Abstract
Purpose: :
Cilia are hair-like organelles protruding from the cell surface of different cell types in many organisms. Recent studies revealed that dysfunction of these axonemal structures results in several severe diseases, the ciliopahties. In photoreceptors, the interconnecting cilium is the only connection between inner and outer segment. All proteins, synthesized in the inner segment need to be transported along this structure by the intraflagellar transport (IFT) machinery. Malfunction of this process by mutations in genes coding for IFT proteins, can lead to the disruption of the IFT along the axonemes of the cilia and to subsequent photoreceptor degeneration. This molecular transport 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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: proteomics • protein purification and characterization • protein structure/function