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Mediated Sorting of INPP5E Into the Cilium Is Determined by Cargo-Carrier Affinity

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Mediated Sorting of INPP5E Into the Cilium Is Determined by Cargo-Carrier Affinity ARTICLE Received 2 Sep 2015 | Accepted 18 Mar 2016 | Published 11 Apr 2016 DOI: 10.1038/ncomms11366 OPEN PDE6d-mediated sorting of INPP5E into the cilium is determined by cargo-carrier affinity Eyad Kalawy Fansa1,*, Stefanie Kristine Ko¨sling1,*, Eldar Zent1, Alfred Wittinghofer1 & Shehab Ismail2 The phosphodiesterase 6 delta subunit (PDE6d) shuttles several farnesylated cargos between membranes. The cargo sorting mechanism between cilia and other compartments is not understood. Here we show using the inositol polyphosphate 50-phosphatase E (INPP5E) and the GTP-binding protein (Rheb) that cargo sorting depends on the affinity towards PDE6d and the specificity of cargo release. High-affinity cargo is exclusively released by the ciliary transport regulator Arl3, while low-affinity cargo is released by Arl3 and its non-ciliary homologue Arl2. Structures of PDE6d/cargo complexes reveal the molecular basis of the sorting signal which depends on the residues at the À 1 and À 3 positions relative to far- nesylated cysteine. Structure-guided mutation allows the generation of a low-affinity INPP5E mutant which loses exclusive ciliary localization. We postulate that the affinity to PDE6d and the release by Arl2/3 in addition to a retention signal are the determinants for cargo sorting and enrichment at its destination. 1 Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany. 2 CR-UK Beatson Institute, Garscube Estate Switchback Road, Glasgow G61 1BD, UK. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to A.W. (email: [email protected]) or to S.I. (email: [email protected]). NATURE COMMUNICATIONS | 7:11366 | DOI: 10.1038/ncomms11366 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11366 rimary cilia are antenna-like microtubule-based cell surface the import into cilia is dependent on the carrier proteins PDE6d, protrusions which can be found on eukaryotic cells and UNC119a and UNC119b and on Arl3 as displacement Pserve as sensory organelles. Genetic disorders affecting factor13,25,28,30,34. However, it has been extensively documented structure or function of cilia result in a large number of diseases that Ras proteins as well as Rheb require PDE6d for their proper collectively termed ciliopathies1,2. While the cilium appears as a localization at the plasma membrane or internal membranes, but protrusion in the plasma membrane that is open to the cell body, do not appear to be localized in cilia15,16. the ciliary content and membrane composition are different than This begs the question about the mechanism of PDE6d- that of the cell body and plasma membrane3,4. This is in part mediated sorting of farnesylated cargo between the cilium and achieved by the presence of a diffusion and transport barrier, other cellular compartments. Thus, we set out to investigate the where entry and exit decisions of ciliary components have to be molecular basis of farnesylated cargo sorting using ciliary INPP5E taken5,6. and non-ciliary Rheb as an example. Here, we show that a PDE6d is a prenyl-binding protein that was originally 100-fold difference in the binding affinity of farnesylated cargo discovered as the delta subunit of rod photoreceptor-specific with PDE6d and the specific release of high-affinity cargo by phosphodiesterase PDE6 (ref. 7). It was found as a solubilizing activated Arl3GTP determines cargo sorting into cilia, while factor for the prenylated subunits of this enzyme and was later low-affinity cargo can be released by both Arl3GTP and shown to be a general prenyl-binding protein (hence also called Arl2GTP and stays outside the cilium. Moreover, we show by PrBP/PDE6d)8–11. PDE6d was shown to bind prenylated peptides structural, biochemical and cell biological approaches, how and or proteins of the Ras subfamily with approximately micromolar why the binding affinity is dependent on the residues at the À 1 affinity12,13 and to play a critical role in their cellular and À 3 positions preceding the farnesylated cysteine and that distribution14–16. Since it is believed to be crucial for the sorting of farnesylated cargo can be manipulated by changing the localization and thus the activity of the oncoprotein Ras, affinity to PDE6d. inhibitors of the Ras-PDE6d complex were actually considered 17 as promising Ras drug candidates . Results INPP5E belongs to the inositol polyphosphate 50-phosphatase INPP5E and Rheb localization and binding affinity to PDE6d. family that hydrolyzes the 50-phosphate of phosphatidylinositols 18,19 Using IMCD3 cells stably expressing either INPP5E or Rheb fused and localizes to primary cilia . The importance of the 35 0 to