The Synthetic Processing (SynPro) system shows that prodomain cleavage can be non-cell-autonomous. (A) The SynPro system comprises an orthogonal secreted protease derived from tobacco etch virus (secTEVp) and a cognate sequence that replaces the endogenous cleavage site of Vg1 (SynPro Vg1, RSRRKR → ENLYFQS). (B) (i) Rescue percentage after 30 hpf of Mvg1 embryos injected with 50 pg of vg1, SynPro vg1, SynPro vg1 and secTEVp, or SynPro vg1 and secTEVp-KDEL mRNAs. (ii) Representative images of 30 hpf Mvg1 embryos for the indicated injection condition. Minor brain and tail defects are noted in embryos transiently rescued with mRNAs of the SynPro system. (Scale bar, 0.5 mm.) (iii) Fluorescence images of Mvg1 embryos at 50 to 60% epiboly that were injected with mRNAs for sfGFP-tagged wild-type Vg1 and secTEVp-sfCherry (Top) or secTEVp-sfCherry-KDEL (Bottom). (Scale bar, 20 μm.) (C) Schematic of transplantation assay. Mvg1 embryos were injected with 50 pg mRNA each of: (i) DONOR: cyc, SynPro vg1, and secTEVp; HOST: none; (ii) DONOR: cyc and SynPro vg1; HOST: secTEVp; (iii) DONOR: cyc and SynPro vg1; HOST: none; (iv) DONOR: none; HOST: secTEVp. All Mvg1 donor embryos were marked by also injecting 50 pg sfGFP mRNA. At high stage, before the onset of Nodal signaling, sfGFP-marked DONOR cells were transplanted to the animal pole of HOST Mvg1 embryos. (D) At 50 to 60% epiboly, chimeric embryos were fixed and immunostained for sfGFP and pSmad2. DAPI, nuclei. (Scale bar, 20 μm.)

Prodomain cleavage affects Vg1-Nodal signaling but not secretion. (A) Live fluorescence imaging of Mvg1 coinjected with 50 pg of vg1-sfGFP or noncleavable vg1-sfGFP (vg1-NC-sfGFP, RSRRKR → SQNTSN) mRNA and 50 pg of sqt or sqt-NC (RRHRR → SQNTS) mRNA. (Scale bar, 17 μm.) (B) Nodal target gene (lefty1) expression at 50% epiboly in WT and Mvg1 embryos injected with 50 pg of sqt, sqt-NC, or cyc mRNA. (C) lefty1 expression in Mvg1 embryos coinjected with 50 pg sqt, sqt-NC, or cyc; and vg1 or vg1-NC mRNA.

Cysteine and N-linked glycosylation sites retain the Vg1 prodomain in the ER. (A) Schematic and primary amino acid sequence of zebrafish Vg1 preproprotein. Cysteines (red, yellow) and asparagines (blue) are highlighted. (B) Alphafold2 model for Vg1 is shown in cartoon representation, whereas the cysteine (red, yellow) and asparagine (blue) residues are shown in spheres (Insets show zoomed-in views of the residues mutated in this study). We make a minor note here that two cysteine residues, C68 and C234 (yellow), are predicted to form a disulfide bond and thus were not further studied. (C–E) Fluorescence images of fixed Mvg1 embryos injected with 50 pg mRNA of sfGFP-tagged vg1 prodomain (proVg1) (C), vg1 mature domain (matVg1) (D), and full-length vg1 (E), with or without the indicated cysteines and asparagines mutated. sfGFP was inserted into vg1 downstream of the predicted basic cleavage site in all constructs. (Scale bar, 20 μm.)

Binding motifs for BiP promote ER retention of the Vg1 mature domain. (A) Amino acid sequence alignment of the mature domains of Vg1 (matVg1) and Cyc (matCyc) and mutant matVg1. Cysteines (red) and a potentially glycosylated asparagine (blue) are highlighted. (B and C) Difference charts of the BiP binding specificity scores (34) between two protein sequences along a sliding window of seven amino acids. Orange fills indicate the matVg1 or matVg1(m1, m2, m3) score (black line) > matCyc score (blue line). Conversely, blue fills indicate matCyc score > matVg1 or matVg1(m1, m2, m3) score. Arrows denote regions where matVg1 score > 0 and matCyc score < 0. Red arrows specifically denote regions that contain cysteines involved in cystine-knot formation. (B) Differences in BiP binding specificity scores between matVg1 (black line) and matCyc (blue line). (C) Differences in BiP binding specificity scores between mutant matVg1(m1, m2, m3) (black line) and matCyc (blue line). Note the loss of orange fills in m1, m2, and m3 regions (pink arrows) when compared to (B). (D) Fluorescence images of fixed Mvg1 embryos injected with 50 pg mRNA of sfGFP-tagged vg1 mature domain variants. sfGFP was inserted upstream of the vg1 mature domain in all constructs. (Scale bar, 20 µm.)

Model of Vg1-Nodal heterodimer formation and processing. Maternal Vg1 is retained in the ER via chaperone-binding motifs; during maternal-zygotic transition, Nodal is produced and induces release of Vg1 from the ER via heterodimer formation; processing of the Vg1 prodomain is required for the activity of Vg1-Nodal heterodimers but can be independent from dimer formation and secretion.