Comparative Proteomics of Plasma and Flagellar Membranes in Chlamydomonas reinhardtii

The flagellar (ciliary) membrane of Chlamydomonas reinhardtii is continuous with the plasma membrane but maintains a distinct protein composition through the ciliary gate mechanism; however, a systematic comparison of the membrane proteomes of these two compartments has not been reported. Here, we performed comparative proteomics on highly enriched plasma and flagellar membrane fractions and identified 2779 proteins, with hundreds differentially abundant. Plasma membrane-enriched proteins were mainly involved in proton transport and ATP synthesis, whereas flagellar membrane-enriched proteins were specialized for cilium movement, axoneme assembly, and intraflagellar transport. Fluorescence localization validated the proteomic data and revealed previously uncharacterized subcellular patterns. Notably, PLD1 showed dual localization at the plasma membrane and punctate cytoplasmic structures, and FAP215 (validated by subflagellar fractionation) displayed a broader flagellar signal than the central microtubule protein FAP178, consistent with its localization in the flagellar matrix or membrane. This work provides the first systematic comparison of plasma and flagellar membrane proteomes in Chlamydomonas, offering a valuable resource for understanding flagellar membrane specialization.