Intramembrane metalloproteases (IMMPs) are conserved from bacteria to humans and control many important signaling pathways, but little is known about how IMMPs interact with their substrates. SpoIVFB is an IMMP that cleaves Pro-K during Bacillus subtilis endospore formation. When catalytically inactive SpoIVFB was coexpressed with C-terminally truncated Pro-K (1-126) (which can be cleaved by active SpoIVFB) in Escherichia coli, the substrate dramatically improved solubilization of the enzyme from membranes with mild detergents. Both the Pro(1-20) and K (21-126) parts contributed to improving SpoIVFB solubilization from membranes, but only the K part was needed to form a stable complex with SpoIVFB in a pulldown assay. The last 10 residues of SpoIVFB were required for improved solubilization from membranes by Pro-K (1-126) and for normal interaction with the substrate. The inactive SpoIVFB⅐Pro-K (1-126)-His 6 complex was stable during affinity purification and gel filtration chromatography. Disulfide cross-linking of the purified complex indicated that it resembled the complex formed in vivo. Ion mobility-mass spectrometry analysis resulted in an observed mass consistent with a 4:2 SpoIVFB⅐Pro-K (1-126)-His 6 complex. Stepwise photobleaching of SpoIVFB fused to a fluorescent protein supported the notion that the enzyme is tetrameric during B. subtilis sporulation. The results provide the first evidence that an IMMP acts as a tetramer, give new insights into how SpoIVFB interacts with its substrate, and lay the foundation for further biochemical analysis of the enzyme⅐substrate complex and future structural studies.Many critical cellular processes are regulated by intramembrane proteolysis (1). Intramembrane proteases (IPs) 3 cleave their substrates within a transmembrane segment (TMS) or near the membrane surface. There are three classes of IPs: rhomboids, aspartyl IPs, and IMMPs (often called site-2 proteases or S2Ps). Rhomboids are serine IPs that promote animal cellular signaling, coordinate bacterial quorum sensing, regulate mitochondrial homeostasis, and control protozoan infection (2-5). Presenilin, an aspartyl IP, is the catalytic component of ␥-secretase, which is involved in the processing of the amyloid precursor protein, Notch, and many other subtrates (6, 7). Dysfunction of ␥-secretase contributes to the pathogenesis of Alzheimer disease (8) and many other diseases (7). Aspartyl IPs also include preflagellin and prepilin peptidases involved in bacterial pathogenesis (9), and signal peptide peptidases, which facilitate the clearance of signal peptides from membranes, participate in viral infection, and generate small peptides as signal molecules for immune systems (10, 11). IMMPs also play critical roles in a wide variety of biological functions. In eukaryotes, cholesterol metabolism, the unfolded protein response, and the acute-phase response are regulated by IMMPs (1, 12, 13). In bacteria, IMMPs control sporulation, envelope stress responses, mating signal production, polar morphogenesis, virulence, ...