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- Open Access
Dissecting the sub-structure of the intraflagellar transport complex B
© Texier et al; licensee BioMed Central Ltd. 2012
- Published: 16 November 2012
- Protein Complex
- Stable Complex
- Gradient Centrifugation
- Spectrometric Analysis
- Mass Spectrometric Analysis
The intraflagellar transport (IFT) machinery is composed of mainly of two major components, IFT complex A and B as well as of motor proteins (kinesins and dyneins). The aim of this study was to identify comprehensively the composition of the IFT complex B as well as the structure of its functional sub-modules. We applied Strep/FLAG-tandem affinity purification (SF-TAP) and yeast-two-hybrid to identify protein complexes and protein-protein interactions within IFT complex B. By combining these methods with the biochemical separation and destabilization of the protein complex B into sub-complexes and mass spectrometric analysis, we further determined its structure. As a first step, we comprehensively identified the composition of the IFT complex B by SF-TAP using several IFT complex B proteins as baits. The sub-complex analysis by SDS-destabilization and sucrose-density gradient centrifugation revealed that this complex is composed of at least two stable sub-complexes. The analysis further revealed that these two sub-complexes are likely to be connected by two IFT complex B proteins that either are present in both sub-complexes, or are excluded from both but act as a linker. These data suggest, that the IFT complex B is not, as previously described, acting as a single stable complex with proteins associated to the core structure. The biochemical analysis of the sub-complex structure shows, that there are two sub-modules that are closely linked. It remains unclear, if these sub-complexes exert one function as a tandem, or if they can act as separated modules within cilia or possibly within other microtubular structures.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.