Peptidyl-prolyl isomerase activity in chloroplast thylakoid lumen is a dispensable function of immunophilins in Arabidopsis thaliana.
Chloroplast thylakoid lumen of Arabidopsis thaliana contains 16 immunophilins, five cyclophilins and 11 FK506-binding proteins (FKBPs), which are considered protein folding catalysts, although only two of them, AtFKBP13 and AtCYP20-2, possess peptidyl-prolyl cis/trans isomerase (PPIase) activity. To address the question of the physiological significance of this activity, we obtained and characterized Arabidopsis mutants deficient in the most active PPIase, AtFKBP13, and a double mutant deficient in both AtFKBP13 and AtCYP20-2. Two-dimensional gel electrophoresis of isolated thylakoid lumen, as well as immunoblotting analyses of major photosynthetic membrane protein complexes did not reveal differences in protein composition between the mutants and the wild type. No changes in the relative content of photosynthetic proteins were found by differential stable isotope labeling and liquid chromatography-mass spectrometry (LC-MS) analyses. PPIase activity was measured in vitro in isolated thylakoid lumen samples using two different synthetic peptide substrates. Depending on the peptide substrate used for the assay, the PPIase activity in the thylakoid lumen of the mutants lacking either AtFKBP13 or both AtFKBP13 and AtCYP20-2 was as low as 10 or 2% of that in the wild type. Residual PPIase activity detected in the double mutant originated from AtCYP20-3, a cyclophilin from chloroplast stroma contaminating thylakoid lumen preparations. None of the mutants differed from the wild-type plants when grown under normal, cold stress or high light conditions. It is concluded that cellular functions of immunophilins in the thylakoid lumen of chloroplasts are not related to their PPIase capacity and should be investigated beyond this enzymatic activity.
Ingelsson
Björn
B
Department of Clinical and Experimental Medicine, Linköping University, SE-581 85 Linköping, Sweden.
Shapiguzov
Alexey
A
Kieselbach
Thomas
T
Vener
Alexander V
AV
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D021983:Cyclophilins / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D055786:Gene Knockout Techniques; D016254:Mutagenesis, Insertional; D009154:Mutation; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D022021:Tacrolimus Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020524:Thylakoids / Q000201:enzymology* / Q000235:genetics
D029681:Arabidopsis Proteins; C517220:FKBP13 protein, Arabidopsis; C523353:CYP20-2 protein, Arabidopsis; D021983:Cyclophilins; D022021:Tacrolimus Binding Proteins; D019696:Peptidylprolyl Isomerase