Functional characterization of the eyespot protein SOUL3 in Chlamydomonas reinhardtii
Thomas Schulze1, Georg Kreimer2 and Maria Mittag1
1) Institute for General Botany and Plant Physiology Friedrich-Schiller University Jena, Am Planetarium 1, 07743 Jena Germany
2) Department of Biology, Friedrich-Alexander University Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen Germany
Chlamydomonas reinhardtii has a primitive visual system, the eyespot, which allows the cell to phototax and thereby respond to environmental light signals. In addition, the phototactic movement is controlled by the endogenous circadian clock that is synchronized by the daily light-dark cycle. Proteomic approaches of the purified eyespot of C. reinhardtii revealed 202 different proteins including photoreceptors, retina-related proteins, as well as members of putative signalling pathways for photo- and chemotaxis (Schmidt et al., 2006, Plant Cell 18:1908-1930). Among them, a protein with similarities to the SOUL-heme binding protein (HBP) family, named SOUL3 was identified. Interestingly, soul mRNA is specifically expressed in the retina and pineal gland in chicken (Zylka and Reppert, 1999, Mol. Brain Res. 74: 174-181). Due to the presence of SOUL-heme binding proteins in the visual system from green algae up to animals, these proteins might be involved in light- and/or circadian signalling. Until now, we could show that His-tagged SOUL3 of C. reinhardtii, which was overexpressed and purified from E. coli, can bind to hemin-agarose indicating its heme binding ability. Additionally, sucrose density gradient experiments revealed that SOUL3 exists in different complexes during subjective day and night. The subcellular localization of SOUL3 was investigated via immunofluorescence and Western analysis of different chloroplast and eyespot fractions with anti-SOUL3 antibodies. The results suggest that SOUL3 is solely localized in the eyespot region. Furthermore, silencing of SOUL3 by RNAi revealed a different phototactic behaviour during the circadian cycle, indicating that SOUL3 is involved in circadian signalling.
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