Peptidyl–Prolyl Isomerase Activity in Chloroplast Thylakoid Lumen is a Dispensable Function of Immunophilins in Arabidopsis thaliana

Abstract

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.

Activation of the Stt7/STN7 Kinase through Dynamic Interactions with the Cytochrome b6f Complex

“…Immunoblotting was performed as described previously in Ingelsson et al (2009) and Shapiguzov et al, (2010) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10% acrylamide gels. Antibodies against HA and P-Thr were purchased from Cell Signaling Technology (Danvers, MA).…”

The FKBP families of higher plants: Exploring the structures and functions of protein interaction specialists

“…Earlier yeast two-hybrid work by Gupta et al [64] found that Rieske interacted specifically with the AtFKBP13 signal peptide and the authors proposed a role for AtFKBP13 as a stromal regulator of Rieske assembly. This has been recently challenged by the absence of Rieske over-accumulation in an atfkbp13 knockout mutant [61] and by our finding that Rieske interacts with the mature, lumenal form of TaFKBP13, and not with its chloroplast precursor [63]. The functional significance of the FKBP13-Rieske interaction is currently unknown.…”

Phosphoproteomics of Arabidopsis chloroplasts reveals involvement of the STN7 kinase in phosphorylation of nucleoid protein pTAC16

“…Isolated proteins were also separated on SDS-PAGE and the presence of pTAC16 in the samples was analyzed by immunoblotting. Immunoblotting was performed as described before [22]. Polyclonal antibody to pTAC16 was raised against a synthetic peptide corresponding to residues 454-467 of the full pTAC16 sequence (Innovagen, Lund, Sweden).…”