An integrated proximity labeling and vesicle reconstitution assay identifies novel regulators of Sonic hedgehog secretion.

Protein trafficking is a fundamental process for cellular organization and signaling. However, identifying the specific machinery that packages cargo into transport vesicles has been a significant challenge. Conventional proximity biotinylation methods often fail to distinguish proteins that are merely near a cargo from those that are functionally co-packaged into the same vesicles, leading to high background noise. To address this limitation, we developed an integrated strategy that combines in vivo proximity biotinylation with an in vitro reconstituted vesicle formation assay. Applying this method to the signaling morphogen Sonic hedgehog (Shh), we successfully enriched and identified proteins co-incorporated into Shh-containing vesicles. Comparative proteomics and subsequent functional validation revealed two novel regulators of Shh secretion: ER-Golgi Intermediate Compartment Protein 2 (ERGIC2), which is essential for efficient ER-to-Golgi transport, and Sec1 Family Domain Containing 2 (SCFD2), which is critical for post-Golgi export. This study establishes a generalizable method to map vesicle-associated interactomes and provides a more comprehensive molecular framework for the regulated secretion of this important morphogen.