Proteomic analysis of the cilia membrane of Paramecium tetraurelia

Channels, pumps, receptors, cyclases and other membrane proteins modulate the motility and sensory function of cilia, but these proteins are generally under-represented in proteomic analyses of cilia. Studies of these ciliary membrane proteins would benefit from a protocol to greatly enrich for integral and lipidated membrane proteins. We used LC–MS/MS to compare the proteomes of unfractionated cilia (C), the ciliary membrane (CM) and the ciliary membrane in the detergent phase (DP) of Triton X-114 phase separation. 55% of the proteins in DP were membrane proteins (i.e. predicted transmembrane or membrane-associated through lipid modifications) and 31% were transmembrane. This is to be compared to 23% membrane proteins with 9% transmembrane in CM and 9% membrane proteins with 3% transmembrane in C. 78% of the transmembrane proteins in the DP were found uniquely in DP, and not in C or CM. There were ion channels, cyclases, plasma membrane pumps, Ca2 + dependent protein kinases, and Rab GTPases involved in the signal transduction in DP that were not identified in the other C and CM preparations. Of 267 proteins unique to the DP, 147 were novel, i.e. not found in other proteomic and genomic studies of cilia.

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► Triton-X 114 phase separation enriches for ciliary membrane proteins.
► LC–MS/MS analysis showed many proteins unique to the detergent phase.
► Novel proteins not previously identified in cilia proteomes among them.