Sorting of lipidated cargo by the Arl2/Arl3 system

Abstract

Arl2 and Arl3 are Arf-like small GTP-binding proteins of the Arf subfamily of the Ras superfamily. Despite their structural similarity and sharing of many interacting partners, Arl2 and Arl3 have different biochemical properties and biological functions. Growing evidence suggest that Arl2 and Arl3 play a fundamental role as regulators of trafficking of lipid modified proteins between different compartments. Here we highlight the similarities and differences between these 2 homologous proteins and discuss the sorting mechanism of lipidated cargo into the ciliary compartment through the carriers PDE6δ and Unc119 and the release factors Arl2 and Arl3.

Keywords: Arl2; Arl3; Arls; PDE6б prenyl transferases; Unc119; cargo release; cilia; myristoylation; prenylation; small GTPases and their effector proteins; sorting signal; structural biology and protein-protein interactions; structure/function studies.

Figure 1.

Type 1 and type 2 effectors of Arl2 and Arl3. Crystal structures of Arl2 and Arl3 in complex with type 1 effectors (BART and BARTL1; PDB codes: 3DOI and 4ZI2), which interact with the switch regions and the N-terminal helix and type 2 effectors (PDE6δ and Unc119a; PDB codes: 1KSG and 4GOJ), which interacts with the switch regions only. Arl2/3 (light blue), type 1 effectors (green), type 2 effectors (pink), Switch I (blue), Switch II (red), GppNHp (a non-hydrolysable GTP analog; yellow).

Figure 2.

The cargo release by Arl2 and Arl3. Left panel; crystal structures of PDE6δ in complex with farnesyl peptide (PDB code: 3T5I) and Unc119 in complex with laurylated peptide (PDB code: 3RBQ). Middle panel; the binding of Arl2/3 to the carrier proteins PDE6δ and Unc119 result in the release of the lipid moiety. Right panel; the N-terminal helix of Arl3 and Arl2 display different conformation in the complex with the carrier proteins.

Figure 3.

The sorting signal of farnesylated cargo. Superimposition of PDE6δ structures in complex with farnesylated INPP5E peptide (PDB code: 5F2U) and Rheb protein (PDB code: 3T5G). The residues at the -1 and -3 positions (arrows) relative to the farnesylated cysteine (F-Cys) from Rheb (green) and INPP5E (yellow) display different interaction patterns with the surrounding residues from PDE6δ (gray).

Figure 4.

Sorting of farnesylated cargo by the Arl2/Arl3 system. The localization of Arl13B (Arl3GEF) inside the cilia and RP2 (Arl3GAP) at the ciliary exit creates an Arl3•GTP domain inside the cilium (the Arl13B domain is shaded blue and the RP2 domain is shaded gray). Farnesylated cargo such as INPP5E which binds to PDE6δ with high affinity cannot be released in the cytosol by Arl2•GTP or the inactive Arl3•GDP. Arl3•GTP releases INPP5E from its complex with PDE6δ inside the cilium. In contrast, Ras which binds to PDE6δ with low affinity can be released by Arl2•GTP in the cytosol. Mislocalization of Ras is observed after knockdown of both PDE6δ and Arl2. After release from PDE6δ, INPP5E is retained inside the cilia by the interaction with the ciliary membrane or specific interacting partners while Ras is retained outside the cilia by the interaction with plasma membrane and endomembranes.