γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aβ that contains longer Aβ; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aβ further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aβ42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.

The γ-secretase complex, consisting of presenilin, nicastrin, presenilin enhancer-2 (PEN-2), and anterior pharynx defective-1 (APH-1) cleaves type I integral membrane proteins like amyloid precursor protein and Notch in a process of regulated intramembrane proteolysis. The regulatory mechanisms governing the multistep assembly of this “proteasome of the membrane” are unknown. We characterize a new interaction partner of nicastrin, the retrieval receptor Rer1p. Rer1p binds preferentially immature nicastrin via polar residues within its transmembrane domain that are also critical for interaction with APH-1. Absence of APH-1 substantially increased binding of nicastrin to Rer1p, demonstrating the competitive nature of these interactions. Moreover, Rer1p expression levels control the formation of γ-secretase subcomplexes and, concomitantly, total cellular γ-secretase activity. We identify Rer1p as a novel limiting factor that negatively regulates γ-secretase complex assembly by competing with APH-1 during active recycling between the endoplasmic reticulum (ER) and Golgi. We conclude that total cellular γ-secretase activity is restrained by a secondary ER control system that provides a potential therapeutic value.

Presenilin-1 is a polytopic membrane protein that assembles with nicastrin, PEN-2, and APH-1 into an active ␥-secretase complex required for intramembrane proteolysis of type I transmembrane proteins. Although essential for a correct understanding of structure-function relationships, its exact topology remains an issue of strong controversy. We revisited presenilin-1 topology by inserting glycosylation consensus sequences in human PS1 and expressing the obtained mutants in a presenilin-1 and 2 knock-out background. Based on the glycosylation status of these variants we provide evidence that presenilin-1 traffics through the Golgi after a conformational change induced by complex assembly. Based on our glycosylation variants of presenilin-1 we hypothesize that complex assembly occurs during transport between the endoplasmic reticulum and the Golgi apparatus. Furthermore, our data indicate that presenilin-1 has a nine-transmembrane domain topology with the COOH terminus exposed to the lumen/extracellular surface. This topology is independently underscored by lysine mutagenesis, cell surface biotinylation, and cysteine derivation strategies and is compatible with the different physiological functions assigned to presenilin-1.␥-Secretase is a multisubunit protease requiring the coordinated action of presenilins (PSs), 4 nicastrin (NCT), PEN-2, and APH-1 (1-3) and is crucial for the intramembrane proteolysis of type I membrane proteins such as the amyloid precursor protein (APP) and Notch (4). The catalytic component, PS1, is a polytopic membrane protein that undergoes endoproteolysis resulting in stable PS1 NH 2 -and COOH-terminal fragments (PS1-NTF and -CTF). According to the Kyte-Doolittle plot, PS1 has ten hydrophobic regions (HR) (5), but it is unclear how many of these cross the lipid bilayer as transmembrane domains (TMDs) (6). A widely accepted model proposes eight TMDs (HR I to VI, VIII, and IX) with the NH 2 -COOH terminus and the hydrophilic loop domain between TMD 6 and 7, all facing the cytosol (Fig. 1A). All published models agree that the first six HR cross the membrane, implying a consensus for the topology of the PS1-NTF. In contrast, divergent proposals exist for the number of TMDs in the PS1-CTF (7-10). Several of these models are difficult to reconcile with the different physiological roles assigned to PS1, such as the location of the aspartate residues in HRVI and VIII or the cytosolic-oriented loop domain required for ␤-catenin binding (4, 11). Knowledge of the exact topology of PS1 is therefore of pivotal importance to understanding its multiple roles.In this report, we revisited the trafficking and topology of PS1 using glycosylation consensus sequences inserted at different positions in human PS1 (hPS1). Expression of these mutants in PS1 and 2 knock-out (KO) mouse embryonic fibroblasts (MEFs) allowed us to evaluate the glycosylation status of these variants, and hence the topology, without interference of endogenous PS. Combined with a cysteine derivation strategy we provide strong evidence f...

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