Intramembrane proteolytic cleavage of a membrane-tethered transcription factor by a metalloprotease depends on ATP

摘要

Regulated intramembrane proteolysis (RIP) involves cleavage of a transmembrane segment of a protein. RIP governs diverse processes in a wide variety of organisms and is carried out by different types of intramembrane proteases (IPs), including a large family of metal-loproteases. The Bacillus subtilis SpolVFB protein is a putative metalloprotease that cleaves membrane-tethered Pro-σ~k, releasing σ~k to direct transcription of genes necessary for spore formation. By attaching an extra transmembrane segment to the N terminus of SpolVFB, expression in E. coli was improved more than 100-fold, facilitating purification and demonstration of metalloprotease activity, which accurately cleaved purified Pro-σ~k. Uniquely for IPs examined so far, SpolVFB activity requires ATP, which binds to the C-terminal cystathionine-β-synthase (CBS) domain of SpolVFB. Deleting just 10 residues from the C-terminal end of SpolVFB nearly eliminated cleavage of coexpressed Pro-σ~k in E. coli. The CBS domain of SpolVFB was shown to interact with Pro-σ~k and ATP changed the interaction, suggesting that ATP regulates substrate access to the active site and renders cleavage sensitive to the cellular energy level, which may be a general feature of CBS-domain-containing IPs. Incorporation of SpolVFB into preformed liposomes stimulated its ability to cleave Pro-σ~k. Cleavage depended on ATP and the correct peptide bond was shown to be cleaved using a rapid and sensitive mass spectrometry assay. A system for biochemical studies of RIP by a metalloprotease in a membrane environment has been established using methods that might be applicable to other IPs.