In plant chloroplasts,photosystem II(PSII)complexes,together with light-harvesting complex II(LHCII),form various PSII-LHCII supercomplexes(SCs).This process likely involves immunophilins,but the underlying regulatory...In plant chloroplasts,photosystem II(PSII)complexes,together with light-harvesting complex II(LHCII),form various PSII-LHCII supercomplexes(SCs).This process likely involves immunophilins,but the underlying regulatory mechanisms are unclear.Here,by comparing Arabidopsis thaliana mutants lacking the chloroplast lumen-localized immunophilin CYCLOPHILIN28(CYP28)to wildtype and transgenic complemented lines,we determined that CYP28 regulates the assembly and accumulation of PSII-LHCII SCs.Compared to the wild type,cyp28 plants showed accelerated leaf growth,earlier flowering time,and enhanced accumulation of high molecular weight PSII-LHCII SCs under normal light conditions.The lack of CYP28 also significantly affected the electron transport rate.Blue native-polyacrylamide gel electrophoresis analysis revealed more Lhcb6 and less Lhcb4 in M-LHCII-Lhcb4-Lhcb6 complexes in cyp28 versus wild-type plants.Peptidyl-prolyl cis/trans isomerase(PPIase)activity assays revealed that CYP28 exhibits weak PPIase activity and that its K113 and E187 residues are critical for this activity.Mutant analysis suggested that CYP28 may regulate PSIILHCII SC accumulation by altering the configuration of Lhcb6 via its PPIase activity.Furthermore,the Lhcb6-P139 residue is critical for PSII-LHCII SC assembly and accumulation.Therefore,our findings suggest that CYP28 likely regulates PSII-LHCII SC assembly and accumulation by altering the configuration of P139 of Lhcb6 via its PPIase activity.展开更多
FKBP23 was found in mouse endoplasmic reticulum (ER) in 1998. It consists of an N-terminal peptidyl-prolyl cis-trans isomerase (PPlase) domain and a C-terminal domain with Ca2+ binding sites. The fusion protein of mou...FKBP23 was found in mouse endoplasmic reticulum (ER) in 1998. It consists of an N-terminal peptidyl-prolyl cis-trans isomerase (PPlase) domain and a C-terminal domain with Ca2+ binding sites. The fusion protein of mouse FKBP23 and glutathione S-transferase (GST), GST-FKBP23, and the fusion protein of BiP, a member of heat shock protein 70 (Hsp 70) in ER, and GST, GST-BiP, were subcloned in E. coli, expressed and purified. The fusion proteins were restrictively digested by Factor Xa (FaXa) to obtain the free cloned proteins FKBP23 and BiP. With the assay of adsorption of free FKBP23 or BiP with GST-BiP or GST-FKBP23 attached to the Glutathione-Sepharose 4B, the adsorbed FKBP23 or BiP could be detected by Immunoblot. It means that FKBP23 binds to BiP. Furthermore, BiP in leukocyte ER-extract can be adsorbed with GST-FKBP23 attached to the glutathione-Sepharose 4B. It shows that FKBP23 binds to natural BiP in ER, too. These experiments show that a PPIase binds to a molecular chaperone of the Hsp70 family.展开更多
基金supported by the National Natural Science Foundation of China(31700206)the Natural Science Basic Research Program of Shaanxi(2016JM3023)the Special Scientific Research Project of Education Department of Shaanxi Province(16JK1792)。
文摘In plant chloroplasts,photosystem II(PSII)complexes,together with light-harvesting complex II(LHCII),form various PSII-LHCII supercomplexes(SCs).This process likely involves immunophilins,but the underlying regulatory mechanisms are unclear.Here,by comparing Arabidopsis thaliana mutants lacking the chloroplast lumen-localized immunophilin CYCLOPHILIN28(CYP28)to wildtype and transgenic complemented lines,we determined that CYP28 regulates the assembly and accumulation of PSII-LHCII SCs.Compared to the wild type,cyp28 plants showed accelerated leaf growth,earlier flowering time,and enhanced accumulation of high molecular weight PSII-LHCII SCs under normal light conditions.The lack of CYP28 also significantly affected the electron transport rate.Blue native-polyacrylamide gel electrophoresis analysis revealed more Lhcb6 and less Lhcb4 in M-LHCII-Lhcb4-Lhcb6 complexes in cyp28 versus wild-type plants.Peptidyl-prolyl cis/trans isomerase(PPIase)activity assays revealed that CYP28 exhibits weak PPIase activity and that its K113 and E187 residues are critical for this activity.Mutant analysis suggested that CYP28 may regulate PSIILHCII SC accumulation by altering the configuration of Lhcb6 via its PPIase activity.Furthermore,the Lhcb6-P139 residue is critical for PSII-LHCII SC assembly and accumulation.Therefore,our findings suggest that CYP28 likely regulates PSII-LHCII SC assembly and accumulation by altering the configuration of P139 of Lhcb6 via its PPIase activity.
基金This work was supported by the National Natural Science Foundation of China(Grant No.30170870)the Ministry of Education of China(Department of Foreign Affairs(99)747).
文摘FKBP23 was found in mouse endoplasmic reticulum (ER) in 1998. It consists of an N-terminal peptidyl-prolyl cis-trans isomerase (PPlase) domain and a C-terminal domain with Ca2+ binding sites. The fusion protein of mouse FKBP23 and glutathione S-transferase (GST), GST-FKBP23, and the fusion protein of BiP, a member of heat shock protein 70 (Hsp 70) in ER, and GST, GST-BiP, were subcloned in E. coli, expressed and purified. The fusion proteins were restrictively digested by Factor Xa (FaXa) to obtain the free cloned proteins FKBP23 and BiP. With the assay of adsorption of free FKBP23 or BiP with GST-BiP or GST-FKBP23 attached to the Glutathione-Sepharose 4B, the adsorbed FKBP23 or BiP could be detected by Immunoblot. It means that FKBP23 binds to BiP. Furthermore, BiP in leukocyte ER-extract can be adsorbed with GST-FKBP23 attached to the glutathione-Sepharose 4B. It shows that FKBP23 binds to natural BiP in ER, too. These experiments show that a PPIase binds to a molecular chaperone of the Hsp70 family.
