Molecular mechanisms controlling the biogenesis of the TGF-β signal Vg1

The TGF-beta signals Vg1 (Dvr1/Gdf3) and Nodal form heterodimers to induce vertebrate mesendoderm. The Vg1 proprotein is a monomer retained in the endoplasmic reticulum (ER) and is processed and secreted upon heterodimerization with Nodal, but the mechanisms underlying Vg1 biogenesis are largely elusive. Here, we clarify the mechanisms underlying Vg1 retention, processing, secretion, and signaling and introduce a Synthetic Processing (SynPro) system that enables the programmed cleavage of ER-resident and extracellular proteins. First, we find that Vg1 can be processed by intra- or extracellular proteases. Second, Vg1 can be processed without Nodal but requires Nodal for secretion and signaling. Third, Vg1-Nodal signaling activity requires Vg1 processing, whereas Nodal can remain unprocessed. Fourth, Vg1 employs exposed cysteines, glycosylated asparagines, and BiP chaperone-binding motifs for monomer retention in the ER. These observations suggest two mechanisms for rapid mesendoderm induction: Chaperone-binding motifs help store Vg1 as an inactive but ready-to-heterodimerize monomer in the ER, and the flexibility of Vg1 processing location allows efficient generation of active heterodimers both intra- and extracellularly. These results establish SynPro as an in vivo processing system and define molecular mechanisms and motifs that facilitate the generation of active TGF-beta heterodimers.