Analysis of the eyespot phosphoproteome from Chlamydomonas reinhardtii

V. Wagner1, K. Ullmann2, A. Mollwo2, M. Kaminski1, M. Mittag1, and G. Kreimer2

1Friedrich-Schiller-University Jena, Institute of General Botany, 07743 Jena, Germany; 2Friedrich-Alexander-University Erlangen, Department Biology, 91058 Erlangen, Germany

In a recent proteomic approach we identified 202 proteins from a fraction enriched in eyespots of C. reinhardtii [Schmidt et al. (2006) Plant Cell 18, 1908-1930]. Among these proteins, 5 protein kinases and 2 protein phosphatases were present, indicating that reversible protein phosphorylation occurs in the eyespot. About 20 major phosphoprotein bands were detected in immunoblots of eyespot proteins with an anti-phosphothreonine antibody. Toward the profiling of the targets of protein kinases in the eyespot fraction, we analyzed its phosphoproteome. The solubilized proteins were treated with two endopeptidases prior to enrichment of phosphopeptides with immobilized metal-ion affinity chromatography. Phosphopeptides were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS/MS as well as neutral-loss-triggered MS/MS/MS spectra. Thereby 68 different phosphopeptides along with 52 precise in-vivo phosphorylation sites corresponding to 32 known proteins of the eyespot fraction were identified. The phosphoproteins belong mainly to 4 functional categories: carotenoid/fatty acid metabolism, (putative) signaling and retina-related proteins, thylakoid/chloroplast envelope-related proteins and conserved/novel proteins of yet unknown functions. Most notably, the photoreceptors channelrhodopsin-1 and channelrhodopsin-2 contain 3 and 1 phosphorylation sites, respectively. Phosphorylation of both occurs in the cytoplasmatic loop next to their seven transmembrane regions in a similar distance to that observed in vertebrate rhodopsins, implying functional importance for their regulation. These results form an efficient basis for further analyzing eyespot related signaling.

e-mail address of presenting author: EMAIL

web page: URL