Analysis of interactions between intraflagellar transport proteins.

Abstract Intraflagellar transport (IFT) involves the movement of large proteinaceous particles or trains along the length of ciliary and flagellar axonemal microtubules. The particles contain multiple copies of two protein complexes. As isolated from the flagellated model organism, Chlamydomonas reinhardtii , IFT A contains 6 distinct gene products while IFT B contains at least 13 distinct gene products. To better understand the architecture of these two complexes, a multifaceted approach has been employed to identify subcomplexes and specific protein–protein interactions. The high biochemical yields afforded with Chlamydomonas preparations have allowed traditional biochemical approaches including chemical cross-linking and disruption of native complexes, which, in the case of IFT B, have revealed a core subcomplex retaining nine of the B subunits. Complementing these results are molecular approaches including two-hybrid screenings and heterologous expression that have identified specific protein–protein interactions. Lastly, genetic approaches utilizing Chlamydomonas IFT mutants have shown how the loss of specific subunits perturb the complexes and, in the case of IFT A, they have revealed a core subcomplex containing half of the A subunits.