Analysis of intramembrane-cleaving proteases and of MEM-superfamily proteases in prokaryotes with Bacillus subtilis as model organism

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There is growing evidence that proteolytic cleavage of membrane spanning regulatory proteins through intramembrane cleaving proteases is involved in a variety of important transmembrane signal-ing processes in bacteria. This so called ‘regulated intramembrane proteolysis’ (RIP) enables bacteria to respond to extracellular signals and stresses, e.g. in pathogenicity. In higher organisms, RIP holds a central role in cellular processes in gene regulation, communication and differentiation. For both, molecular mechanisms that regulate protease activity in RIP are less understood. In our work, we will examine RIP of membrane-spanning regulatory proteins in bacteria at the example of the Gram-positive model bacterium Bacillus subtilis in detail. Previously, we have identified and analyzed the first (‘site-1’) and second (‘site-2’) protease catalyzing RIP of a specific B. subtilis transcription factor. Here, we will focus on the investigation of the so far unknown mechanisms that modulate proteolytic activity of both of these enzymes. We aim to identify the molecular signal(s) and the putative factor(s) that activate the site-1 protease. Also, the mechanism that prevents premature cleavage through the site-2 protease will be analyzed. Furthermore, the role of intramembrane proteolysis in the regulation of additional B. subtilis transcription factors will be examined. Finally, we intend to assign function to other intramembrane proteases. In summary, our work will significantly contribute to unravel the gen-eral mechanisms that regulate activity of proteases involved in RIP.