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theses

Wnts are secreted glycolipoproteins fundamental for embryonic development and adult tissue homeostasis. Abnormal Wnt signal transduction is associated with human diseases, most notably colon cancers. Wnts act as extracellular ligands that bind specific surface receptors in Wnt-responding cells. In contrast to our comprehensive understanding of Wnt signal transduction in the ligand-responding cells, there is still a deficiency in the knowledge about the cellular mechanism that regulates Wnt secretion from the ligand-producing cells. A multi-pass transmembrane protein Wntless (Wls) binds and transports Wnt ligands for secretion, however, the mechanism underlying the initial assembly of Wnt secretory vesicles has not been defined. Using proteomic and mutagenic analysis in mammalian cells and primary mouse intestinal organoids, we have delineated a sophisticated regulatory mechanism that controls the initial assembly of Wnt secretory vesicles at ER membrane. Binding of lipidated Wnt ligands to Wls in ER promotes an association between Wls and SEC12, an ER membrane-localized guanine nucleotide-exchanging factor (GEF) that activates the small GTPase SAR1. Compared with Wnt molecules that lack lipid modification, mature ligands drive a stronger Wls-SEC12 interaction that was biochemically mapped to cytosolic segments of both proteins. Remarkably, Wls utilizes a well conserved but separate protein motif in its C-terminus to engage with SAR1 for assembly of COPII ER-exiting vesicles. Mutating Wls at responsible amino acids or disrupting SAR1 activity accumulated Wls at ER exiting sites. Unlike the wild type Wls, mutant transporter failed to facilitate Wnt secretion and to support stem cell maintenance in mouse intestinal organoids. Additional study suggests that GOLD domain-containing proteins may participate in subsequent transport of Wls/Wnt complex through COPII vesicles. Specifically, TMED10 binds Wnts and may act as a cargo adaptor to incorporate Wnt/Wls complex into the COPII vesicles. ACBD3, another GOLD domain-containing protein, may contribute to selectively transport WNT7A for ER-to Golgi delivery. Our results suggest that Wnt exocytosis is carefully corroborated, at several distinct protein complex interfaces, from early steps of the secretory pathway. These regulations may ensure effective export of mature and functional ligands. Due to the relatively simplified regulators, e.g., Wls, that control Wnt production, the molecular mechanism uncovered for Wnt secretion regulation may be useful to intervening Wnt production and signaling activity in certain cancers.

ETD_8463

Ph.D.

Includes bibliographical references

by Jiaxin Sun

doi:10.7282/T3TH8QT6

ETD doctoral

The author owns the copyright to this work.