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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.

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Flu is caused by the influenza virus that, due to mutations, keeps our body vulnerable for infections, making early diagnosis essential. Although immuno-based diagnostic tests are available, they have low sensitivity and reproducibility. In this paper, the prospect of detecting influenza A virus using digital ELISA has been studied. To appropriately select bioreceptors for this bioassay, seven commercial antibodies against influenza A nucleoprotein were methodically tested for their reactivity and binding affinity. The study has been performed on two markedly different platforms, being an enzyme-linked immunosorbent assay and a surface plasmon resonance system. The selected antibodies displayed completely different behavior on the two platforms and in various assay configurations. Surprisingly, the antibodies that showed overall good reactivity on both platforms had the highest dissociation constant among the tested antibodies, suggesting that, although important, binding affinity is not the only parameter to be considered when selecting antibodies. Moreover, only one antibody had the capacity to capture the nucleoprotein directly in lysis buffer used for releasing this viral protein, which might pose a huge advantage when developing assays with a fast time-to-result. This antibody was implemented on an in-house developed digital ELISA platform for ultrasensitive detection of recombinant nucleoprotein, reaching a detection limit of 4 ± 1 fM in buffer and 10 ± 2 fM in 10-fold diluted nasopharyngeal swabs, which is comparable to currently available fast molecular detection techniques. These results point to a great potential for ultrasensitive immuno-based influenza detection.

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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...

occurs under tight control of ER-Golgi recycling regulators, which allows defined quantities of complexes to reach postGolgi compartments, where ␥-secretase activity is regulated by multiple other factors. 3D-EM rendering reveals a complex with a translucent inner space, suggesting the presence of a water-filled cavity required for intramembrane proteolysis. Despite huge efforts, we are now only beginning to unravel the assembly, stoichiometry, activation and subcellular location of ␥-secretase. Presenilin at the heart of ␥-secretase Mammalian PSs come in two flavours, PS1 and PS2, which are highly homologous. Both are polytopic membrane proteins and have ten hydrophobic domains, of which nine are proposed to span the membrane (Laudon et al., 2005;Oh and Turner, 2005;Spasic et al., 2006) (Fig. 1). PSs are initially translocated in the ER as fulllength proteins but get converted by endoproteolysis within hydrophobic domain 7 to stable N-and C-terminal fragments (NTFs and CTFs, respectively) that associate to form a heterodimer. Although both fragments are part of the catalytic ␥-secretase, endoproteolysis is not a requirement for activity (Li et al., 2000;Steiner et al., 1999) (reviewed in Dillen and Annaert, 2006).Two highly conserved aspartate residues (Asp257 and Asp385 in human PS1) within transmembrane domain 6 (TMD6) and TMD7 constitute the core of the catalytic site. Mutation of either abolishes ␥-secretase activity Wolfe et al., 1999). Together with surrounding residues, they mark a highly conserved YD/GxGD consensus motif (Steiner et al., 2000). Tyr389 may also contribute to the catalytic site . The conformation of the active site also depends on more remote sequences within PS1, such as the C-terminal PAL motif and Cys residues in TMD1 and TMD8 (Kornilova et al., 2006). Binding of an active-site-directed inhibitor prevents the disulphide cross-linking between TMD1 and TMD8, implicating their close proximity to or allosteric interaction with the catalytic site. Finally, catalytic activity must be sensitive to subtle changes in the overall TMD conformation, given the effects of the scattered FAD-linked mutations.These interactions between remote parts of the molecule fit with the idea that PS1 adopts a ring-like topology (Annaert et al., 2001). This model is based on the identification of APP-binding regions in PS1 -TMD1 (extending to part of the first luminal loop domain) and the C-terminus -and led to the idea that the substrate must first bind to a remote docking site at the heterodimer interface prior to entering the active site. Indeed, subsequent imaging and biochemical studies have demonstrated that transition-state ␥-secretase inhibitors that bind to PS1 do not affect the interaction between PS1 and the APP C-terminal fragment (Berezovska et al., 2003; Esler et al., 2002). Similarly, inhibitors based on the substrate TMD do not prevent labelling of the complex by active-sitedirected inhibitors (Kornilova et al., 2003). Interestingly, these peptide inhibitors label only PS heterodimers and not th...