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Combining RNA interference mutants and comparative proteomics to identify protein component interactions and dependencies in a eukaryotic flagellum.

Portman, Neil and Lacomble, Sylvain and Thomas, Benjamin and McKean, Paul G. and Gull, Keith (2009) Combining RNA interference mutants and comparative proteomics to identify protein component interactions and dependencies in a eukaryotic flagellum. Journal of Biological Chemistry, 284 (9). pp. 5610-5619. ISSN 1083-351X

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Abstract

Eukaryotic flagella from organisms such as Trypanosoma brucei can be isolated and their protein components identified by mass spectrometry. Here we used a comparative approach utilizing two-dimensional difference gel electrophoresis and isobaric tags for relative and absolute quantitation to reveal protein components of flagellar structures via ablation by inducible RNA interference mutation. By this approach we identified 20 novel components of the paraflagellar rod (PFR). Using epitope tagging we validated a subset of these as being present within the PFR by immunofluorescence. Bioinformatic analysis of the PFR cohort reveals a likely calcium/calmodulin regulatory/signaling linkage between some components. We extended the RNA interference mutant/comparative proteomic analysis to individual novel components of our PFR proteome, showing that the approach has the power to reveal dependences between subgroups within the cohort.

Item Type: Article
Journal or Publication Title: Journal of Biological Chemistry
Subjects: Q Science > QH Natural history > QH301 Biology
Departments:
ID Code: 26044
Deposited By: Dr Paul G McKean
Deposited On: 11 Mar 2009 11:49
Refereed?: Yes
Published?: Published
Last Modified: 23 Jul 2014 14:48
Identification Number:
URI: http://eprints.lancs.ac.uk/id/eprint/26044

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