Abstract
The chloroplast Deg1 protein performs proteolytic cleavage of the photodamaged D1 protein of the photosystem II (PSII) reaction center, PSII extrinsic subunit PsbO and the soluble electron carrier plastocyanin. Using biochemical, immunological and mass spectrometry approaches we showed that the heterogeneously expressed Deg1 protease from Arabidopsis thaliana can be responsible for the degradation of the monomeric light-harvesting complex antenna subunits of PSII (LHCII), CP26 and CP29, as well as PSII-associated PsbS (CP22/NPQ4) protein. The results may indicate that cytochrome b 6 protein and two previously unknown thylakoid proteins, Ptac16 and an 18.3-kDa protein, may be the substrates for Deg1. The interaction of Deg1 with the PsbS protein and the minor LHCII subunits implies its involvement in the regulation of both excess energy dissipation and state transition adaptation processes.
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Abbreviations
- BSA:
-
Bovine serum albumin
- Chl:
-
Chlorophyll
- EDTA:
-
Ethylene-diamine-tetraacetic acid
- Fv/Fm:
-
Ratio of variable to maximum chlorophyll fluorescence
- IPTG:
-
Isopropylthio-b-d-galactoside
- LHCII:
-
Light harvesting complex II
- OG:
-
n-Octyl-β-d-glucoside
- PMSF:
-
Phenylmethylsulfonylfluoride
- Pn:
-
Net photosynthesis rate
- PPFD:
-
Photosynthetic photon flux density
- PSII:
-
Photosystem II
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Acknowledgments
The work was supported by the Grant N303605438 from the Polish Ministry of Science and Higher Education. We would like to thank Professor Zach Adam for providing the Deg1 antibody and a plasmid containing the A. thaliana Deg1 transgene. We are especially grateful to Dr. Joanna Kargul for helpful comments and correction of the manuscript.
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425_2011_1505_MOESM2_ESM.tif
Suppl. Fig. S1. Ni-NTA purification of recombinant Deg1 of A. thaliana. (a) SDS-PAGE analysis of Deg1 expression. Samples were resolved on SDS-PAGE and stained with Coomassie Blue. Lanes 1 and 2, crude bacterial extracts before and after induction of Deg1 overexpression, respectively; lane 3, purified Deg1 protein. (b) Mass spectrometric analysis of purified A. thaliana Deg1. A band containing the His-tagged Deg1 protein was excised from the gel and analyzed using mass spectrometry. Identified peptides that matched the Deg1 amino acid sequence are shown in bold. Matching peptides covered 43% of the Deg1 amino acid sequence. (TIFF 133 kb)
425_2011_1505_MOESM3_ESM.tif
Suppl. Fig. S2. Degradation of β-casein by purified Deg1 (wild type) and Deg1S280G (mutant). Fraction eluted from the Ni-NTA agarose column (3 'g) was incubated with β-casein (8 'g) for 1h at 30°C. At the end of the incubation, reaction mixtures were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. Similar results were obtained in three independent experiments. (TIFF 606 kb)
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Zienkiewicz, M., Ferenc, A., Wasilewska, W. et al. High light stimulates Deg1-dependent cleavage of the minor LHCII antenna proteins CP26 and CP29 and the PsbS protein in Arabidopsis thaliana . Planta 235, 279–288 (2012). https://doi.org/10.1007/s00425-011-1505-x
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DOI: https://doi.org/10.1007/s00425-011-1505-x