title: Arabidopsis cotyledon chloroplast biogenesis factor CYO1 uses glutathione as an electron donor and interacts with PSI (A1 and A2) and PSII (CP43 and CP47) subunits.
abstract: CYO1 is required for thylakoid biogenesis in cotyledons of Arabidopsis thaliana. To elucidate the enzymatic characteristics of CYO1, we analyzed the protein disulfide isomerase (PDI) activity of CYO1 using dieosin glutathione disulfide (Di-E-GSSG) as a substrate. The reductase activity of CYO1 increased as a function of Di-E-GSSG, with an apparent K(m) of 824nM and K(cat) of 0.53min(-1). PDI catalyzes dithiol/disulfide interchange reactions, and the cysteine residues in PDI proteins are very important. To analyze the significance of the cysteine residues for the PDI activity of CYO1, we estimated the kinetic parameters of point-mutated CYO1 proteins. C117S, C124S, C135S, and C156S had higher values for K(m) than did wild-type CYO1. C158S had a similar K(m) but a higher K(cat), and C138S and C161S had similar K(m) values but lower K(cat) values than did wild-type CYO1. These results suggested that the cysteine residues at positions 138 and 161 were important for PDI activity. Low PDI activity of CYO1 was observed when NADPH or NADH was used as an electron donor. However, PDI activity was observed with CYO1 and glutathione, suggesting that glutathione may serve as a reducing agent for CYO1 in vivo. Based on analysis with the split-ubiquitin system, CYO1 interacted with the A1 and A2 subunits of PSI and the CP43 and CP47 subunits of PSII. Thus, CYO1 may accelerate the folding of cysteine residue--containing PSI and PSII subunits by repeatedly breaking and creating disulfide bonds.
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Atsuko
authors.initials: A
authors.lastname: Muranaka
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Watanabe
authors.forename: Atsushi
authors.initials: A
authors.lastname: Sakamoto
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Shimada
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism*; D018548:Cotyledon / Q000378:metabolism*; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives* / Q000378:metabolism; D014644:Genetic Variation; D005838:Genotype; D005978:Glutathione / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000031:analogs & derivatives* / Q000378:metabolism; D009154:Mutation; D030821:Plants, Genetically Modified; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*
chemical_list: C523201:dieosin glutathione disulfide; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D004801:Eosine Yellowish-(YS); D019803:Glutathione Disulfide