PDE6δ-mediated sorting of INPP5E into the cilium is determined by cargo-carrier affinity

Abstract

The phosphodiesterase 6 delta subunit (PDE6δ) 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 5′-phosphatase E (INPP5E) and the GTP-binding protein (Rheb) that cargo sorting depends on the affinity towards PDE6δ 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 PDE6δ/cargo complexes reveal the molecular basis of the sorting signal which depends on the residues at the −1 and −3 positions relative to farnesylated cysteine. Structure-guided mutation allows the generation of a low-affinity INPP5E mutant which loses exclusive ciliary localization. We postulate that the affinity to PDE6δ and the release by Arl2/3 in addition to a retention signal are the determinants for cargo sorting and enrichment at its destination.

Sorting of lipidated cargo by the Arl2/Arl3 system

“…INPP5E showed an approximately 100 fold higher binding affinity to PDE6d with a dissociation constant in the nanomolar range as compared to submicromolar affinities for Ras and Rheb. 46 Stimulated by the findings that only Arl3 can release high affinity ciliary cargo from Unc119 12,15 and from PDE6d, 38 we could confirm that under identical concentrations only Arl3 is able to release a C-terminal prenylated INPP5E peptide from its complex with PDE6d, while Arl2 did not. 46 Kinetic studies showed that the stimulated release of farnesylated INPP5E peptide by Arl3 GTP is 600fold faster than by Arl2 GTP.…”

“…46 Stimulated by the findings that only Arl3 can release high affinity ciliary cargo from Unc119 12,15 and from PDE6d, 38 we could confirm that under identical concentrations only Arl3 is able to release a C-terminal prenylated INPP5E peptide from its complex with PDE6d, while Arl2 did not. 46 Kinetic studies showed that the stimulated release of farnesylated INPP5E peptide by Arl3 GTP is 600fold faster than by Arl2 GTP. 46 Structural and biochemical analysis of Arl2/3 complexes with PDE6d and Unc119 showed that the major determinant for this difference is the structure and dynamics of the N-terminal amphiphatic helix which is absolutely required for the release activity of Arl3.…”

“…Differences in the interaction pattern due to the difference in sequence could be observed here. 46 The structures suggested that the residues -1 and -3 relative to the farnesylated cysteine are responsible for the difference in affinity (Fig. 3).…”

“…46 Structural and biochemical analysis of Arl2/3 complexes with PDE6d and Unc119 showed that the major determinant for this difference is the structure and dynamics of the N-terminal amphiphatic helix which is absolutely required for the release activity of Arl3. The N-terminal helix of Arl3 but not Arl2 folds into a hydrophobic pocket on the surface of the protein and contributes to the release of the bound lipidated cargo 15,24,46 (Fig. 2).…”

“…Binding experiments with INPP5E(KS) and Rheb(SI) showed that we could reverse the binding affinities of these peptides to PDE6d. 46 That the difference in affinity of the cargo-carrier complex is the major factor for ciliary entry was demonstrated by expressing the mutated proteins INPP5E(KS) and Rheb(SI) as GFP fusion proteins from a stable promoter in IMCD3 cells. This resulted in a dramatic turnaround in the specificity of localization of the proteins.…”