Dataset Information

Proteomic analysis of the cilia membrane of Paramecium tetraurelia.

ABSTRACT: 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, Ca(2+) 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.

SUBMITTER: Yano J

PROVIDER: S-EPMC3667161 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Proteomic analysis of the cilia membrane of Paramecium tetraurelia.

Yano Junji J Rajendran Anbazhagan A Valentine Megan S MS Saha Madhurima M Ballif Bryan A BA Van Houten Judith L JL

Journal of proteomics 20121110

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- ...[more]

PMID: 23146917

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Project description:BackgroundA Paramecium tetraurelia pilot genome project, the subsequent sequencing of a Megabase chromosome as well as the Paramecium genome project aimed at gaining insight into the genome of Paramecium. These cells display a most elaborate membrane trafficking system, with distinct, predictable pathways in which actin could participate. Previously we had localized actin in Paramecium; however, none of the efforts so far could proof the occurrence of actin in the cleavage furrow of a dividing cell, despite the fact that actin is unequivocally involved in cell division. This gave a first hint that Paramecium may possess actin isoforms with unusual characteristics. The genome project gave us the chance to search the whole Paramecium genome, and, thus, to identify and characterize probably all actin isoforms in Paramecium.ResultsThe ciliated protozoan, P. tetraurelia, contains an actin multigene family with at least 30 members encoding actin, actin-related and actin-like proteins. They group into twelve subfamilies; a large subfamily with 10 genes, seven pairs and one trio with > 82% amino acid identity, as well as three single genes. The different subfamilies are very distinct from each other. In comparison to actins in other organisms, P. tetraurelia actins are highly divergent, with identities topping 80% and falling to 30%. We analyzed their structure on nucleotide level regarding the number and position of introns. On amino acid level, we scanned the sequences for the presence of actin consensus regions, for amino acids of the intermonomer interface in filaments, for residues contributing to ATP binding, and for known binding sites for myosin and actin-specific drugs. Several of those characteristics are lacking in several subfamilies. The divergence of P. tetraurelia actins and actin-related proteins between different P. tetraurelia subfamilies as well as with sequences of other organisms is well represented in a phylogenetic tree, where P. tetraurelia sequences only partially cluster.ConclusionAnalysis of different features on nucleotide and amino acid level revealed striking differences in isoforms of actin and actin-related proteins in P. tetraurelia, both within the organism and in comparison to other organisms. This diversification suggests unprecedented specification in localization and function within a unicellular eukaryote.

Dataset Information

Proteomic analysis of the cilia membrane of Paramecium tetraurelia.

Publications

Proteomic analysis of the cilia membrane of Paramecium tetraurelia.

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