Proteomics gives insight into the regulatory function of chloroplast thioredoxins.
Thioredoxins are small multifunctional redox active proteins widely if not universally distributed among living organisms. In chloroplasts, two types of thioredoxins (f and m) coexist and play central roles in regulating enzyme activity. Reduction of thioredoxins in chloroplasts is catalyzed by an iron-sulfur disulfide enzyme, ferredoxin-thioredoxin reductase, that receives photosynthetic electrons from ferredoxin, thereby providing a link between light and enzyme activity. Chloroplast thioredoxins function in the regulation of the Calvin cycle and associated processes. However, the relatively small number of known thioredoxin-linked proteins (about 16) raised the possibility that others remain to be identified. To pursue this opportunity, we have mutated thioredoxins f and m, such that the buried cysteine of the active disulfide has been replaced by serine or alanine, and bound them to affinity columns to trap target proteins of chloroplast stroma. The covalently linked proteins were eluted with DTT, separated on gels, and identified by mass spectrometry. This approach led to the identification of 15 potential targets that function in 10 chloroplast processes not known to be thioredoxin linked. Included are proteins that seem to function in plastid-to-nucleus signaling and in a previously unrecognized type of oxidative regulation. Approximately two-thirds of these targets contained conserved cysteines. We also identified 11 previously unknown and 9 confirmed target proteins that are members of pathways known to be regulated by thioredoxin. In contrast to results with individual enzyme assays, specificity for thioredoxin f or m was not observed on affinity chromatography.
Balmer
Yves
Y
Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley 94720, USA.
Koller
Antonius
A
del Val
Gregorio
G
Manieri
Wanda
W
Schürmann
Peter
P
Buchanan
Bob B
BB
D002736:Chloroplasts / Q000235:genetics*; D003001:Cloning, Molecular; D003624:Darkness; D004798:Enzymes / Q000378:metabolism; D008027:Light; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000096:biosynthesis; D040901:Proteomics; D011994:Recombinant Proteins / Q000378:metabolism; D018724:Spinacia oleracea / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics* / Q000378:metabolism
D004798:Enzymes; D010940:Plant Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins