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
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    • "The highly conserved Arl3 À only present in ciliated organisms – localizes throughout the cell and is enriched in the primary cilium (Avidor- Reiss et al., 2004; Blacque et al., 2005; Zhou et al., 2006). While RP2 functions as an Arl3 GAP and is thus important for the import of lipidated cargo by recycling Arl3 and its effectors (Schwarz et al., 2012; Wright et al., 2011; Zhang et al., 2015), the guanine nucleotide exchange factor (GEF) that activates Arl3 remains unknown. We had anticipated that in order for Arl3 to mediate cargo release inside cilia, an Arl3-specific GEF should be localized there as well. "
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    ABSTRACT: Small G-proteins of the ADP-ribosylation-factor-like (Arl) subfamily have been shown to be crucial to ciliogenesis and cilia maintenance. Active Arl3 is involved in targeting and releasing lipidated cargo proteins from their carriers PDE6δ and UNC119a/b to the cilium. However, the Guanine-Nucleotide-Exchange-factor (GEF) which activates Arl3 is unknown. Here we show that the ciliary G-protein Arl13B mutated in Joubert-Syndrome is the GEF for Arl3 and its function is conserved in evolution. The GEF activity of Arl13B is mediated by the G-domain plus an additional C-terminal helix. The switch regions of Arl13B are involved in the interaction with Arl3. Overexpression of Arl13B in mammalian cell lines leads to an increased Arl3•GTP level, whereas Arl13B Joubert-Syndrome patient mutations impair GEF activity and thus Arl3 activation. We anticipate that through Arl13B's exclusive ciliary localization, Arl3 activation is spatially restricted and thereby an Arl3•GTP compartment generated where ciliary cargo is specifically released.
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    • "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). "
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    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.
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    • "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). "
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