Rhomboid-catalyzed intramembrane proteolysis requires hydrophobic matching with the surrounding lipid bilayer.

Membrane thinning by rhomboid proteins has been proposed to reduce hydrophobic mismatch, providing a unique environment for important functions ranging from intramembrane proteolysis to retrotranslocation in protein degradation. We show by in vitro reconstitution and solid-state nuclear magnetic resonance that the lipid environment of the rhomboid protease GlpG influences its activity with an optimal hydrophobic membrane thickness between 24 and 26 Å. While phosphatidylcholine membranes are only negligibly altered by GlpG, in an -relevant lipid mix of phosphatidylethanolamine and phosphatidylglycerol, a thinning by 1.1 Å per leaflet is observed. Protease activity is strongly correlated with membrane thickness and shows no lipid headgroup specificity. We infer from these results that, by adjusting the thickness of specific membrane domains, membrane proteins shape the bilayer for their specific needs.