Zhu, M.; Raj, Y.; Bradshaw, G. A.; Mick, D. U.; Kalocsay, M.; Nachury, M. V.

Signaling receptors can exit cilia either by retrieval back into the cell or by secretion in extracellular vesicles (EVs), a process known as ectocytosis. The mechanisms that govern ectocytosis remain poorly understood. Here, we leverage quantitative proteomic profiling to identify the Hedgehog signaling receptor Smoothened (SMO) as a major cargo of cilia-derived EVs. Surprisingly, ligands that promote ciliary accumulation of SMO strongly suppressed its packaging into EVs, indicating that ectocytosis selectively packages specific conformational states of SMO. We further find that SMO packaged into EVs is extensively modified with K63-linked ubiquitin chains. Preventing SMO ubiquitination or selectively removing K63-linked ubiquitin chains from SMO markedly reduced its secretion into EVs and caused SMO accumulation within cilia. Together, these results identify K63-linked ubiquitin chains as a sorting signal for ciliary ectocytosis and indicate that ubiquitin-dependent packaging of inactive SMO contributes to the dynamic redistribution of SMO during Hedgehog signaling.