Intramembrane proteolytic cleavage of a membrane-tethered transcription factor by a metalloprotease depends on ATP.
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-sigma(K), releasing sigma(K) 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-sigma(K). Uniquely for IPs examined so far, SpoIVFB activity requires ATP, which binds to the C-terminal cystathionine-beta-synthase (CBS) domain of SpoIVFB. Deleting just 10 residues from the C-terminal end of SpoIVFB nearly eliminated cleavage of coexpressed Pro-sigma(K) in E. coli. The CBS domain of SpoIVFB was shown to interact with Pro-sigma(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 SpoIVFB into preformed liposomes stimulated its ability to cleave Pro-sigma(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.
Zhou
Ruanbao
R
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Cusumano
Christina
C
Sui
Dexin
D
Garavito
R Michael
RM
Kroos
Lee
L
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D015151:Immunoblotting; D013058:Mass Spectrometry / Q000379:methods; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D011498:Protein Precursors / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D011498:Protein Precursors; D014157:Transcription Factors; C084190:sigma K; D000255:Adenosine Triphosphate; D010450:Endopeptidases; C071442:spoIVFB protein, Bacillus subtilis