Collaborative Research: Mechanism of Ste24, a Novel Integral Membrane Zinc Metalloprotease that Promotes Catalysis Within an Intramembrane Chamber

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Drs. Christine Hrycyna from Purdue University and Mark Distefano from the University of Minnesota to define how the yeast S. cerevisiae integral membrane protein Ste24 functions. Ste24 is the founding member of a novel class of membrane proteases, enzymes that catalyze the hydrolysis of proteins; these reactions cleave the original proteins. What is unique to Ste24 is that it cleaves proteins at two different sites one after the other (rather than cleaving the proteins at only one site as the majority proteases do) and it only cleaves proteins that are attached to specific lipids. Ste24 is unique not only in reactivity but also in structure; for example, in contrast to the other of intramembrane proteases described to date, it has a large, water-filled, intramembrane barrel-shaped reaction chamber that is capped at both ends. The proposed studies examine how a substrate enters and exits the chamber of Ste24, how it is being recognized, and how Ste24 mediates the proteolysis. This work creates opportunities for graduate students to both understand the structure and biochemical function of the Ste24 protease, as well as become versed in the use of methods and tools for working with membrane proteins, studies of which are central to an emerging frontier of science. Furthermore, the research program is integrated with an outreach program that exposes high school students from economically-disadvantaged backgrounds and their teachers to modern questions and techniques in membrane protein biochemistry and molecular biology via participation in hands-on research.

This research project aims to understand the mechanism of action of Ste24 at the molecular level using an array of novel chemical probes and biochemical methods. The manner in which Ste24 binds and mediates the proteolysis of a-factor substrate precursors inside the intramembrane chamber is determined using structure-guided mutagenesis, enzymatic assays, synthetic peptide substrates and inhibitors, photoaffinity substrate-analog probes, time-resolved fluorescence spectroscopy, and computational modeling. Furthermore, the research focuses on the identification of which of the four large portals that lead to the interior of the chamber are used for the entry and exit of a-factor from the enzyme using site-directed mutagenesis and biochemical assays. These studies provide a better understanding of the mechanism of action and substrate specificity of Ste24 and reveal new fundamental properties unique to this conserved family of proteases.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.