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.

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Available from: Diane C Slusarski, Oct 09, 2015
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    • "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. "
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    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
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    • "A key aspect is the role of the small GTPase Arl3 in targeting myristoylated and prenylated proteins, such as NPHP3, to the primary cilium via UNC119 and PDE6D effectors. Some of this work has already been published (Wright et al., 2011). We thank all the speakers for their participation and contributions and the discussion participants for making this Minisymposium such a great event. "
    Molecular biology of the cell 03/2013; 24(6):670. DOI:10.1091/mbc.E12-12-0871 · 4.47 Impact Factor
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    • "Both Arl2 and Arl3 have in addition to the N-terminal amphipathic helix a totally conserved glycine in position 2 however they are apparently not myirstoylated (Bologna et al, 2004). Although not much is known about membrane interactions of Arl2 and Arl3 a recent study has shown that the constitutively active Arl3 fractionates to both soluble and membranes fractions which is most likely due to the week affinity to membranes (Wright et al, 2011). "
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    ABSTRACT: Access to the ciliary membrane for trans-membrane or membrane-associated proteins is a regulated process. Previously, we have shown that the closely homologous small G proteins Arl2 and Arl3 allosterically regulate prenylated cargo release from PDEδ. UNC119/HRG4 is responsible for ciliary delivery of myristoylated cargo. Here, we show that although Arl3 and Arl2 bind UNC119 with similar affinities, only Arl3 allosterically displaces cargo by accelerating its release by three orders of magnitude. Crystal structures of Arl3 and Arl2 in complex with UNC119a reveal the molecular basis of specificity. Contrary to previous structures of GTP-bound Arf subfamily proteins, the N-terminal amphipathic helix of Arl3·GppNHp is not displaced by the interswitch toggle but remains bound on the surface of the protein. Opposite to the mechanism of cargo release on PDEδ, this induces a widening of the myristoyl binding pocket. This leads us to propose that ciliary targeting of myristoylated proteins is not only dependent on nucleotide status but also on the cellular localization of Arl3.
    The EMBO Journal 09/2012; 31(20):4085-94. DOI:10.1038/emboj.2012.257 · 10.43 Impact Factor
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