Environment of the active site region of RseP, an Escherichia coli regulated intramembrane proteolysis protease, assessed by site-directed cysteine alkylation.
Regulated intramembrane proteolysis (RIP) plays crucial roles in both prokaryotic and eukaryotic organisms. Proteases for RIP cleave transmembrane regions of substrate membrane proteins. However, the molecular mechanisms for the proteolysis of membrane-embedded transmembrane sequences are largely unknown. Here we studied the environment surrounding the active site region of RseP, an Escherichia coli S2P ortholog involved in the sigma(E) pathway of extracytoplasmic stress responses. RseP has two presumed active site motifs, HEXXH and LDG, located in membrane-cytoplasm boundary regions. We examined the reactivity of cysteine residues introduced within or in the vicinity of these two active site motifs with membrane-impermeable thiol-alkylating reagents under various conditions. The active site positions were inaccessible to the reagents in the native state, but many of them became partially modifiable in the presence of a chaotrope, while requiring simultaneous addition of a chaotrope and a detergent for full modification. These results suggest that the active site of RseP is not totally embedded in the lipid phase but located within a proteinaceous structure that is partially exposed to the aqueous milieu.
Koide
Kayo
K
Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.
Maegawa
Saki
S
Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.
Ito
Koreaki
K
Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.
Akiyama
Yoshinori
Y
Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan. Electronic address: yakiyama@virus.kyoto-u.ac.jp.
D000478:Alkylation; D020816:Amino Acid Motifs; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology*; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism
D029968:Escherichia coli Proteins; D008563:Membrane Lipids; D008565:Membrane Proteins; D010450:Endopeptidases; C440001:RseP protein, E coli; D003545:Cysteine