Intramembrane proteolytic cleavage of a membrane-tethered transcription factor by a metalloprotease depends on ATP
R. Zhou, C. Cusumano, D. Sui
Sep 22, 2009
Citations
35
Citations
Journal
Proceedings of the National Academy of Sciences
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Key Takeaway Key Takeaway: Bacillus subtilis SpoIVFB protein cleaves membrane-tethered Pro-K, releasing K to direct transcription for spore formation, with ATP regulating substrate access and cleavage sensitive to cellular energy levels.
Abstract
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 metalloproteases. The Bacillus subtilis SpoIVFB protein is a putative metalloprotease that cleaves membrane-tethered Pro-σK, releasing σK to direct transcription of genes necessary for spore formation. By attaching an extra transmembrane segment to the N terminus of SpoIVFB, expression in E. coli was improved more than 100-fold, facilitating purification and demonstration of metalloprotease activity, which accurately cleaved purified Pro-σK. Uniquely for IPs examined so far, SpoIVFB activity requires ATP, which binds to the C-terminal cystathionine-β-synthase (CBS) domain of SpoIVFB. Deleting just 10 residues from the C-terminal end of SpoIVFB nearly eliminated cleavage of coexpressed Pro-σK in E. coli. The CBS domain of SpoIVFB was shown to interact with Pro-σK 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 SpoIVFB into preformed liposomes stimulated its ability to cleave Pro-σK. 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.