guanosine-triphosphate and Ciliopathies

UNC119 and PDEδ are lipid-binding proteins and are thought to form diffusible complexes with transducin-α and prenylated OS proteins, respectively, to mediate their trafficking to photoreceptor outer segments. Here, we investigate mechanisms of trafficking which are controlled by Arf-like protein 3 (Arl3), a small GTPase. The activity of ARL3 is regulated by a GEF (ARL13b) and a GAP (RP2). In a mouse germline knockout of RP2, ARL3-GTP is abundant as its intrinsic GTPase activity is extremely low. High levels of ARL3-GTP impair binding and trafficking of cargo to the outer segment. Germline knockout of ARL3 is embryonically lethal generating a syndromic ciliopathy-like phenotype. Retina- and rod-specific knockout of ARL3 allow to determine the precise mechanisms leading to photoreceptor degeneration. The knockouts reveal binary functions of ARL3-GTP as a key molecule in late-stage photoreceptor ciliogenesis and cargo displacement factor.

Topics: Adaptor Proteins, Signal Transducing; ADP-Ribosylation Factors; Animals; Ciliopathies; Cone-Rod Dystrophies; Cyclic Nucleotide Phosphodiesterases, Type 6; Genes, Lethal; GTP-Binding Proteins; Guanosine Triphosphate; Lipoproteins; Membrane Proteins; Mice; Mice, Knockout; Organ Specificity; Protein Prenylation; Protein Transport; Pyrophosphatases; Rod Cell Outer Segment

Highly conserved intraflagellar transport (IFT) protein complexes direct both the assembly of primary cilia and the trafficking of signaling molecules. IFT complexes initially accumulate at the base of the cilium and periodically enter the cilium, suggesting an as-yet-unidentified mechanism that triggers ciliary entry of IFT complexes. Using affinity-purification and mass spectrometry of interactors of the centrosomal and ciliopathy protein, CEP19, we identify CEP350, FOP, and the RABL2B GTPase as proteins organizing the first known mechanism directing ciliary entry of IFT complexes. We discover that CEP19 is recruited to the ciliary base by the centriolar CEP350/FOP complex and then specifically captures GTP-bound RABL2B, which is activated via its intrinsic nucleotide exchange. Activated RABL2B then captures and releases its single effector, the intraflagellar transport B holocomplex, from the large pool of pre-docked IFT-B complexes, and thus initiates ciliary entry of IFT.

Topics: Animals; Cell Cycle Proteins; Centrioles; Cilia; Ciliopathies; Flagella; Gene Knockout Techniques; Guanosine Triphosphate; Mice, Inbred C57BL; Mice, Knockout; Multiprotein Complexes; Nucleotides; Phenotype; Protein Binding; Protein Stability; Protein Transport; rab GTP-Binding Proteins; Reproducibility of Results