In vivo characterization of the bacterial intramembrane-cleaving protease RseP using the heme binding tag-based assay iCliPSpy.

ABSTRACT: Regulated intramembrane proteolysis (RIP) describes the protease-dependent cleavage of transmembrane proteins within the hydrophobic core of cellular membranes. Intramembrane-cleaving proteases (I-CliPs) that catalyze these reactions are found in all kingdoms of life and are involved in a wide range of cellular processes, including signaling and protein homeostasis. I-CLiPs are multispanning membrane proteins and represent challenging targets in structural and enzyme biology. Here we introduce iCLiPSpy, a simple assay to study I-CLiPs in vivo. To allow easy detection of enzyme activity, we developed a heme-binding reporter based on TNFα that changes color after I-CLiP-mediated proteolysis. Co-expression of the protease and reporter in Escherichia coli (E. coli) results in white or green co

SUBMITTER: Kupke T 

PROVIDER: S-EPMC10024687 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature