The role of Kin5, a homodimeric kinesin-2 in intraciliary transport in Tetrahymena thermophila
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Two Tetrahymena kinesin-like proteins (klps) of the kinesin-2 subfamily, Kin1 and Kin2, were identified by Brown et al. (1999; Mol Biol Cell 10:3081-3096) and shown to be involved in ciliary morphogenesis, probably as molecular motors in i&barbelow;ntrac&barbelow;iliary t&barbelow;ransport (ICT). We cloned another kinesin-2 subfamily member (Kin5), which is most closely related to Osm3, a homodimeric C. elegans kinesin-2. Osm3 and Kin5 have a 56% identity overall and a 45% identity in a 60 amino acid region of the C-terminal FERM domain, not present in Kin1 or Kin2, thought to be a critical domain either for dimerization or for cargo recognition in ICT. Kin5 is upregulated upon deciliation. An antibody to a peptide sequence from the tail region of Kin5 localizes in a punctate pattern along the ciliary axoneme, colocalizing with an antibody to the raft protein IFT139. These findings suggest that Kin5 is an ICT motor like Osm3.;To test this hypothesis, the effect of knocking down Kin5 on ciliogenesis, cell motility and survival, and transport of a putative cargo protein was examined. An RNA interference (RNAi) construct specific for KIN5 was shown to be stably integrated into the genome, such that with appropriate induction, Kin5 is severely downregulated and disappears from the cilia. Because Kin5 is an essential component, cell motility decreases by 90% and knockdown is lethal by 12 h. However, unlike kin1/kin2 knockout, Kin5p knockdown does not cause ciliary resorption, while deciliation in the knockdown condition reveals that cilia regrow in the absence of ciliary Kin5. Next, a specific intraciliary peripheral membrane protein that was a putative Kin5 cargo was examined to see whether it would be affected by the knockdown. Gef1, a putative guanine nucleotide exchange factor, localizes along the cilium and Gef1 coimmunoprecipitates with Kin5. Nevertheless, Gef1 remains in the cilium in the knockdown cells. However, upon deciliation, Gef1 immunofluorescence disappears in the regenerating cilia of the knockdown cells. These results suggest that Gef1 is transported into the cilium by Kin5, and is released into a position along the cilium, where it affects signal transduction specific to motility and/or viability.
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