γ-Cleavage Is Dependent on ζ-Cleavage during the Proteolytic Processing of Amyloid Precursor Protein within Its Transmembrane Domain*

β-Amyloid precursor protein apparently undergoes at least three major cleavages, γ-, ϵ-, and the newly identified ζ-cleavage, within its transmembrane domain to produce secreted β-amyloid protein (Aβ). However, the roles of ϵ- and ζ-cleavages in the formation of secreted Aβ and the relationship among these three cleavages, namely ϵ-, ζ-, and γ-cleavages, remain elusive. We investigated these issues by attempting to determine the formation and turnover of the intermediate products generated by these cleavages, in the presence or absence of known γ-secretase inhibitors. By using a differential inhibition strategy, our data demonstrate that Aβ₄₆ is an intermediate precursor of secreted Aβ. Our co-immunoprecipitation data also reveal that, as an intermediate, Aβ₄₆ is tightly associated with presenilin in intact cells. Furthermore, we identified a long Aβ species that is most likely the long sought after intermediate product, Aβ₄₉, generated by ϵ-cleavage, and this Aβ₄₉ is further processed by ζ- and γ-cleavages to generate Aβ₄₆ and ultimately the secreted Aβ_40/42. More interestingly, our data demonstrate that γ-cleavage not only occurs last but also depends on ζ-cleavage occurring prior to it, indicating that ζ-cleavage is crucial for the formation of secreted Aβ. Thus, we conclude that the C terminus of secreted Aβ is most likely generated by a series of sequential cleavages, namely first ϵ-cleavage which is then followed by ζ- and γ-cleavages, and that Aβ₄₆ produced by ζ-cleavage is the precursor of secreted Aβ_40/42.