Differential role of Rab proteins in ciliary trafficking: Rab23 regulates Smoothened levels
Differential role of Rab proteins in ciliary trafficking: Rab23 regulates Smoothened levels
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DOI：
10.1242/jcs.058883
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发表时间：
2010-05-01
影响因子：
4
通讯作者：
Kuehn, E. Wolfgang
中科院分区：
文献类型：
--
作者：
Boehlke, Christopher;Bashkurov, Mikhail;Kuehn, E. Wolfgang
初级纤毛作为感觉细胞器的结构和功能依赖于运动蛋白驱动的鞭毛内运输系统（IFT）；IFT缺陷导致视网膜变性和多种疾病，如Bardet Biedl综合征（BBS）和hedgehog基因信号缺陷（HH）。蛋白质转运到纤毛涉及到拉布gtp酶。Rab8与BBS蛋白的多蛋白复合物一起，向基底体招募货物以运输到纤毛。小鼠中Rab23的缺失再现了HH表型，但其在HH信号传导中的功能尚不清楚。在这里，我们建立了一种新的方案，基于光漂白后荧光恢复（FRAP），允许定量分析蛋白质转运到MDCK细胞的纤毛。我们比较了Rab8、Rab5和Rab23对hh相关的跨膜受体Smoothened、微管尖端蛋白EB1和受体蛋白Kim1纤毛转运的影响。纤毛FRAP证实了Rab8在蛋白质进入纤毛中的作用。Rab5显性阴性对Smoothened或EB1纤毛转运无影响，但减缓了纤毛中根尖蛋白Kim1的恢复。Rab23的缺失或显性阴性Rab23的表达降低了Smoothened纤毛的稳态，但对EB1和Kim1没有影响，这表明Rab23在纤毛蛋白转换中起作用。
The structure and function of the primary cilium as a sensory organelle depends on a motor-protein-powered intraflagellar transport system (IFT); defective IFT results in retinal degeneration and pleiotropic disorders such as the Bardet Biedl syndrome (BBS) and defective hedgehog (HH) signaling. Protein transport to the cilium involves Rab GTPases. Rab8, together with a multi protein complex of BBS proteins, recruits cargo to the basal body for transport to the cilium. Loss of Rab23 in mice recapitulates the HH phenotype but its function in HH signaling is unknown. Here we established a novel protocol, based on fluorescence recovery after photo-bleaching (FRAP), allowing the quantitative analysis of protein transport into the cilium of MDCK cells. We compared the effect of Rab8, Rab5 and Rab23 on the ciliary transport of the HH-associated transmembrane receptor Smoothened, the microtubular tip protein EB1, and the receptor protein Kim1. Ciliary FRAP confirmed the role of Rab8 in protein entry to the cilium. Dominant negative Rab5 had no impact on the ciliary transport of Smoothened or EB1, but slowed the recovery of the apical protein Kim1 in the cilium. Depletion of Rab23 or expression of dominant-negative Rab23 decreased the ciliary steady state specifically of Smoothened but not EB1 or Kim1, suggesting a role of Rab23 in protein turnover in the cilium.