Abstract
The small GTP-binding proteins Arl2 and Arl3, which are close homologs, share a number of interacting partners and act as displacement factors for prenylated and myristoylated cargo. Nevertheless, both proteins have distinct biological functions. Whereas Arl3 is considered a ciliary protein, Arl2 has been reported to be involved in tubulin folding, mitochondrial function, and Ras signaling. How these different roles are attained by the two homolog proteins is not fully understood. Recently, we showed that the N-terminal amphipathic helix of Arl3, but not that of Arl2, regulates the release of myristoylated ciliary proteins from the GDI-like solubilizing factor UNC119a/b. In the biophysical study presented here, both proteins are shown to exhibit a preferential localization and clustering in liquid-disordered domains of phase-separated membranes. However, the membrane interaction behavior differs significantly between both proteins with regard to their nucleotide loading. Whereas Arl3 and other Arf proteins with an N-terminal amphipathic helix require GTP loading for the interaction with membranes, Arl2 binds to membranes in a nucleotide-independent manner. In contrast to Arl2, the N-terminal helix of Arl3 increases the binding affinity to UNC119a. Furthermore, UNC119a impedes membrane binding of Arl3, but not of Arl2. Taken together, these results suggest an interplay among the nucleotide status of Arl3, the location of the N-terminal helix, membrane fluidity and binding, and the release of lipid modified cargos from carriers such as UNC119a. Since a specific Arl3-GEF is postulated to reside inside cilia, the N-terminal helix of Arl3•GTP would be available for allosteric regulation of UNC119a cargo release only inside cilia.