Cytosolic extensions directly regulate a rhomboid protease by modulating substrate gating

Intramembrane proteases catalyze the signal-generating step of various cell signaling pathways, and continue to be implicated in diseases ranging from malaria infection to Parkinsonian neurodegeneration(1–3). Despite playing such decisive roles, it remains unclear whether or how these membrane-immersed enzymes might be regulated directly. To address this limitation, we sought intramembrane proteases containing domains known to exert regulatory functions in other contexts, and focused on rhomboid proteases that harbor calcium-binding EF-hands. We found calcium potently stimulates proteolysis by endogenous rhomboid-4 in Drosophila cells, and, remarkably, when rhomboid-4 was purified and reconstituted in liposomes. Interestingly, deleting the amino-terminal EF-hands activated proteolysis prematurely, while residues in cytoplasmic loops connecting distal transmembrane segments mediated calcium stimulation. Rhomboid regulation was not orchestrated by either dimerization or substrate interactions. Instead, calcium increased catalytic rate by promoting substrate gating. Substrates with cleavage sites outside the membrane could be cleaved but lost the capacity to be regulated. These observations indicate substrate gating is not an essential step in catalysis, but instead evolved as a mechanism for regulating proteolysis inside the membrane. Moreover, these insights provide new approaches for studying rhomboid functions by investigating upstream inputs that trigger proteolysis.