Optimized immunoprecipitation identifies candidate proteins associated with eyespot protein EYE2 in Chlamydomonas reinhardtii

​The EYE2 protein is essential for eyespot assembly and localization in Chlamydomonas reinhardtii, yet the molecular mechanisms underlying its function remain poorly understood. In this study, we present a systematic evaluation and optimization of an immunoprecipitation (IP)-based workflow for isolating EYE2 and its associated protein complexes. Key methodological variables including strain selection, flagella removal, and detergent concentration were compared using western blotting to determine optimal extraction conditions. The combination of the cell wall-deficient cw15 strain, 4% digitonin extraction, and pH shock-mediated deflagellation significantly enhanced IP specificity and reproducibility. Using this optimized protocol, EYE2 and its potential interaction partners were isolated and analyzed by liquid chromatography-tandem mass spectrometry, resulting in the identification of 23 candidate proteins. These included chaperones, translation-associated factors, metabolic enzymes, and a subunit of a circadian RNA-binding complex, suggesting that EYE2 may serve as a multifunctional scaffold linking photoreception, metabolism, and subcellular organization. The methodological advances reported here not only improve the recovery of membrane-associated proteins in C. reinhardtii but also establish a framework for studying chloroplast-localized protein networks in algal systems. These findings provide novel insight into the broader functional landscape of EYE2 and pave the way for future validation and functional studies.