Journal Article

Intramembrane Proteolytic Cleavage of a Membrane-Tethered Transcription Factor by a Metalloprotease Depends on ATP

Kroos, Lee, Garavito, R. Michael, Sui, Dexin, Zhou, Ruanbao, Cusumano, Christina, Losick, Richard M.

2009

Overview

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 SpolVFB protein is a putative metalloprotease that cleaves membrane-tethered ${\\rm{Pro - \\sigma }}^{\\rm{K}}$, releasing ${\\rm{\\sigma }}^{\\rm{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 ${\\rm{Pro - \\sigma }}^{\\rm{K}}$. Uniquely for IPs examined so far, SpolVFB activity requires ATP, which binds to the Cterminal cystathionine-ß-synthase (CBS) domain of SpolVFB. Deleting just 10 residues from the C-terminal end of SpolVFB nearly eliminated cleavage of coexpressed ${\\rm{Pro - \\sigma }}^{\\rm{K}}$ in E. coli. The CBS domain of SpolVFB was shown to interact with ${\\rm{Pro - \\sigma }}^{\\rm{K}}$ 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 ${\\rm{Pro - \\sigma }}^{\\rm{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.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: active sites / Adenosine triphosphatase / adenosine triphosphate / Adenosine Triphosphate - metabolism / ATP / Bacillus subtilis / Bacteria / Bacterial proteins / Bacterial Proteins - genetics / Bacterial Proteins - metabolism / Binding sites / Biochemistry / Biological Sciences / Catalytic Domain - genetics / Cobalt / cystathionine beta-synthase / E coli / Electrophoresis, Polyacrylamide Gel / Endopeptidases - genetics / Endopeptidases - metabolism / energy / Enzyme kinetics / Enzymes / Escherichia coli / Gene expression / genes / Hydrolysis / Immunoblotting / Lipids / Liposomes / Mass spectrometry / Mass Spectrometry - methods / Membrane Proteins - genetics / Membrane Proteins - metabolism / Membranes / metalloproteinases / Models, Biological / Mother cells / Mutation / Protein Precursors - metabolism / Proteins / proteolysis / Signal Transduction / spores / Sporulation / transcription factors / Transcription Factors - metabolism / Zinc