The long-term goal of this project is to understand how intramembrane metalloproteases (IMMPs) function in bacteria. IMMPs are broadly conserved membrane-embedded enzymes that cleave their substrates within a membrane or near the membrane surface. Bacterial IMMPs play important roles during infections, stress responses, cell division, sporulation, mating, and polar morphogenesis. Understanding how IMMPs function in bacteria could lead to the development of new antibiotics. In eukaryotes, IMMPs cleave transcription factors that regulate lipid metabolism, responses to endoplasmic reticulum stress, and skin and neurological development. These pathways are critical for human health. Knowledge about bacterial IMMPs will facilitate studies of eukaryotic IMMPs, which could lead to the development of novel therapeutics. Little is known about how IMMPs select their substrates or how IMMP activity can be regulated. To fill this knowledge gap, the project focuses on two IMMPs of Bacillus subtilis that represent two large subfamilies of IMMPs. SpoIVFB represents the CBS domain subfamily. CBS domains sense cellular energy levels and regulate the activity of a variety of proteins. SpoIVFB cleaves Pro-K during B. subtilis sporulation when cells have been starving for hours. The cleavage reaction requires ATP, which binds to the CBS domain of SpoIVFB. We hypothesize that the ATP level rises in the mother cell compartment after the completion of forespore engulfment and the destruction of channels through which the mother cell feeds the forespore. ATP binding to the CBS domain is proposed to induce a conformational change in SpoIVFB that positions Pro-K for cleavage. To test this model, a combination of genetic, biochemical, and structural approaches is proposed. Likewise, a combination of approaches is proposed to achieve a molecular understanding of the mechanism of SpoIVFB inhibition by its natural inhibitors, the proteins BofA and SpoIVFA. Knowledge from studies of SpoIVFB inhibition by BofA and SpoIVFA, and activation by ATP, could guide efforts to develop IMMP regulators that benefit human health. The second B. subtilis IMMP is RasP and it represents the PDZ domain subfamily. RasP functions in stress responses and cell division. In stress responses, RasP cleaves an anti- transmembrane segment after initial cleavage of the anti- extracytoplasmic domain. However, our genetic evidence suggests that RasP cleaves a cell division protein without a prior cleavage. Biochemical experiments are proposed to test this potential new paradigm. The known substrates of RasP do not account for certain defects of a rasP mutant or for the effects of RasP depletion. Innovative approaches are proposed to identify the unknown substrate(s) of RasP. Knowledge from studies of RasP is expected to advance understanding of substrate selectivity by PDZ domain-containing IMMPs and facilitate studies of related eukaryotic IMMPs that function in pathways critical for human health.