ALIX and ITCH localize to the base of primary cilia and negatively regulate ciliary Polycystin-2 levels.

Primary cilia are antenna-like organelles that function as cellular hubs for signaling pathways, including Sonic hedgehog and signaling mediated by the Polycystin-1/Polycystin-2 cation channel complex. Proper regulation of signaling output depends on the dynamic control of ciliary protein composition, which involves intraflagellar transport-mediated trafficking, protein retrieval, and the shedding of extracellular vesicles from cilia. Here we identify ALIX, a protein previously linked to the biogenesis of small extracellular vesicles, as a novel component localized at the base of primary cilia in cultured mammalian cells. We show that ALIX retention at this site requires the ciliary kinesin-3 motor protein KIF13B, which physically interacts with ALIX and the E3 ubiquitin ligase ITCH. In turn, ITCH is enriched at the ciliary base and is essential for ALIX stability. Depletion of either ALIX or ITCH results in elevated ciliary levels of Polycystin-2, while ITCH loss additionally leads to constitutive accumulation of Smoothened, a key Sonic hedgehog effector, within the cilium. Collectively, our findings establish ALIX and ITCH as critical regulators of ciliary membrane protein homeostasis and signaling, acting in coordination with KIF13B to maintain proper ciliary function.