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We have conducted a protein interaction study of components within a specific sub-compartment of a eukaryotic flagellum. The trypanosome flagellum contains a para-crystalline extra-axonemal structure termed the paraflagellar rod (PFR) with around forty identified components. We have used a Gateway cloning approach coupled with yeast two-hybrid, RNAi and 2D DiGE to define a protein-protein interaction network taking place in this structure. We define two clusters of interactions; the first being characterised by two proteins with a shared domain which is not sufficient for maintaining the interaction. The other cohort is populated by eight proteins, a number of which possess a PFR domain and sub-populations of this network exhibit dependency relationships. Finally, we provide clues as to the structural organisation of the PFR at the molecular level. This multi-strand approach shows that protein interactome data can be generated for insoluble protein complexes.

Original publication

DOI

10.1371/journal.pone.0007685

Type

Journal article

Journal

PLoS One

Publication Date

03/11/2009

Volume

4

Keywords

Animals, Cloning, Molecular, Epitopes, Flagella, Genetic Techniques, Genetic Vectors, Genomics, Microscopy, Electron, Models, Biological, Open Reading Frames, Protein Interaction Mapping, Proteomics, RNA Interference, Trypanosoma brucei brucei, Two-Hybrid System Techniques