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In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination
Published on: January 2, 2026
K63-linked ubiquitin chains mark inactive Smoothened for packaging into ciliary extracellular vesicles
Mingli Zhu 祝明莉1,2, Yash Raj1,2, Gary A Bradshaw3
1Department of Ophthalmology, University of California San Francisco; CA 94143, USA.
Biorxiv : the Preprint Server for Biology
|July 29, 2026
Summary
Extracellular vesicle (EV) secretion, or ectocytosis, removes signaling proteins from cilia. This study reveals K63-linked ubiquitin chains act as a signal to package the Hedgehog pathway receptor Smoothened (SMO) into EVs, regulating its levels.
Area of Science:
- Cell Biology
- Molecular Biology
- Signal Transduction
Background:
- Signaling receptors can be retrieved into the cell or secreted via extracellular vesicles (EVs) in a process called ectocytosis.
- The mechanisms governing ectocytosis, particularly for ciliary receptors, are not well understood.
- The Hedgehog signaling pathway relies on precise regulation of its components, including the Smoothened (SMO) receptor.
Purpose of the Study:
- To investigate the mechanisms of ectocytosis for ciliary signaling receptors.
- To identify the role of Smoothened (SMO) in cilia-derived EV secretion.
- To determine the molecular signals that regulate SMO packaging into EVs.
Main Methods:
- Quantitative proteomic profiling of cilia-derived EVs.
- Analysis of Smoothened (SMO) localization and EV packaging under different signaling conditions.
- Investigation of SMO ubiquitination status and its impact on EV secretion using genetic and biochemical approaches.
Main Results:
- Smoothened (SMO), a Hedgehog pathway receptor, was identified as a major cargo of cilia-derived EVs.
- Ligand-induced ciliary accumulation of SMO suppressed its packaging into EVs, suggesting selective sorting.
- K63-linked ubiquitin chains on SMO were identified as a critical sorting signal for its secretion into EVs, and their absence led to SMO accumulation within cilia.
Conclusions:
- K63-linked ubiquitination serves as a specific sorting signal for the ectocytosis of Smoothened (SMO) from cilia.
- Ubiquitin-dependent packaging of inactive SMO into EVs contributes to the dynamic regulation of Hedgehog signaling.
- This mechanism highlights a novel pathway for controlling signaling receptor levels and activity at the cilium.
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