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-σE protein 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.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Hizukuri Y,Akiyama K,Akiyama Ydoi
10.1016/bs.mie.2016.09.044subject
Has Abstractpub_date
2017-01-01 00:00:00pages
1-33eissn
0076-6879issn
1557-7988pii
S0076-6879(16)30317-2journal_volume
584pub_type
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