Reconstitution of intramembrane proteolysis in vitro reveals that pure rhomboid is sufficient for catalysis and specificity

Sinisa Urban, Michael S. Wolfe

Research output: Contribution to journalArticlepeer-review

151 Scopus citations


Intramembrane proteolysis is a new paradigm in biology that controls signaling events throughout evolution. Hydrolysis of peptide bonds is thought to occur within the normally hydrophobic membrane environment, but insights into this unusual activity have been lacking because of difficulty in recapitulating activity in vitro. We have reconstituted intramembrane proteolysis with a pure recombinant substrate and rhomboid proteins in both detergent micelles and artificial membrane environments. Rhomboid proteins from diverse organisms including two model bacteria, a pathogen, an extremophile, and an animal were robustly active in pure form, proving that rhomboids are a new class of enzymes and do not require cofactors to catalyze intramembrane proteolysis. Rhomboid proteins directly recognized their substrates in vitro by the top of the substrate transmembrane domain, displaying specificity apparently reciprocal to that of γ-secretase, the only other activity known to cleave type-I transmembrane domains. Rhomboid proteases represent a different evolutionary path to a serine protease mechanism and exhibited an inhibitor profile unlike other serine proteases. Intriguingly, activity was dramatically modulated by different membrane phospholipid environments, suggesting a mechanism for regulating these proteases. This analysis promises to help reveal the biochemical mechanisms and biological roles of this most widely conserved membrane protein family.

Original languageEnglish (US)
Pages (from-to)1883-1888
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number6
StatePublished - Feb 8 2005
Externally publishedYes


  • Cell signaling
  • Presenilin
  • Regulated intramembrane proteolysis
  • Signal peptide peptidase
  • Site-2 protease

ASJC Scopus subject areas

  • General


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