Direct interactions of intraflagellar transport complex B proteins IFT88, IFT52, and IFT46

Department of Microbiology, Center for Reproductive Biology, University of Idaho, Moscow, Idaho 83844, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2010; 285(28):21508-18. DOI: 10.1074/jbc.M110.106997
Source: PubMed


Intraflagellar transport (IFT) particles of Chlamydomonas reinhardtii contain two distinct protein complexes, A and B, composed of at least 6 and 15 protein subunits, respectively. As isolated
from C. reinhardtii flagella, IFT complex B can be further reduced to a ∼500-kDa core that contains IFT88, 2× IFT81, 2× IFT74/72, IFT52, IFT46,
IFT27, IFT25, and IFT22. In this study, yeast-based two-hybrid analysis was combined with bacterial coexpression to show that
three of the core B subunits, IFT88, IFT52, and IFT46, interact directly with each other and, together, are capable of forming
a ternary complex. Chemical cross-linking results support the IFT52-IFT88 interaction and provide additional evidence of an
association between IFT27 and IFT81. With previous studies showing that IFT81 and IFT74/72 interact to form a (IFT81)2(IFT74/72)2 heterotetramer and that IFT27 and IFT25 form a heterodimer, the architecture of complex B is revealing itself. Last, electroporation
of recombinant IFT46 was used to rescue flagellar assembly of a newly identified ift46 mutant and to monitor in vivo localization and movement of the IFT particles.

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Available from: Ben F Lucker, Jul 17, 2014
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    • "However, most IFT protein domains not required for IFT complex stability (the ADs) are also highly conserved in sequence, likely reflecting important functions such as ciliary cargo interactions. A good example of the PD/AD division is IFT46, a core component of IFT-B, where only the IFT46 C-terminal domain is required for the stability of the IFT complex via interaction with the C-terminal domain of IFT52 [25,35], while the N-terminal domain is involved in the ciliary transport of outer dynein arms (ODAs) [24,41,42]. Similarly, IFT52 interacts directly with at least four different IFT proteins (IFT74/81, IFT46, IFT70 and IFT88) via its middle and C-terminal domains, while the conserved N-terminal domain is not required for IFT-B complex formation and thus likely constitutes an AD [25,35]. "
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    • "Whether Qilin is a core component of IFT B complex or it functions as a peripheral regulator of this complex remains unclear at this stage. Biochemical purification of IFT complexes, as those done in Chlamydomonas [34], in both zebrafish and C. elegans, will provide direct evidence for the role of Qilin in IFT complexes. "
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    ABSTRACT: Cilia are microtubule-based protrusions from the cell surface that are involved in a number of essential signaling pathways, yet little is known about many of the proteins that regulate their structure and function. A number of putative cilia genes have been identified by proteomics and comparative sequence analyses, but functional data are lacking for the vast majority. We therefore monitored the effects in three cell lines of small interfering RNA (siRNA) knockdown of 40 of these genes by high-content analysis. We assayed cilia number, length, and transport of two different cargoes (membranous serotonin receptor 6-green fluorescent protein [HTR6-GFP] and the endogenous Hedgehog [Hh] pathway transcription factor Gli3) by immunofluorescence microscopy; and cilia function using a Gli-luciferase Hh signaling assay. Hh signaling was most sensitive to perturbations, with or without visible structural cilia defects. Validated hits include Ssa2 and mC21orf2 with ciliation defects; Ift46 with short cilia; Ptpdc1 and Iqub with elongated cilia; and Arl3, Nme7, and Ssna1 with distinct ciliary transport but not length defects. Our data confirm various ciliary roles for several ciliome proteins and show it is possible to uncouple ciliary cargo transport from cilia formation in vertebrates.
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