Supporting Information

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Fig. 5. External hydrophobic patch (EHP) sequences are found in the C-terminal propeptides of different zona pellucida (ZP) domain proteins. A multiple sequence alignment is shown of the C-terminal propeptides of Ascidian sperm receptor HrVC120 and mammalian urinary Tamm-Horsfall protein (THP)/uromodulin, major pancreatic membrane glycoprotein GP-2, deleted in malignant brain tumor-1 (DMBT1), integral membrane-associated protein-1 (ITMAP-1), TGF-b receptor III/betaglycan, inner ear a - and b -tectorins and liver LZP/EF-9. Note that, similarly to fish vitelline envelope (VE) precursors, mammalian DMBT1 and LZP have short propeptides lacking a transmembrane domain (TMD), but containing an EHP. Conventions are as in Fig. 1B.

Fig. 6. The common phenotype of EHP and internal hydrophobic patch (IHP) mutants is influenced by the presence of a TMD. Whereas full-length constructs mutated in either the EHP or IHP are efficiently secreted (Upper), no secretion is observed for corresponding mutants lacking a TMD (Lower). However, in both cases, mutation of either hydrophobic patch inhibits protein incorporation into the extracellular matrix. Conventions are as in Figs. 1-4.