Toward a protein map of the green algal eyespot: Analysis of eyespot globule-associated proteins

Despite the well-established biophysical function of the green algal eyespot as a combined absorption/reflection screen for the retinal-based photoreceptors, only little is known about structural eyespot proteins and proteins involved in its assembly. A recently developed method for preparation of eyespot globules from Spermatozopsis similis (Chlorophyceae) has allowed now a detailed characterisation of this specific subset of eyespot proteins. Five antisera were raised against eyespot globule-associated proteins (GAPs). Two-dimensional gel electrophoresis and subsequent immunoblotting were combined to analyse the GAPs in detail. Approximately 180 spots were resolved in silver-stained two-dimensional gels, indicating the complexity of proteins needed for formation and maintenance of the highly ordered eyespot globule plate and its interactions with the chloroplast envelope and thylakoid. Topological analyses identified 19 proteins possessing domains hidden in the globule matrix or otherwise not susceptible to thermolysin treatment. These GAPs might be involved in globule stabilisation. Antibody screening of a cDNA expression library resulted in the isolation of a full-length clone coding for a novel basic proline- and alanine-rich protein of 727 amino acids. The deduced amino acid sequence revealed the presence of a putative N-terminal signal peptide, followed by a hydrophobic region and four TPR motifs in the C terminal region. This protein, termed GAP1, might thus be involved in mediating protein-protein interactions in a protein scaffold around the hydrophobic globule core. This study paves the way for future molecular identification of GAPs by systematic proteomic approaches.