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Plant and Cell Physiology Advance Access originally published online on August 28, 2009
Plant and Cell Physiology 2009 50(10):1801-1814; doi:10.1093/pcp/pcp122
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© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

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

Björn Ingelsson1, Alexey Shapiguzov1,3, Thomas Kieselbach2 and Alexander V. Vener1,*

1Department of Clinical and Experimental Medicine, Linköping University, SE-581 85 Linköping, Sweden
2Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden

*Corresponding author: E-mail, alexander.vener{at}liu.se; Fax, +46-13-224314.


  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.

Keywords: Arabidopsis thaliana - Chloroplast thylakoid lumen - Cyclophilin - FKBP - Immunophilin - Peptidyl-prolyl isomerase activity

Abbreviations: CsA, cyclosporin A; FKBP, FK506-binding protein; FPLC, fast protein liquid chromatography; ICPL, isotope-coded protein labeling; LC-MS, liquid chromatography–mass spectrometry; PPIase, peptidyl–prolyl cis/trans isomerase; RT-PCR, reverse transcription–PCR.

3Present address: Departments of Molecular Biology and of Plant Biology, University of Geneva, 30 Quai E. Ansermet, CH-1211 Geneva 4, Switzerland.

(Received July 10, 2009; Accepted August 24, 2009)
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