In recent years the field of intramembrane proteolysis has generated great discoveries about the unique process by which proteins are cut within cell membranes. The intramembrane metalloprotease (IMMP) family of intramembrane proteases has provided a number of excellent models for study in bacteria. The PDZ domain-containing IMMPs, RseP (Escherichia coli) and RasP (Bacillus subtilis) are the best understood IMMPs, and their study has have laid the groundwork for studying the role of IMMPs in a number of important bacterial pathogens. Work described in this dissertation has advanced the study of RasP. Better methods for heterologous expression of RasP and its substrates in E. coli are presented. Site-1 cleavage of the RasP substrate RsiW was required, while the substrate FtsL was cleaved directly in E. coli. Importantly, RasP is only the third IMMP to be purified and have in vitro activity demonstrated on a substrate (RsiW). Surprisingly, FtsL was not cleaved in vitro. The work also advanced knowledge about the cystathione beta synthase (CBS) domain-containing IMMP, SpoIVFB. IMMPs with CBS domains use ATP binding to regulate activity. To test for physiological conditions that change the ATP concentration, a luciferase-based ATP sensor was developed for B. subtilis. ATP levels change significantly during sporulation and in response to channels or "feeding tubes" present in B. subtilis cells during endospore formation. Interestingly, two different treatments that artificially lower ATP levels to the same extent affected processing of the substrate (Pro-sigmaK) differently, suggesting other adenine nucleotides may bind the CBS domain. Indeed, an assay for conformational change upon ATP binding demonstrated that AMP may modulate SpoIVFB conformational changes. Critical residues of the CBS domain were identified by substitutional analysis in E. coli and B. subtilis, and additional residues of interest were identified in a suppressor screen. Outstanding questions and future directions related to these projects are presented.
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