BBSome trains remove activated GPCRs from cilia by enabling passage through the transition zone

A diffusion barrier at the transition zone enables the compartmentalization of signaling molecules by cilia. The BBSome and the small GTPase Arl6, which triggers BBSome coat polymerization, are required for the exit of activated signaling receptors from cilia, but how diffusion barriers are crossed when membrane proteins exit cilia remains to be determined. Here we found that activation of the ciliary GPCRs Smoothened and SSTR3 drove the Arl6-dependent assembly of large, highly processive and cargo-laden retrograde BBSome trains. Single-molecule imaging revealed that the assembly of BBSome trains enables the lateral transport of ciliary GPCRs across the transition zone. Yet, the removal of activated GPCRs from cilia was inefficient because a second, periciliary diffusion barrier was infrequently crossed. We conclude that exit from cilia is a two-step process in which BBSome/Arl6 trains first moves activated GPCRs through the transition zone before a periciliary barrier can be crossed.\n\nSummaryUpon activation, GPCRs must exit cilia for appropriate signal transduction. Using bulk imaging of BBSome and single molecule imaging of GPCRs, Ye et al. demonstrate that retrograde BBSome trains assemble on-demand upon GPCR activation and ferry GPCRs across the transition zone. Yet, ciliary exit often fails because of a second diffusion barrier.