Immunophilin AtFKBP13 sustains all peptidyl-prolyl isomerase activity in the thylakoid lumen from Arabidopsis thaliana deficient in AtCYP20-2.
The physiological roles of immunophilins are unclear, but many possess peptidyl-prolyl isomerase (PPIase) activity, and they have been found in all organisms examined to date, implying that they are involved in fundamental, protein-folding processes. The chloroplast thylakoid lumen of the higher plant Arabidopsis thaliana contains up to 16 immunophilins (five cyclophilins and 11 FKBPs), but only two of them, AtCYP20-2 and AtFKBP13, have been found to be active PPIases, indicating that the other immunophilins in this cellular compartment may have lost their putative PPIase activities. To assess this possibility, we characterized two independent Arabidopsis knockout lines lacking AtCYP20-2 in enzymological and quantitative proteomic analyses. The PPIase activity in thylakoid lumen preparations of both mutants was equal to that of corresponding wild-type preparations, and comparative two-dimensional difference gel electrophoresis analyses of the lumenal proteins of the mutants and wild type showed that none of the potential PPIases was more abundant in the AtCYP20-2 deficient plants. Enzymatic analyses established that all PPIase activity in the mutant thylakoid lumen was attributable to AtFKBP13, and oxidative activation of this enzyme compensated for the lack of AtCYP20-2. Accordingly, sequence analyses of the potential catalytic domains of lumenal cyclophilins and FKBPs demonstrated that only AtCYP20-2 and AtFKBP13 possess all of the amino acid residues found to be essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. Thus, none of the immunophilins in the chloroplast thylakoid lumen of Arabidopsis except AtCYP20-2 and AtFKBP13 appear to possess prolyl isomerase activity toward peptide substrates.
Edvardsson
Anna
A
Division of Cell Biology, Linköping University, SE-581 85 Linköping, Sweden.
Shapiguzov
Alexey
A
Petersson
Ulrika A
UA
Schröder
Wolfgang P
WP
Vener
Alexander V
AV
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D021983:Cyclophilins / Q000235:genetics / Q000378:metabolism; D023281:Genomics; D020104:Immunophilins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D019696:Peptidylprolyl Isomerase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D013379:Substrate Specificity; D022021:Tacrolimus Binding Proteins / Q000032:analysis / Q000378:metabolism*; D020524:Thylakoids / Q000201:enzymology*
D029681:Arabidopsis Proteins; C517220:FKBP13 protein, Arabidopsis; C523353:CYP20-2 protein, Arabidopsis; D021983:Cyclophilins; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins; D019696:Peptidylprolyl Isomerase