The Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase （GAPDH） and phosphoribulokinase （PRK） can form under oxidizing conditions a supramolecular complex with the regulatory protein CP12. Both GAPDH and...The Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase （GAPDH） and phosphoribulokinase （PRK） can form under oxidizing conditions a supramolecular complex with the regulatory protein CP12. Both GAPDH and PRK activities are inhibited within the complex, but they can be fully restored by reduced thioredoxins （TRXs）. We have investigated the interactions of eight different chloroplast thioredoxin isoforms （TRX f1, m1, m2, m3, m4, y1, y2, x） with GAPDH （A4, B4, and B8 isoforms）, PRK and CP12 （isoform 2）, all from Arabidopsis thaliana. In the complex, both A4-GAPDH and PRK were promptly activated by TRX f1, or more slowly by TRXs ml and m2, but all other TRXs were ineffective. Free PRK was regulated by TRX f1, m1, or m2, while B4- and Bs-GAPDH were absolutely specific for TRX fl. Interestingly, reductive activation of PRK caged in the complex was much faster than reductive activation of free oxidized PRK, and activation of A4-GAPDH in the complex was much faster （and less demanding in terms of reducing potential） than activation of free oxidized B4- or BB-GAPDH. It is proposed that CP12-assembled supramolecular complex may represent a reservoir of inhibited enzymes ready to be released in fully active conformation following reduction and dissociation of the complex by TRXs upon the shift from dark to low light. On the contrary, autonomous redox-modulation of GAPDH （B- containing isoforms） would be more suited to conditions of very active photosynthesis.展开更多
Thiol-based redox post-translational modifications have emerged as important mechanisms of signaling and regulation in all organisms, and thioredoxin plays a key role by controlling the thiol-disulfide status of targe...Thiol-based redox post-translational modifications have emerged as important mechanisms of signaling and regulation in all organisms, and thioredoxin plays a key role by controlling the thiol-disulfide status of target proteins. Recent redox proteomic studies revealed hundreds of proteins regulated by glutathio- nylation and nitrosylation in the unicellular green alga Chlamydomonas reinhardtii, while much less is known about the thioredoxin interactome in this organism. By combining qualitative and quantitative proteomic analyses, we have comprehensively investigated the Chlamydomonas thioredoxome and 1188 targets have been identified. They participate in a wide range of metabolic pathways and cellular pro- cesses. This study broadens not only the redox regulation to new enzymes involved in well-known thiore- doxin-regulated metabolic pathways but also sheds light on cellular processes for which data supporting redox regulation are scarce （aromatic amino acid biosynthesis, nuclear transport, etc）. Moreover, we char- acterized 1052 thioredoxin-dependent regulatory sites and showed that these data constitute a valuable resource for future functional studies in Chlamydomonas. By comparing this thioredoxome with proteomic data for glutathionylation and nitrosylation at the protein and cysteine levels, this work confirms the existence of a complex redox regulation network in Chlamydomonas and provides evidence of a tremendous selectivity of redox post-translational modifications for specific cysteine residues.展开更多
文摘The Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase （GAPDH） and phosphoribulokinase （PRK） can form under oxidizing conditions a supramolecular complex with the regulatory protein CP12. Both GAPDH and PRK activities are inhibited within the complex, but they can be fully restored by reduced thioredoxins （TRXs）. We have investigated the interactions of eight different chloroplast thioredoxin isoforms （TRX f1, m1, m2, m3, m4, y1, y2, x） with GAPDH （A4, B4, and B8 isoforms）, PRK and CP12 （isoform 2）, all from Arabidopsis thaliana. In the complex, both A4-GAPDH and PRK were promptly activated by TRX f1, or more slowly by TRXs ml and m2, but all other TRXs were ineffective. Free PRK was regulated by TRX f1, m1, or m2, while B4- and Bs-GAPDH were absolutely specific for TRX fl. Interestingly, reductive activation of PRK caged in the complex was much faster than reductive activation of free oxidized PRK, and activation of A4-GAPDH in the complex was much faster （and less demanding in terms of reducing potential） than activation of free oxidized B4- or BB-GAPDH. It is proposed that CP12-assembled supramolecular complex may represent a reservoir of inhibited enzymes ready to be released in fully active conformation following reduction and dissociation of the complex by TRXs upon the shift from dark to low light. On the contrary, autonomous redox-modulation of GAPDH （B- containing isoforms） would be more suited to conditions of very active photosynthesis.
文摘Thiol-based redox post-translational modifications have emerged as important mechanisms of signaling and regulation in all organisms, and thioredoxin plays a key role by controlling the thiol-disulfide status of target proteins. Recent redox proteomic studies revealed hundreds of proteins regulated by glutathio- nylation and nitrosylation in the unicellular green alga Chlamydomonas reinhardtii, while much less is known about the thioredoxin interactome in this organism. By combining qualitative and quantitative proteomic analyses, we have comprehensively investigated the Chlamydomonas thioredoxome and 1188 targets have been identified. They participate in a wide range of metabolic pathways and cellular pro- cesses. This study broadens not only the redox regulation to new enzymes involved in well-known thiore- doxin-regulated metabolic pathways but also sheds light on cellular processes for which data supporting redox regulation are scarce （aromatic amino acid biosynthesis, nuclear transport, etc）. Moreover, we char- acterized 1052 thioredoxin-dependent regulatory sites and showed that these data constitute a valuable resource for future functional studies in Chlamydomonas. By comparing this thioredoxome with proteomic data for glutathionylation and nitrosylation at the protein and cysteine levels, this work confirms the existence of a complex redox regulation network in Chlamydomonas and provides evidence of a tremendous selectivity of redox post-translational modifications for specific cysteine residues.
