Although activity-based protein profiling (ABPP) has been used to study a variety of enzyme classes, its application to intramembrane proteases is still in its infancy. Intramembrane proteolysis is an important biochemical mechanism for activating proteins residing within the membrane in a dormant state. Rhomboid proteases (intramembrane serine proteases) are embedded in the lipid bilayers of membranes and occur in all phylogenetic domains. The study of purified rhomboid proteases has mainly been performed in detergent micelle environments. Here we report on the reconstitution of rhomboids in liposomes. Using ABPP, we have been able to detect active rhomboids in large and giant unilamellar vesicles. We have found that the inhibitor profiles of rhomboids in micelles and liposomes are similar, thus validating previous inhibitor screenings. Moreover, fluorescence microscopy experiments on the liposomes constitute the first steps towards activity-based imaging of rhomboid proteases in membrane environments.

Center for Integrated Protein Science Munich Lehrstuhl für Chemie der Biopolymere Technische Universität München Weihenstephaner Berg 3 85354 Freising

Department of Cellular and Molecular Medicine University of Leuven Herestraat 49 Box 802 3000 Leuven

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Flemingovo n 2 Prague 166 10

Leibniz Institut für Analytische Wissenschaften ISAS e 5 Otto Hahn Strasse 6b 44227 Dortmund

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