In an article published online 29 November 2004 in the Journal of Biological Chemistry, Dennis Selkoe's team reports further evidence placing the mature γ-secretase complex at the cell surface, and implicating it in cleavage of amyloid-β precursor protein (AβPP) to produce amyloid-β (Aβ) at that site.
A great deal of evidence supports this model of Aβ production (see, e.g., Kaether et al., 2002), but naysayers point out that presenilin seems to prefer the environs of the endoplasmic reticulum, and γ-secretase regulars Aph-1 and Pen-2 have not been spotted at the cell surface (see ARF related news story and a report from the 2004 Society for Neuroscience meeting). In their current article, first author Jay Chyung, Daniel Raper, and Selkoe first report experiments in which they fished out biotinylated versions of each of the four essential γ-secretase elements—Aph-1 and Pen-2, along with presenilin N-terminal fragment (PS-NFT) and nicastrin (Nic)—from γ-30 CHO cell line. The γ-secretase was determined to be intact, despite being overexpressed in these cells. In HeLa cells, the researchers found a biotinylated complex containing PS, Nic, and Aph-1, though Pen-2 could not be identified in the complex. The authors suggest this might be due to Pen-2's N-terminus being inaccessible to surface biotinylation. Using antibodies targeted to PS-NFT, the researchers were also able to fish out the biotinylated γ-secretase substrates C83 and C99.
Chyung and colleagues then set out to show that the biotinylated γ-secretase complex they isolated was proteolytically active on the cell membrane. In vitro assays using biotinylated proteins from γ-30 cells showed that the complex cleaved C100 into Aβ and the Aβ intracellular domain (AICD). It also cleaved one of the other γ-secretase substrates, Notch (see ARF related news story). "These data directly establish the proteolytic function of γ-secretase on the plasma membrane, independent of a hypothesized substrate trafficking role," conclude the authors.—Hakon Heimer
- γ-Secretase Activity is Mostly Intracellular—Needs PEN-2 C-terminus
- San Diego: γ-Secretase Takes Scientists on a Wild Ride
- γ-Secretase Cuts Not Just Notch, but Ligands Delta and Jagged, Too
- Kaether C, Lammich S, Edbauer D, Ertl M, Rietdorf J, Capell A, Steiner H, Haass C. Presenilin-1 affects trafficking and processing of betaAPP and is targeted in a complex with nicastrin to the plasma membrane. J Cell Biol. 2002 Aug 5;158(3):551-61. PubMed.
- Chyung JH, Raper DM, Selkoe DJ. Gamma-secretase exists on the plasma membrane as an intact complex that accepts substrates and effects intramembrane cleavage. J Biol Chem. 2005 Feb 11;280(6):4383-92. PubMed.