thaliana.SA enhances GSH oxidation and protein glutathionylation both in vitro and in vivo.To visualize glutathionylated proteins under native conditions,we developed CamLog,a transgene-free,click-activated metabolic labeling method.Using CamLog method,we revealed that SA induces glutathionylated protein condensates that strongly colocalize with SG markers but not with processing bodies.These condensates share more than 77% of their components with canonical SGs,with translation-related proteins being particularly enriched,and pharmacological analyses confirmed their identity as oxidative stressinduced SGs.Glutathionylation of the SG marker RBP47B regulates its mobility and SA responsiveness,and global inhibition of glutathionylation abolishes SG formation.Conversely,SGs sequester glutathionylated proteins,including components of the translation machinery,forming a reductive niche that prevents oxidation-induced proteolysis.SGs also sequester GSH1,the rate-limiting enzyme in GSH biosynthesis,suggesting a mechanism for fine-tuning GSH metabolism rather than fully counteracting oxidation.Taken together,these findings uncover an organelle-level role for SGs in shaping cytosolic redox heterogeneity and reveal a spatial antioxidative strategy critical for maintaining proteostasis in oxidation-vulnerable systems.
