Assembly of IFT B core subcomplexes using heterologous coexpression
Robert H. Behal1, Ben F. Lucker1, Thomas Esplin1, Micah Ferrell1, Hongmin Qin2, and Douglas G. Cole1
1) Dept. Microbiol, Molec Biol & Biochem, Univ of Idaho, Moscow, ID 83844, USA
2) Dept. Biology, Texas A&M Univ, College Station, TX 77843, USA
Intraflagellar transport (IFT) particles consist of multiple copies of two distinct complexes, A and B. The less stable of the two, the Chlamydomonas B complex undergoes partial dissociation in 300 mM NaCl to reveal a salt-stable core containing IFT88, IFT81, IFT74, IFT72, IFT52, IFT46, IFT27, IFT25 and IFT22. Several pair-wise interactions between the B core subunits have been previously reported: IFT81-IFT81 and IFT81-IFT74/72 (Lucker et al., 2005; JBC 280:27688); IFT52-IFT46, IFT88-IFT52 and IFT88-IFT46 (Chlamy 2008 abstracts) and IFT27-25 (Wang et al., 2009; PLoS One 4:e5384). In order to characterize higher order complexes, we have undertaken the coexpression of three or more B core proteins in E. coli. To facilitate these studies, we have modified bacterial protein expression plasmids so that we can express up to six different proteins simultaneously, with up to four different epitope tags suitable for tandem affinity purification. For example, coexpression of IFT88, MBP-IFT52 and His6-IFT46 yields a stable ternary complex. More recently, the coexpression of IFT88, IFT81, IFT74/72, IFT52 and IFT46 resulted in an oligomeric structure containing all five subunits. Sequential purification on different affinity resins specific for different epitope tags resulted in copurification of all five subunits. Experiments are underway to further characterize these multimeric complexes and to include the remaining core subunits. Supported by GM61920 (DGC) and P20RR016454 (DGC).
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