Structural Biology Group, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, D-44227 Dortmund, Germany.
Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, D-44227 Dortmund, Germany.
Biol Chem. 2018 Feb 23;399(3):277-292. doi: 10.1515/hsz-2017-0226.
The inositol polyphosphate 5'-phosphatase E (INPP5E) localizes to cilia. We showed that the carrier protein phosphodiesterase 6 delta subunit (PDE6δ) mediates the sorting of farnesylated INPP5E into cilia due to high affinity binding and release by the ADP-ribosylation factor (Arf)-like protein Arl3·GTP. However, the dynamics of INPP5E transport into and inside the ciliary compartment are not fully understood. Here, we investigate the movement of INPP5E using live cell fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) analysis. We show that PDE6δ and the dynein transport system are essential for ciliary sorting and entry of INPP5E. However, its innerciliary transport is regulated solely by the intraflagellar transport (IFT) system, independent from PDE6δ activity and INPP5E farnesylation. By contrast, movement of Arl3 into and within cilia occurs freely by diffusion and IFT-independently. The farnesylation defective INPP5E CaaX box mutant loses the exclusive ciliary localization. The accumulation of this mutant at centrioles after photobleaching suggests an affinity trap mechanism for ciliary entry, that in case of the wild type is overcome by the interaction with PDE6δ. Collectively, we postulate a three-step mechanism regulating ciliary localization of INPP5E, consisting of farnesylation- and PDE6δ-mediated targeting, INPP5E-PDE6δ complex diffusion into the cilium with transfer to the IFT system, and retention inside cilia.
肌醇多磷酸 5′-磷酸酶 E（INPP5E）定位于纤毛。我们表明，载体蛋白磷酸二酯酶 6 德尔塔亚基（PDE6δ）通过与 ADP-核糖基化因子（Arf）样蛋白 Arl3·GTP 的高亲和力结合和释放，介导棕榈酰化 INPP5E 分选到纤毛中。然而，INPP5E 转运到纤毛隔室内部的动力学尚未完全了解。在这里，我们使用活细胞荧光显微镜和荧光恢复后光漂白（FRAP）分析研究 INPP5E 的运动。我们表明，PDE6δ 和动力蛋白运输系统对于 INPP5E 的纤毛分选和进入是必不可少的。然而，其内部纤毛内运输仅受鞭毛内运输（IFT）系统调节，与 PDE6δ 活性和 INPP5E 棕榈酰化无关。相比之下，Arl3 进入和在纤毛内的运动通过扩散和 IFT 独立地自由发生。无棕榈酰化功能的 INPP5E CaaX 框突变体失去了纤毛的特异性定位。光漂白后该突变体在中心粒的积累表明存在一种亲和陷阱机制来进入纤毛，而在野生型中，这种机制是通过与 PDE6δ 的相互作用来克服的。总的来说，我们提出了一个三步机制来调节 INPP5E 的纤毛定位，包括棕榈酰化和 PDE6δ 介导的靶向、INPP5E-PDE6δ 复合物扩散进入纤毛并转移到 IFT 系统，以及在纤毛内保留。
