Rhomboid proteolysis: why inside the membrane?

Rhomboid proteins are membrane-immersed enzymes with the remarkable ability to hydrolyze protein segments within the membrane. This form of intramembrane proteolysis plays key roles in cell communication including EGF signalling during animal development, and quorum sensing during bacterial growth. We have been investigating rhomboid protease mechanism using a pure enzyme reconstitution system that we developed, coupled with high-resolution crystal structures of a model bacterial rhomboid protease (1, 2). Intramembrane proteolysis is catalyzed within a hydrated cavity that opens to the outside of the cell and allows water to enter, but is protected laterally from membrane lipids by a ring of protein segments. Using a structure-function approach, we subsequently identified one transmembrane helix and overlying loop that provide a rate-limiting movement to gate substrate entry laterally from the membrane (3). By integrating reconstitution into defined bilayer environments with spectroscopic interrogation, we recently discovered that rhomboid proteases rely on the natural biophysical constraints of the membrane to achieve site-specific proteolysis. Since rhomboid proteins play critical roles in the infective cycles of several protozoan parasites (4,5,6), understanding the protease mechanism may provide therapeutic opportunities (7).