bioRxiv preprint doi: https://doi.org/10.1101/2020.07.27.224220; this version posted July 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 2 Insights into the catalytic properties of the mitochondrial rhomboid protease PARL 3 4 5 Laine Lysyk1, Raelynn Brassard1, Elena Arutyunova1, Verena Siebert2, Zhenze Jiang3, 6 Emmanuella Takyi1, Melissa Morrison1, Howard S. Young1, Marius K. Lemberg2, Anthony J. 7 O’Donoghue3, M. Joanne Lemieux1* 8 9 1 Department of Biochemistry, Faculty of Medicine and Dentistry, Membrane Protein Disease 10 Research Group, University of Alberta, Edmonton T6G 2R3, Alberta, Canada 11 2 Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, 12 Im Neuenheimer Feld 282, 69120 Heidelberg, Germany 13 3 Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San 14 Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA 15 16 *Correspondence: e-mail:
[email protected] 17 18 19 Abstract 20 The rhomboid protease PARL is a critical regulator of mitochondrial homeostasis through its 21 cleavage of substrates such as PINK1, PGAM5, and Smac, which have crucial roles in 22 mitochondrial quality control and apoptosis. To gain insight into the catalytic properties of the 23 PARL protease, we expressed human PARL in yeast and used FRET-based kinetic assays to 24 measure proteolytic activity in vitro. We show PARL activity in detergent is enhanced by 25 cardiolipin. Significantly higher turnover rates are observed for PARL reconstituted in 26 proteoliposomes, with Smac being cleaved most rapidly at a rate of 1 min-1.