Rhomboid proteases were co-reconstituted with substrates into liposomes using the inducible reconstitution system that we described recently16 (link). Bacterial rhomboid proteases were reconstituted in liposomes formed from an E. coli polar lipid extract, while DmRho4 was reconstituted in liposomes formed from a yeast polar lipid extract or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipids (Avanti Polar Lipids). All enzymes were assayed for 1 hour at 37°C, except DmRho4, which was assayed at 25°C for 2–4 hours. APP+Spi7-Flag reaction products were resolved by SDS-PAGE and quantified by anti-Flag western analysis using an Odyssey infrared laser scanner (LiCor Biosciences), while products for the real-time assay using FITC-TatA were quantified using a Synergy H4 Hybrid plate reader (Biotek) scanning once per minute. Proteolysis assays were supplemented with 0.5 mM calcium unless otherwise indicated. For calcium titration experiments, 10 pmol of DmRho4 and 200 pmol FITC-TatA were co-reconstituted into 30 µg yeast liposomes and total calcium was titrated from 0 to 1 mM. For kinetic analysis, 10 pmol of Rho4 was titrated against 15-600 pmol FITC-TatA substrate in the presence or absence of 0.5 mM calcium, initial reaction rates were extracted and fit to a Michaelis-Menten model using Prism software.