a localization and tandem affinity purification (LAP) tag ,we 5 -phosphatase activity for ciliary function is underscored by can show that INPP5E localizes almost exclusively to the primary the finding that INPP5E is mutated in Joubert syndrome, a 18–20 cilium with very small fraction in the cell body (Fig. 1a; upper), ciliopathy characterized by motor and intellectual disabilities , which is consistent with previous reports18,19,26. In contrast, Rheb and that the gene mutated in the OCRL (Oculocerebrorenal) or mainly localizes to endomembranes (Fig. 1a; lower), this Lowe syndrome also encodes an inositol polyphosphate observation is consistent with previous reports13,36. Given that 50-phosphatase21,22. INPP5E contains a C-terminal CaaX motif 23 the prenyl-binding protein PDE6d is the shuttle factor mediating where the C-terminal residue Cys644 is farnesylated . A mutation the localization of INPP5E and Rheb13,16,18,25,26, we set out to encoding a stop codon near to the CaaX motif (Q627) of INPP5E 18 characterize the interaction of PDE6d with INPP5E and Rheb. was identified in a family with MORM syndrome , a ciliopathy Previously we have shown that farnesylated C-terminal peptides characterized by intellectual disability, obesity, retinal dystrophy derived from Rheb or KRas bind to PDE6d in exactly the same and micropenis24. This mutation was shown to affect INPP5E 25 way and with similar affinities as the full-length farnesylated ciliary localization, which in combination with other reports proteins12,13. Hence, we used a fluorescently labelled C-terminal indicates the importance of the C-terminus and its farnesylation farnesylated and carboxy-methylated peptide of INPP5E (residues for the ciliary localization of INPP5E (ref. 18). 637–644) and Rheb (residues 175–181) to measure the affinity to Recently, PDE6d was co-purified with INPP5E and siRNA- PDE6d by fluorescence polarization. Figure 1b (left) shows that d mediated knockdown of PDE6 resulted in impaired ciliary PDE6d binds to INPP5E peptide with low nanomolar affinity localization of INPP5E (ref. 26). Moreover, a PDE6d deletion (Kd ¼ 3.7 nM±0.2,±indicates s.d., n ¼ 9). In contrast, the affinity mutation, which was identified in Joubert syndrome, was between PDE6d and the farnesylated C-terminal peptide of Rheb shown to impair the targeting of farnesylated INPP5E protein falls into the submicromolar range (K ¼ 445±83 nM,±indicates 25 d d to the primary cilium . Knockdown of PDE6 also impeded the s.d., n ¼ 10) (Fig. 1b; right), which is in the same range with the transport of GRK1 and PDE6 catalytic subunits to photoreceptor previously described values12,13. These data raised the question, outer segments, which are considered specialized forms of whether the almost 100-fold higher affinity of INPP5E towards cilia27,28. PDE6d as compared to Rheb is involved in the sorting The homologous small Arf-like GTP-binding proteins Arl2 mechanism of these two proteins to different destinations. and Arl3 have been shown to act as nucleotide-dependent-specific release factors of farnesylated cargo from PDE6d in vitro and in vivo. Structural and kinetic analyses have shown that Arl2/3 act High-affinity cargo is specifically released by Arl3GTP. allosterically to increase the dissociation rate constants for cargo- Towards an explanation for the possible sorting mechanism that carrier complexes13,15,29,30. In contrast, it was shown recently by leaves some PDE6d-cargo in the cell body but allows others to be pull-down experiments with cellular extracts that Arl3 but enriched in the cilia we turned to the release activities of Arl2 and not Arl2 can efficiently release INPP5E from its complex with Arl3. Both GTP-binding proteins in their active conformation PDE6d (ref. 25). have been shown to be responsible for releasing cargo from In analogy to nuclear localization signals a number of different PDE6d. While Arl2 is a non-ciliary protein, Arl3 localizes along ciliary localization signals have been identified for different the length of the cilium37. Using fluorescence polarization, we transmembrane proteins31–33. However, not much is known measured the release of INPP5E and Rheb peptides from PDE6d about the molecular mechanism of how these signals are by the addition of Arl2 or Arl3 bound to the non-hydrolysable recognized and how decisions on ciliary entry based on these GTP analogue GppNHp. The data show that Rheb peptide can be signals are made. For certain membrane-associated, post- released by both Arl2GppNHp and Arl3GppNHp (Fig. 2a), translationally modified proteins carrying an N-terminal supporting earlier observations13. In contrast, INPP5E peptide myristoyl or a C-terminal prenyl motif, it has been shown that can only be released by Arl3GppNHp under the same conditions 2 NATURE COMMUNICATIONS | 7:11366 | DOI:
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