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Turkish Journal of Botany

Abstract

​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.

Author ORCID Identifier

ESRA ÖZTÜRK: 0000-0001-7115-5323

TELSA MITTELMEIER: 0000-0003-2894-3986

CAROL DIECKMANN: 0000-0002-6913-6913

ATAKAN SUKATAR: 0000-0002-1895-0165

DOI

10.55730/1300-008X.2906

Keywords

Immunoprecipitation, protein-protein interaction, eyespot proteins, chloroplast proteins

First Page

290

Last Page

302

Publisher

The Scientific and Technological Research Council of Türkiye (TÜBİTAK)

Creative Commons License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

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