The power of functional proteomics: Components of the green algal eyespot and its light signaling pathway(s).

One of the key modifications of proteins that can affect protein functions, activities, stabilities, localizations and interactions, represents phosphorylation. For functional phosphoproteomics, phosphopeptides are enriched from isolated sub-cellular fractions of interest and analyzed by liquid chromatography-electrospray ionization-mass spectrometry. Such an approach was recently applied to the eyespot apparatus of the green flagellate alga Chlamydomonas reinhardtii, which represents a primordial visual system. Thereby, 32 phosphoproteins of known eyespot proteins along with 52 precise in vivo phosphorylation sites were identified. They include enzymes of carotenoid and fatty acid metabolism, (putative) light signaling components and proteins with unknown function. Strikingly, the two unique green algal photoreceptors, channelrhodopsin-1 and -2 were found to be phosphorylated in the cytoplasmic loop next to their seven transmembrane regions in a similar distance as observed in vertebrate rhodopsins.