Architectural analysis of intraflagellar transport complex B

Ben F. Lucker1, Mark S. Miller1, Slawomir Dziedzic1, Hongmin Qin2, Phillip Blackmarr1, Joel L. Rosenbaum3, and Douglas G. Cole1

1Dept of MMBB, LSS142, University of Idaho, Moscow, ID, 83844-3052, USA; 2Dept of Biology, Texas A&M University, 3258 TAMU, College Station, TX 77843-3258, USA; 3Dept of MCDB, Yale University, 266 Whitney Ave, New Haven, CT 06520, USA.

Intraflagellar transport (IFT) is a strongly conserved process required for the assembly and function of eukaryotic cilia and flagella. Chlamydomonas IFT particles consist of multiple copies of two separable complexes, A and B. Previous characterization of complex B architecture indicates that several B subunits dissociate from the Chlamydomonas complex in moderate ionic strength (300 mM NaCl) revealing a salt-stable core containing IFT88, IFT81, IFT74, IFT72, IFT52, IFT46, IFT27, IFT25 and IFT22. Additionally, we reported IFT81, IFT74, and IFT72 appear to form a higher order oligomer within the complex B core at a ratio of 2:1:1, respectively. Current two-hybrid analysis combined with heterologous co-expression reveal direct interactions can occur between IFT46 and IFT52, between IFT46 and IFT88 and between IFT52 and IFT88. These three subunits could form a ternary complex within the B core. Lastly, we have used protein electroporation of tagged IFT46 to show that loss of the N-terminal 25 amino acids does not affect rescue of flagellar assembly in an ift46 mutant, whereas the loss of 50 amino acids from the N-terminus does not rescue the mutant phenotype. These results suggest that the amino acids between position 25 and 50 are required for flagellar assembly. Supported by GM61920 (DGC), P20RR016454 (DGC) and GM14642 (JLR).

e-mail address of presenting author: blucker@wsu.edu