A Nine-transmembrane Domain Topology for Presenilin 1

Abstract

Presenilin (PS) provides the catalytic core of the ␥-secretase complex. ␥-Secretase activity leads to generation of the amyloid ␤-peptide, a key event implicated in the pathogenesis of Alzheimer disease. PS has ten hydrophobic regions, which can all theoretically form membrane-spanning domains. Various topology models have been proposed, and the prevalent view holds that PS has an eighttransmembrane (TM) domain organization; however, the precise topology has not been unequivocally determined. Previous topological studies are based on non-functional truncated variants of PS proteins fused to reporter domains, or immunocytochemical staining. In this study, we used a more subtle N-linked glycosylation scanning approach, which allowed us to assess the topology of functional PS1 molecules. Glycosylation acceptor sequences were introduced into full-length human PS1, and the results showed that the first hydrophilic loop is oriented toward the lumen of the endoplasmic reticulum, whereas the N terminus and large hydrophilic loop are in the cytosol. Although this is in accordance with most current models, our data unexpectedly revealed that the C terminus localized to the luminal side of the endoplasmic reticulum. Additional studies on the glycosylation pattern after TM domain deletions, combined with computer-based TM protein topology predictions and biotinylation assays of different PS1 mutants, led us to conclude that PS1 has nine TM domains and that the C terminus locates to the lumen/extracellular space.Most proteases operate in aqueous environments, but ␥-secretase, along with other intramembrane cleaving proteases, is unusual in the sense that it executes proteolysis in the lipid bilayer (1). ␥-Secretase is a high molecular weight protein complex composed of presenilin (PS) 2 1 or 2, nicastrin, Aph-1, and Pen-2 (2-5). Growing evidence suggests that the polytopic PS1 protein is a TM aspartyl protease and that two of its aspartate residues, Asp-257 in hydrophobic domain (HD) 6 and Asp-385 in HD 8, form the catalytic site of the ␥-secretase complex (6 -8). An endoproteolytic cleavage within HD 7 of PS1 generates an N-terminal fragment (NTF) and a C-terminal fragment (CTF) (9). The NTF/ CTF heterodimer is believed to be the biologically active form of PS. Nicastrin, Aph-1, and Pen-2 are all essential cofactors in the ␥-secretase complex, but their precise biochemical functions are not fully known. Nicastrin and Aph-1 have been proposed to form a subcomplex that can stabilize full-length PS, and the final addition of Pen-2 has been suggested to trigger PS endoproteolysis (10) and stabilize the NTF/CTF heterodimer (11). ␥-Secretase cleavage occurs within the lipid bilayer of the membrane through a mechanism referred to as "regulated intramembrane proteolysis" (12). Recently, not only APP, but a large number of other type-1 TM proteins, such as the Notch receptors, CD44 and E-cadherin, have been shown to undergo PS-dependent regulated intramembrane proteolysis (reviewed in Ref. 13). Given that target proteins of ␥-secret...

Deducing the Transmembrane Domain Organization of Presenilin-1 in γ-Secretase by Cysteine Disulfide Cross-Linking

“…Human PS1 contains 5 native cysteines. According to currently accepted PS1 topology (17,18), 3 cysteines are in transmembrane domains and 2 are exposed to cytosol ( Fig. 2A).…”

Presenilin Transmembrane Domain 8 Conserved AXXXAXXXG Motifs Are Required for the Activity of the γ-Secretase Complex

“…The assembly of the ␥-secretase complex requires the association of either PS1 or PS2 with three other membrane proteins: nicastrin (NCT), anterior pharynx defective 1 (APH-1a or APH-1b), and presenilin enhancer 2 (PEN-2) (13,14). Presenilins have nine transmembrane domains (TMDs) (15), and the two catalytic aspartic acid residues are located on its TMD6 and -7, both required for substrate hydrolysis. ␥-Secretase assembly is accompanied by an activating step in which presenilin is endoproteolysed and converted into a heterodimer formed by its N-and C-terminal fragments (PS-NTF/PS-CTF) (16 -18).…”

Evidence That the “NF” Motif in Transmembrane Domain 4 of Presenilin 1 Is Critical for Binding with PEN-2

“…The most widely accepted topology model for PS1 is one in which the molecule spans the membrane eight times with amino and carboxyl termini as well as the large hydrophilic loop between TMDs 6 and 7 being cytosolic (21,22). However, recent studies have offered support for a 9-TMD model for PS1 in which the carboxyl terminus is luminal (23). Notwithstanding the differences in the topology models, PS1 endoproteolysis unequivocally occurs within the large hydrophilic loop to generate stable NTF and CTF derivatives.…”