Abstract
The primary cilium is a sensory organelle, defects in which cause a wide range of human diseases including retinal degeneration, polycystic kidney disease and birth defects. The sensory functions of cilia require specific receptors to be targeted to the ciliary subdomain of the plasma membrane. Arf4 has been proposed to sort cargo destined for the cilium at the Golgi complex and deemed a key regulator of ciliary protein trafficking. In this work, we show that Arf4 binds to the ciliary targeting sequence (CTS) of fibrocystin. Knockdown of Arf4 indicates that it is not absolutely required for trafficking of the fibrocystin CTS to cilia as steady-state CTS levels are unaffected. However, we did observe a delay in delivery of newly synthesized CTS from the Golgi complex to the cilium when Arf4 was reduced. Arf4 mutant mice are embryonic lethal and die at mid-gestation shortly after node formation. Nodal cilia appeared normal and functioned properly to break left-right symmetry in Arf4 mutant embryos. At this stage of development Arf4 expression is highest in the visceral endoderm but we did not detect cilia on these cells. In the visceral endoderm, the lack of Arf4 caused defects in cell structure and apical protein localization. This work suggests that while Arf4 is not required for ciliary assembly, it is important for the efficient transport of fibrocystin to cilia, and also plays critical roles in non-ciliary processes.
Highlights
Cilia play diverse motility and sensory functions throughout the eukaryotic kingdom, but play especially critical roles in vertebrates where severe defects lead to embryonic lethality while mild defects cause a wide range of syndromes affecting every organ system
The list of membrane proteins found in the ciliary compartment is constantly growing; among the most studied ciliary proteins are the polycystins and fibrocystin that are defective in human polycystic kidney disease, rhodopsins and opsins that are critical for vision and the patched and smoothened receptors of the hedgehog pathway
In support of this idea, we found that the fibrocystin ciliary targeting sequence (CTS) interacted with Rab8, a protein widely recognized as important to ciliary membrane protein trafficking [7,8,9]
Summary
Cilia play diverse motility and sensory functions throughout the eukaryotic kingdom, but play especially critical roles in vertebrates where severe defects lead to embryonic lethality while mild defects cause a wide range of syndromes affecting every organ system. The mechanism that targets membrane proteins to the ciliary compartment is an active area of study but very little is definitively known [3] It appears that ciliary membrane proteins contain cis-acting motifs that cause them to be localized to cilia. Like many other CTSs, the fibrocystin CTS contains lipid-modified residues that target the protein to lipid rafts, which appears to be part of the ciliary trafficking pathway We proposed that this sequence might interact with proteins that are important for sorting or transport to the ciliary membrane compartment. In the present work we asked if the fibrocystin CTS could interact with Arf as work of Deretic and colleagues has shown that this protein interacts with the CTS of opsin and is important for the formation of rhodopsin carrier vesicles at the Golgi complex [10,11]
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