Active γ-secretase complexes contain only one of each component

Harvard University, Cambridge, Massachusetts, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 12/2007; 282(47):33985-93. DOI: 10.1074/jbc.M705248200
Source: PubMed

ABSTRACT Gamma-secretase is an intramembrane aspartyl protease complex that cleaves type I integral membrane proteins, including the amyloid beta-protein precursor and the Notch receptor, and is composed of presenilin, Pen-2, nicastrin, and Aph-1. Although all four of these membrane proteins are essential for assembly and activity, the stoichiometry of the complex is unknown, with the number of presenilin molecules present being especially controversial. Here we analyze functional gamma-secretase complexes, isolated by immunoprecipitation from solubilized membrane fractions and able to produce amyloid beta-peptides and amyloid beta-protein precursor intracellular domain. We show that the active isolated protease contains only one presenilin per complex, which excludes certain models of the active site that require aspartate dyads formed between two presenilin molecules. We also quantified components in the isolated complexes by Western blot using protein standards and found that the amounts of Pen-2 and nicastrin were the same as that of presenilin. Moreover, we found that one Aph-1 was not co-immunoprecipitated with another in active complexes, evidence that Aph-1 is likewise present as a monomer. Taken together, these results demonstrate that the stoichiometry of gamma-components presenilin:Pen-2:nicastrin:Aph-1 is 1:1:1:1.

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    • "Following cleavages sites are the γ-sites which produce Aβ species of 43, 42, 40, 39, 38, 37 amino acid long following the rule of trior tetrapeptide release (Takami et al., 2009; for review see Karran et al., 2011). The γ-secretase is a multiprotein complex composed of at least four proteins, Presenilin, Pen-2, Aph-1, Nicastrin, and one molecule of each is necessary and sufficient to form an active enzymatic complex (Edbauer et al., 2003; Kimberly et al., 2003; Takasugi et al., 2003; Sato et al., 2007). The α-and β-secretases are sheddases releasing the extracellular domain of APP as well as several others type I transmembrane proteins . "
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    • "Additional proteins might, however, be involved in the regulation of the activity or subcellular localization of the complex (Chen et al. 2006; Wakabayashi et al. 2009; He et al. 2010). The stoichiometry of the g-secretase complex is likely 1:1:1:1, based on molecular mass estimates in blue native electrophoresis (Kimberly et al. 2003), quantitative western blot analysis (Sato et al. 2007), and electron microscopy (EM) studies of the purified complex (Osenkowski et al. 2009). Thus, as there are two different PSEN genes and two different Aph1 genes (Aph1a and Aph1b) encoded in the human genome, it follows that at least four different g-secretase complexes exist (De Strooper 2003). "
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