Mechanism of intramembrane proteolysis investigated with purified rhomboid proteases

Marius K Lemberg, Javier Menendez, Angelika Misik, Maite Garcia, Christopher M Koth, Matthew Freeman

Author Affiliations

  1. Marius K Lemberg1,
  2. Javier Menendez2,
  3. Angelika Misik2,
  4. Maite Garcia2,
  5. Christopher M Koth*,2 and
  6. Matthew Freeman*,1
  1. 1 MRC Laboratory of Molecular Biology, Cambridge, UK
  2. 2 Ontario Center for Structural Proteomics, University of Toronto, Toronto, Ontario, Canada
  1. *Corresponding authors: Cell Biology Division, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. Tel.: +44 1223 402351; Fax: +44 1223 412142; E‐mail: mf1{at} Center for Structural Proteomics, University of Toronto, 112 College Street, Toronto, Ontario, Canada M5G 1L6. Tel.: +1 416 946 0074; E‐mail: chris.koth{at}
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Intramembrane proteases have the unusual property of cleaving peptide bonds within the lipid bilayer, an environment not obviously suited to a water‐requiring hydrolysis reaction. These enzymes include site‐2 protease, γ‐secretase/presenilin, signal peptide peptidase and the rhomboids, and they have a wide range of cellular functions. All have multiple transmembrane domains and, because of their high hydrophobicity, have been difficult to purify. We have now developed an in vitro assay to monitor rhomboid activity in the detergent solubilised state. This has allowed us to isolate for the first time a highly pure rhomboid with catalytic activity. Our results suggest that detergent‐solubilised rhomboid activity mimics its activity in biological membranes in many aspects. Analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad. This analysis also suggests that other conserved residues participate in subsidiary functions like ligand binding and water supply. We identify a motif shared between rhomboids and the recently discovered derlins, which participate in translocation of misfolded membrane proteins.

  • Received November 23, 2004.
  • Accepted December 8, 2004.
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