Interaction analysis of the Chlamydomonas intraflagellar transport protein, IFT81

Ewelina Betleja, Robert H. Behal, Christina Blasick, Mark S. Miller, and Douglas G. Cole

Microbiology, Molecular Biology and Biochemistry, LSS142, University of Idaho, Moscow, ID, 83844, USA

Intraflagellar transport (IFT) is a strongly conserved process required for the assembly and function of eukaryotic cilia and flagella. Excluding the IFT motors (kinesin-2 and cytoplasmic dynein 1b/2), IFT particles consist of multiple copies of two separable complexes, A and B. We previously published that two subunits of IFT81 appear to combine with IFT72 and IFT74 to form a heterotetrameric subcomplex within the B particle (Lucker et al., 2005, J. Biol. Chem.). In an effort to identify additional IFT81 interactors, we have probed Stephen Miller's (Univ. of Maryland, USA) Volvox two-hybrid library using the full length Chlamydomonas IFT81 as our bait. In our screen, four known flagellar proteins were identified as potential IFT81 interactors. As hoped, the Volvox IFT74/72 was identified proving that the Volvox two-hybrid library could be successfully probed with Chlamydomonas bait proteins. One of the other positive clones encoded a portion of the IFT retrograde motor, cytoplasmic dynein 1b heavy chain (DHC1b). In an effort to independently verify this association, we co-expressed both DHC1b and IFT81 in bacteria but were unable to demonstrate a direct interaction. We have also used a fusion protein consisting of the dynein fused to maltose binding protein (MBP-DHC1b) to create an affinity matrix. Although the MBP-DHC1b resin failed to bind to either IFT complex, the resin specifically pulled down other flagellar proteins that are in the process of being characterized. At this point, however, we do not have the supporting evidence needed to conclude that IFT81 and DHC1b interact directly. Supported by GM61920 (DGC) and P20RR016454 (DGC).

e-mail address of corresponding author: dcole@uidaho.edu