Substrate recognition and binding by RseP, an Escherichia coli intramembrane protease.
Escherichia coli RseP belongs to the S2P family of intramembrane cleaving proteases. RseP catalyzes proteolytic cleavage of the membrane-bound anti-sigma(E) protein RseA as an essential step in transmembrane signal transduction in the sigma(E) extracytoplasmic stress response pathway. RseP cleaves transmembrane segments of membrane proteins, but the molecular mechanisms of its substrate recognition and proteolytic action remain largely unknown. Here we analyzed interaction between RseP and substrate membrane proteins. Co-immunoprecipitation assays showed that helix-destabilizing residues in a substrate transmembrane segment, which were previously shown to be required for efficient proteolysis of the substrate by RseP, stabilize the substrate-RseP interaction. Substitutions of certain amino acid residues, including those evolutionarily conserved, in the third transmembrane region (TM3) of RseP weakened the RseP-substrate interaction. Specific combinations of Cys substitutions in RseP TM3 and in the RseA transmembrane segment led to the formation of disulfide bonds upon oxidation, suggesting that TM3 of RseP directly binds the substrate. These results provide insights into the mechanism of membrane protein proteolysis by RseP.
Koide
Kayo
K
Institute for Virus Research, Kyoto University, Kawara-chu, Shogoin, Kyoto 606-8507, Japan.
Ito
Koreaki
K
Akiyama
Yoshinori
Y
D019943:Amino Acid Substitution; D004220:Disulfides / Q000378:metabolism; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D048168:Escherichia coli K12 / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013379:Substrate Specificity / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C433802:RseA protein, E coli; D012808:Sigma Factor; D014157:Transcription Factors; D010450:Endopeptidases; C440001:RseP protein, E coli