A C-terminal region of signal peptide peptidase defines a functional domain for intramembrane aspartic protease catalysis.
Intramembrane proteolysis is now firmly established as a prominent biological process, and structure elucidation is emerging as the new frontier in the understanding of these novel membrane-embedded enzymes. Reproducing this unusual hydrolysis within otherwise water-excluding transmembrane regions with purified proteins is a challenging prerequisite for such structural studies. Here we show the bacterial expression, purification, and reconstitution of proteolytically active signal peptide peptidase (SPP), a membrane-embedded enzyme in the presenilin family of aspartyl proteases. This finding formally proves that, unlike presenilin, SPP does not require any additional proteins for proteolysis. Surprisingly, the conserved C-terminal half of SPP is sufficient for proteolytic activity; purification and reconstitution of this engineered fragment of several SPP orthologues revealed that this region defines a functional domain for an intramembrane aspartyl protease. The discovery of minimal requirements for intramembrane proteolysis should facilitate mechanistic and structural analysis and help define general biochemical principles of hydrolysis in a hydrophobic environment.
Narayanan
Saravanakumar
S
Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
Sato
Toru
T
Wolfe
Michael S
MS
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D004331:Drosophila melanogaster; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D053763:Presenilins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology*
D053763:Presenilins; D011993:Recombinant Fusion Proteins; D016282:Aspartic Acid Endopeptidases; C460085:signal peptide peptidase