Abstract
Several lines of evidence have indicated that the presenilin proteins function within macromolecular complexes and are necessary for the regulated intramembranous proteolysis of certain type 1 transmembrane proteins, including the amyloid precursor protein, Notch, and p75. Data from multiple complementary experiments now suggest that there may be several distinct presenilin complexes. We show here that presenilin mutations and certain detergents affect the abundance and componentry of the presenilin complexes, and these structural effects correlate with their effects on gamma-secretase activity. Our data suggest that there are at least three complexes, including a approximately 150-kDa nicastrin-aph-1 complex (which is likely to be a precursor complex). There is a stable and abundant intermediate complex of approximately 440 kDa, which contains aph-1, pen-2, nicastrin, and PS1. However, it is the very low abundance, high mass (>/=670 kDa) heteromeric complexes that are associated with the highest gamma-secretase-specific activity.
Highlights
There is a stable and abundant intermediate complex of ϳ440 kDa, which contains aph-1, pen-2, nicastrin, and PS1. It is the very low abundance, high mass (>670 kDa) heteromeric complexes that are associated with the highest ␥-secretase-specific activity. After their initial cloning as the site of mutations causing familial Alzheimer disease [1,2,3], numerous functional studies have shown that the presenilin proteins are necessary for an unusual form of proteolytic cleavage in which selected type 1 transmembrane proteins are cleaved within their hydrophobic transmembrane domains
Based upon the fact that aph-1 is relatively stable in the absence of PS1 and binds to immature nicastrin, we have previously suggested that aph-1 is an initial scaffolding molecule upon which the functional complex is built [8]
This is supported by the fact that in PS1Ϫ/Ϫ cells, the ϳ150 kDa aph-1-nicastrin complex is stable and contains immature nicastrin, whereas the ϳ440- and Ն670-kDa complexes are de
Summary
Immunocytochemistry—KNS human glioma cells, mouse cerebellar glial cells (A3), and mouse cerebellar granule neurons were cultured and fixed as described previously [8, 12]. Cells were harvested and homogenized in buffer containing 20 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, and Complete protease inhibitor mixture (Roche Applied Science). The postnuclear supernatant was collected by centrifugation of the cell homogenates at 1000 ϫ g for 5 min in 1.5-ml Eppendorf tubes. Microsomal membranes were prepared as described above and were solubilized with ice-cold BN-lysis buffer containing 1% (w/v) detergent (either digitonin or CHAPSO), 500 mM 6-aminocaproic acid, 50 mM Bis-Tris, pH 7.0, plus Complete protease inhibitor mixture (Roche Applied Science). The lysate (100 l) was centrifuged at 100,000 ϫ g for 20 min, and the supernatant.
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