An ARL3-UNC119-RP2 GTPase cycle targets myristoylated NPHP3 to the primary cilium

Genentech Inc., South San Francisco, California 94080, USA.
Genes & development (Impact Factor: 10.8). 11/2011; 25(22):2347-60. DOI: 10.1101/gad.173443.111
Source: PubMed


The membrane of the primary cilium is a highly specialized compartment that organizes proteins to achieve spatially ordered signaling. Disrupting ciliary organization leads to diseases called ciliopathies, with phenotypes ranging from retinal degeneration and cystic kidneys to neural tube defects. How proteins are selectively transported to and organized in the primary cilium remains unclear. Using a proteomic approach, we identified the ARL3 effector UNC119 as a binding partner of the myristoylated ciliopathy protein nephrocystin-3 (NPHP3). We mapped UNC119 binding to the N-terminal 200 residues of NPHP3 and found the interaction requires myristoylation. Creating directed mutants predicted from a structural model of the UNC119-myristate complex, we identified highly conserved phenylalanines within a hydrophobic β sandwich to be essential for myristate binding. Furthermore, we found that binding of ARL3-GTP serves to release myristoylated cargo from UNC119. Finally, we showed that ARL3, UNC119b (but not UNC119a), and the ARL3 GAP Retinitis Pigmentosa 2 (RP2) are required for NPHP3 ciliary targeting and that targeting requires UNC119b myristoyl-binding activity. Our results uncover a selective, membrane targeting GTPase cycle that delivers myristoylated proteins to the ciliary membrane and suggest that other myristoylated proteins may be similarly targeted to specialized membrane domains.

Download full-text


Available from: Diane C Slusarski,
    • "We repeatedly identified peptides of CCDC104 by mass spectrometry analysis of TAP eluates (Table S1). Although CCDC104 was previously identified in a TAP using constitutively active Arl3 Q71L (Wright et al., 2011), we speculated, based on our findings, that CCDC104 might be a GEF for Arl3. In assessing this role, however, CCDC104 showed no GEF activity toward Arl3 (Figure S1). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Cilia are small antenna-like cellular protrusions critical for many developmental signaling pathways. The ciliary protein Arl3 has been shown to act as a specific release factor for myristoylated and farnesylated ciliary cargo molecules by binding to the effectors Unc119 and PDE6δ. Here we describe a newly identified Arl3 binding partner, CCDC104/CFAP36. Biochemical and structural analyses reveal that the protein contains a BART-like domain and is called BARTL1. It recognizes an LLxILxxL motif at the N-terminal amphipathic helix of Arl3, which is crucial for the interaction with the BART-like domain but also for the ciliary localization of Arl3 itself. These results seem to suggest a ciliary role of BARTL1, and possibly link it to the Arl3 transport network. We thus speculate on a regulatory mechanism whereby BARTL1 aids the presentation of active Arl3 to its GTPase-activating protein RP2 or hinders Arl3 membrane binding in the area of the transition zone.
    Structure 10/2015; DOI:10.1016/j.str.2015.08.016 · 5.62 Impact Factor
  • Source
    • "Myristoylation is essential for CIL-7 function, including its association with EVs (Figure 2, D–F) and its role in PC-dependent mating behaviors (Figure 1K). N-myristoylation is used by proteins for membrane anchoring and for ciliary localization of proteins in Trypanosome flagella, C. elegans sensory neurons, mammalian photoreceptors, and retinal pigment epithelial cells (Ramulu and Nathans, 2001; Evans et al., 2010; Maric et al., 2010; Wright et al., 2011). In Jurkat T-cells, myristoylation signals target proteins to EVs (Shen et al., 2011). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The cilium both releases and binds to extracellular vesicles (EVs). EVs may be used by cells as a form of intercellular communication and mediate a broad range of physiological and pathological processes. The mammalian polycystins (PCs) localize to cilia, as well as to urinary EVs released from renal epithelial cells. PC ciliary trafficking defects may be an underlying cause of autosomal dominant polycystic kidney disease (PKD), and ciliary-EV interactions have been proposed to play a central role in the biology of PKD. In Caenorhabditis elegans and mammals, PC1 and PC2 act in the same genetic pathway, act in a sensory capacity, localize to cilia, and are contained in secreted EVs, suggesting ancient conservation. However, the relationship between cilia and EVs and the mechanisms generating PC-containing EVs remain an enigma. In a forward genetic screen for regulators of C. elegans PKD-2 ciliary localization, we identified CIL-7, a myristoylated protein that regulates EV biogenesis. Loss of CIL-7 results in male mating behavioral defects, excessive accumulation of EVs in the lumen of the cephalic sensory organ, and failure to release PKD-2::GFP-containing EVs to the environment. Fatty acylation, such as myristoylation and palmitoylation, targets proteins to cilia and flagella. The CIL-7 myristoylation motif is essential for CIL-7 function and for targeting CIL-7 to EVs. C. elegans is a powerful model with which to study ciliary EV biogenesis in vivo and identify cis-targeting motifs such as myristoylation that are necessary for EV-cargo association and function.
    Molecular biology of the cell 06/2015; 26(15). DOI:10.1091/mbc.E15-01-0009 · 4.47 Impact Factor
  • Source
    • "Humbert et al. (2012) reported the involvement of another ARL family small GTPase, ARL13B, in the INPP5E ciliary targeting network. The authors suggest that ARL13B releases INPP5E from PDE6D by binding to INPP5E rather than PDE6D, which differs from the cargo-release mechanism described biochemically and structurally used by ARL3 for both PDE6D and UNC119B [Ismail et al., 2011, 2012; Wright et al., 2011]. It will be of interest to further determine the role of ARL13B in PDE6D-dependent protein targeting. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Joubert syndrome (JS) is characterized by a distinctive cerebellar structural defect, namely the « molar tooth sign ». JS is genetically heterogeneous, involving 18 genes identified to date, which are all required for cilia biogenesis and/or function. In a consanguineous family with JS associated with optic nerve coloboma, kidney hypoplasia and polydactyly, combined exome sequencing and mapping identified a homozygous splice site mutation in PDE6D, encoding a prenyl-binding protein. We found that pde6d depletion in zebrafish leads to renal and retinal developmental anomalies and wild-type but not mutant PDE6D is able to rescue this phenotype. Proteomic analysis identified INPP5E, whose mutations also lead to JS or MORM syndromes, as novel prenyl-dependent cargo of PDE6D. Mutant PDE6D shows reduced binding to INPP5E, which fails to localize to primary cilia in patient fibroblasts and tissues. Furthermore, mutant PDE6D is unable to bind to GTP-bound ARL3, which acts as a cargo-release factor for PDE6D-bound INPP5E. Altogether, these results indicate that PDE6D is required for INPP5E ciliary targeting and suggest a broader role for PDE6D in targeting other prenylated proteins to the cilia. This study identifies PDE6D as a novel JS disease gene and provides the first evidence of prenyl-binding dependent trafficking in ciliopathies. This article is protected by copyright. All rights reserved.
    Human Mutation 02/2014; 35(1). DOI:10.1002/humu.22470 · 5.14 Impact Factor
Show more