title: Biochemical Characterization of Function and Structure of RseP, an Escherichia coli S2P Protease.
abstract: Intramembrane-cleaving proteases (I-CLiPs) are a group of membrane-associated proteases with a unique feature: they are believed to cleave their substrate within the hydrophobic lipid bilayer, even though peptide bond hydrolysis requires a water molecule. Escherichia coli RseP, which belongs to the S2P zinc metalloprotease family of I-CLiPs, plays an essential role in activation of a cell envelope stress response through cleavage of anti-σEprotein RseA, a single-span transmembrane protein. A recent study showed that it also cleaves remnant signal peptides generated upon membrane translocation of secretory proteins. Here, we describe several methods for characterization of the proteolytic functions and structure of RseP mainly in vivo, including a proteolytic activity assay using model substrates, an in vitro analysis of cleavage of signal peptides in a detergent solution and in the membrane vesicles, structural analysis of membrane-embedded RseP based on the thiol modifiability of introduced cysteine residues, and the protein interaction analysis by in vivo cross-linking protocols.
authors.affiliation: Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Hizukuri
authors.affiliation: Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Akiyama
authors.affiliation: Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan. Electronic address: yakiyama@virus.kyoto-u.ac.jp.
authors.forename: Y
authors.initials: Y
authors.lastname: Akiyama
mesh_terms: D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C433802:RseA protein, E coli; D014157:Transcription Factors; D010450:Endopeptidases; C440001:RseP protein, E coli
keywords: Bacteria; Escherichia coli; I-CLiP; In vivo cross-linking; Membrane; Protein purification; RIP; Thiol modification