Journal Article

Topological Analysis of the Extrinsic PsbO, PsbP and PsbQ Proteins in a Green Algal PSII Complex by Cross-Linking with a Water-Soluble Carbodiimide

Ryo Nagao, Takehiro Suzuki, Akinori Okumura, Ayako Niikura, Masako Iwai, Naoshi Dohmae, Tatsuya Tomo, Jian-Ren Shen, Masahiko Ikeuchi and Isao Enami

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 51, issue 5, pages 718-727
Published in print May 2010 | ISSN: 0032-0781
Published online April 2010 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcq042
Topological Analysis of the Extrinsic PsbO, PsbP and PsbQ Proteins in a Green Algal PSII Complex by Cross-Linking with a Water-Soluble Carbodiimide

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The close association of the extrinsic PsbO, PsbP and PsbQ proteins with PSII core subunits in oxygen-evolving PSII complexes from a green alga, Chlamydomonas reinhardtii, was examined by cross-linking experiments with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The green algal PSII complexes treated with EDC were washed with alkaline Tris to remove the non-cross-linked extrinsic proteins, and then applied to Blue-Native-PAGE to prepare PSII core complexes. The extrinsic proteins cross-linked with PSII core complexes were detected by immunoblotting analysis using antibodies against extrinsic proteins and PSII core subunits. The results showed that the PsbO, PsbP and PsbQ proteins directly associated with CP47, the α subunit of cytochrome b559 and a small subunit in PSII core complexes, respectively, through electrostatic interactions. In addition, a cross-linked product between the PsbP and PsbQ proteins was found in alkaline Tris extracts of EDC-treated PSII complexes, and its cross-linked site was examined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS) after digestions with trypsin and endoproteinase Asp-N. The results demonstrated that the positively charged amino group of K176 on the PsbP protein electrostatically interacts with the negatively charged carboxyl group of D28 on the PsbQ protein. These binding properties of the extrinsic proteins in the green algal PSII were compared with those in higher plant PSII.

Keywords: Chlamydomonas reinhardtii; Cross-linking; Extrinsic protein; Oxygen evolution; PSII

Journal Article.  6164 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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