Molecular mechanism ofreguLaled inframembrane proteolysis (RIP)

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Regulated intramembrane proteolysis (RIP) plays key roles in the regulation of various cellular processes. During RIP, a target membrane protein is believed to receive limited cleavage within the transmembrane segment. E. coli has two RIP proteases, RseP and GlpG. We studied these proteins to understand their physiological roles and the mechanism of RIP. We found that RseP cleaved RseA, a negative regulator of transcription factor σ_E, within its transmembrane region and that RseP was thereby essential for the activation of the σ_E pathway of ESR. We showed that the active site of RseP is sequestered form the surrounding lipidic and aqueous environments and exists in a folded protein domain. We also characterized the proteolytic activity of RseP. Although the physiological function of GlpG remains unknown, we constructed a model substrate and demonstrated that GlpG possesses proteolytic activity against model membrane proteins. We showed that although the GlpG active site exists within a hydrophilic cavity open to the periplasm, which is consistent with the recently reported crystal structures of GlpG, it cleaves a substrate at a region located outside the membrane. We identified two sequence elements in a model substrate required for cleavage by GlpG and suggested a model of the protease action of GlpG. We also analyzed the structure and function of the L1 periplasmic loop of GlpG, and suggested that it protrudes from the GlpG core region and plays a regulatory role for the GlpG activity.