IFT121 and IFT122 bind to IFT43 and are required for flagellar assembly
Robert H. Behal, Mark S. Miller, Alexis Jones, and Douglas G. Cole
1. Dept. Microbiol, Molec Biol & Biochem, Univ of Idaho, Moscow, ID 83844, USA
Essential for the assembly and maintenance of eukaryotic flagella, intraflagellar transport (IFT) particles consist of multiple copies of two distinct complexes, A and B. In numerous studies, complex B has been associated with anterograde IFT while complex A has often been associated with retrograde transport. Preliminary reports from our laboratory (ASCB Meeting, 2008) revealed a specific interaction between A components, IFT43 and the C-terminal half of IFT121. In addition, we reported that mutant Chlamydomonas strains disrupted in either the IFT121 or IFT122 gene displayed strong flagellar assembly phenotypes. In order to better understand the structure and function of complex A, we have extended the protein-protein interaction and mutant analysis studies. We report here the bacterial coexpression and copurification of IFT43 and IFT122 indicates that these two A subunits can directly interact in the absence of IFT121 and the other A subunits. A more ambitious coexpression analysis of IFT43, IFT121 and IFT22 is currently underway. To address how the loss of IFT121 or IFT122 might affect assembly of complex A, we have probed mutant extracts using different anti-A subunit affinity resins to pull down specific A subunits. In brief, clarified whole-cell extracts (e.g. ift121 or ift122) were incubated with anti-43, anti-122, or anti-140 antibody resins. All three antibody resins pulled down intact complex A from wild-type cell extract. When probing the ift121 mutant extract, however, anti-122 and anti-140 resins pulled down IFT144 and IFT140 but neither resin appeared to pull down IFT121 or IFT139; anti-43 resin failed to pull down IFT144 or IFT140. These results suggest that IFT121 is required for IFT139 and IFT43 to assemble onto complex A. Analogous pull down experiments with the ift122 mutant strain were unable to selectively purify any of the complex A subunits, consistent with the idea that IFT122 is required for complex A stability. Supported by GM61920 (DGC) and P20RR016454 (DGC).
e-mail address of presenting author: dcole@uidaho.edu