The cytoplasmic tail of fibrocystin contains a ciliary targeting sequence

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605
The Journal of Cell Biology (Impact Factor: 9.69). 01/2010; 188(1):21-8. DOI: 10.1083/jcb.200910096
Source: PubMed

ABSTRACT Sensory functions of primary cilia rely on ciliary-localized membrane proteins, but little is known about how these receptors are targeted to the cilium. To further our understanding of this process, we dissected the ciliary targeting sequence (CTS) of fibrocystin, the human autosomal recessive polycystic kidney disease gene product. We show that the fibrocystin CTS is an 18-residue motif localized in the cytoplasmic tail. This motif is sufficient to target green fluorescent protein (GFP) to cilia of ciliated cells and targets GFP to lipid rafts if the cells are not ciliated. Rab8, but not several other Rabs implicated in ciliary assembly, binds to the CTS in a coimmunoprecipitation assay. Dominant-negative Rab8 interacts more strongly than wild-type or constitutively active Rab8, and coexpression of this dominant-negative mutant Rab8 blocks trafficking to the cilium. This suggests that the CTS functions by binding regulatory proteins like Rab8 to control trafficking through the endomembrane system and on to the cilium.

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    ABSTRACT: Cilia dysfunction underlies a class of human diseases with variable penetrance in different organ systems. Across eukaryotes, intraflagellar transport (IFT) facilitates cilia biogenesis and cargo trafficking, but our understanding of mammalian IFT is insufficient. Here we perform live analysis of cilia ultrastructure, composition and cargo transport in native mammalian tissue using olfactory sensory neurons. Proximal and distal axonemes of these neurons show no bias towards IFT kinesin-2 choice, and Kif17 homodimer is dispensable for distal segment IFT. We identify Bardet-Biedl syndrome proteins (BBSome) as bona fide constituents of IFT in olfactory sensory neurons, and show that they exist in 1:1 stoichiometry with IFT particles. Conversely, subpopulations of peripheral membrane proteins, as well as transmembrane olfactory signalling pathway components, are capable of IFT but with significantly less frequency and/or duration. Our results yield a model for IFT and cargo trafficking in native mammalian cilia and may explain the penetrance of specific ciliopathy phenotypes in olfactory neurons.
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    ABSTRACT: In most cells, the cilium is formed within a compartment separated from the cytoplasm. Entry into the ciliary compartment is regulated by a specialized gate located at the base of the cilium in a region known as the transition zone. The transition zone is closely associated with multiple structures of the ciliary base, including the centriole, axoneme, and ciliary membrane. However, the contribution of these structures to the ciliary gate remains unclear. Here we report that, in Drosophila spermatids, a conserved module of transition zone proteins mutated in Meckel-Gruber syndrome (MKS), including Cep290, Mks1, B9d1, and B9d2, comprise a ciliary gate that continuously migrates away from the centriole to compartmentalize the growing axoneme tip. We show that Cep290 is essential for transition zone composition, compartmentalization of the axoneme tip, and axoneme integrity and find that MKS proteins also delimit a centriole-independent compartment in mouse spermatids. Our findings demonstrate that the ciliary gate can migrate away from the base of the cilium, thereby functioning independently of the centriole and of a static interaction with the axoneme to compartmentalize the site of axoneme assembly. Copyright © 2014 Elsevier Ltd. All rights reserved.
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    ABSTRACT: Primary cilia in biology and disease gained considerable importance now that their involvement in a wide range of human ciliopathies has been abundantly documented. However, detailed molecular mechanisms for specific targeting of sensory receptors to primary cilia are still unknown. Here, we show that the Arf/Rab11 effector FIP3 promotes the activity of Rab11a and the Arf GAP ASAP1 in the Arf4-dependent ciliary transport of the sensory receptor rhodopsin. During its passage out of the photoreceptor Golgi/Trans Golgi Network (TGN), rhodopsin indirectly interacts with FIP3 through Rab11a and ASAP1. FIP3 competes with rhodopsin for binding to ASAP1 and displaces it from the ternary complex with Arf4•GTP and ASAP1. Resembling the lack of ASAP1, ablation of FIP3 abolishes ciliary targeting and causes rhodopsin mislocalization. FIP3 coordinates interactions of ASAP1 and Rab11a with the Rab8 guanine nucleotide exchange factor Rabin8. Our study implies that FIP3 functions as a crucial targeting regulator, which impinges on rhodopsin-ASAP1 interactions and shapes the binding pocket for Rabin8 within the ASAP1-Rab11a-FIP3 targeting complex, thus facilitating the orderly assembly and activation of the Rab11-Rabin8-Rab8 cascade during ciliary receptor trafficking.

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May 22, 2014