JXB Advance Access originally published online on March 7, 2005
Journal of Experimental Botany 2005 56(414):1239-1244; doi:10.1093/jxb/eri119
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RESEARCH PAPER |
Isolation of phosphorylated and dephosphorylated forms of the CP43 internal antenna of photosystem II in Hordeum vulgare L.
1Dipartimento di Scienze Ambientali e della Vita, Università del Piemonte Orientale ‘Amedeo Avogadro’, Via Bellini 25/G, I-15100 Alessandria, Italy
2Dipartimento di Biologia, Università di Padova, Via Bassi 58/B, I-35131 Padova, Italy
* To whom correspondence should be addressed. Fax: +39 0131 360390; E-mail: roberto.barbato{at}unipmn.it
As a consequence of variation in environmental factors, light being the most important one, a number of photosystem II polypeptides may be reversibly phosphorylated by thylakoid-bound kinase(s). Among them, the reaction centre D1 and D2 polypeptides, the PsbH subunit, and the inner antenna CP43. Here, the separation of two forms of CP43 by high-resolution denaturing polyacrylamide gel electrophoresis is reported. By means of immunoblotting with antibody to phosphothreonine-containing proteins and authentic CP43 and limited proteolysis, these two bands could be identified as the phosphorylated and dephosphorylated forms of CP43. Using non-denaturing isoelectrofocusing, a chromatographically derived CP43-enriched fraction could be resolved into three different native forms of CP43. Among them, one was found to be a phosphorylated form, whereas the other two were dephosphorylated forms of the protein. With respect to other methods, the procedure described here allows the isolation, for the first time, of a fully homogeneous population of this chlorophyll–protein complex, opening the way to the study of the role of phopshorylation on functional properties of this core antenna protein.
Key words: Hordeum vulgare L., photosynthesis, photosystem II, thylakoid phosphoproteins