Abstract
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.
Highlights
FEBRUARY 16, 2007 VOLUME 282 NUMBER 7 which regulated cleavages are introduced into transmembrane segments of membrane proteins
The 2 His residues in the HEXXH motif are supposed to act as ligands for zinc coordination while the Glu residue is supposed to be directly involved in catalysis [5]
Whereas HA-MBP-RseA(LacYTM1)140 was detected as the full-length product (UC) in cells without RseP or in cells with the active site-mutated RseP, it was efficiently converted to the cleaved form (CL) in the presence of wild type RseP, Cys-less RseP, or a complementation-positive single cysteine RseP variant (Fig. 2, A and B)
Summary
Several elements, including the periplasmic PDZ domain of RseP, the Gln-rich regions in the RseA periplasmic domain, and periplasmic protein RseB, are involved in this negative regulation [21,22,23]. Helix-destabilizing residues in transmembrane sequences of substrates facilitate the proteolysis by RseP [9]. Extensive studies have characterized the biological functions of the RIP proteases, including those of the S2P family proteins, their molecular mechanisms remain largely unknown. RIP proteases have their active site residues located in predicted transmembrane segments and they are believed to catalyze proteolysis within the lipid bilayer [3, 4]. This notion poses a question of how hydrolysis of a peptide bond is facilitated in the lipid environment with limited availability of water. Our results suggest that the proteolytic active site is not completely embedded in the lipid phase but is in a folded structure that is either partly integrated into or stabilized by the lipid bilayer
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