Aph-1 Contributes to the Stabilization and Trafficking of the γ-Secretase Complex through Mechanisms Involving Intermolecular and Intramolecular Interactions

Abstract

␥-Secretase cleaves type I transmembrane proteins, including ␤-amyloid precursor protein and Notch, and requires the formation of a protein complex comprised of presenilin, nicastrin, Aph-1, and Pen-2 for its activity. Aph-1 is implicated in the stabilization of this complex, although its precise mechanistic role remains unknown. Substitution of the first glycine within the transmembrane GXXXG motif of Aph-1 causes a loss-of-function phenotype in Caenorhabditis elegans. Here, using an untranslated region-targeted RNA interference/rescue strategy in Drosophila Schneider 2 cells, we show that Aph-1 contributes to the assembly of the ␥-secretase complex by multiple mechanisms involving intermolecular and intramolecular interactions depending on or independent of the conserved glycines. Aph-1 binds to nicastrin forming an early subcomplex independent of the conserved glycines within the endoplasmic reticulum. Certain mutations in the conserved GXXXG motif affect the interaction of the Aph-1⅐nicastrin subcomplex with presenilin that mediates trafficking of the presenilin⅐Aph-1⅐nicas-trin tripartite complex to the Golgi. The same mutations decrease the stability of Aph-1 polypeptides themselves, possibly by affecting intramolecular associations through the transmembrane domains. Our data suggest that the proper assembly of the Aph-1⅐nicastrin subcomplex with presenilin is the prerequisite for the trafficking as well as the enzymatic activity of the ␥-secretase complex and that Aph-1 functions as a stabilizing scaffold in the assembly of this complex. Mutations in presenilin (PS)1 genes account for the majority of early onset familial Alzheimer's disease cases, causing an overproduction of those amyloid ␤ peptides (A␤) ending at position 42 (A␤42) that most readily form amyloid deposits (1). A␤ is derived from the ␤-amyloid precursor protein (␤APP) through sequential cleavages by ␤-and ␥-secretases (2). Genetic and biochemical studies suggest that PS is essential for the ␥-secretase-mediated intramembrane cleavage of ␤APP. Additional type 1 transmembrane proteins (e.g. notch, ErbB4, and CD44) also are cleaved by ␥-secretase to release intracellular domains that harbor biological activities (3). These data suggest that PS-dependent ␥-cleavage is involved in a novel mode of intramembrane proteolysis-dependent signal transduction.PS is a highly conserved, polytopic integral membrane protein that spans the membrane eight times and undergoes endoproteolysis to generate amino-and carboxyl-terminal fragments (reviewed in Ref. 4). The endoproteolytic fragments of PS form a heterodimer and are then incorporated into a highly stabilized, high molecular weight (HMW) protein complex, whereas the PS holoprotein forms a low molecular weight complex and is rapidly degraded (5, 6). We have shown by a systematic mutational analysis that the stabilization and HMW complex formation of PS are required for ␥-secretase activity, which is mediated by the carboxyl terminus of PS (7-9). PS carries two highly conserved intramembrane aspartates ...

SRG3 Interacts Directly with the Major Components of the SWI/SNF Chromatin Remodeling Complex and Protects Them from Proteasomal Degradation

“…TAF1, one of the subunits of the TFIID holocomplex, has been reported to be a scaffold component of TFIID and contributes to stability of the complex (44). In addition, Aph-1, one component of the ␥-secretase complex that cleaves type I transmembrane proteins, functions as a scaffold and stabilizing component of the assembly of the complex (45).…”

An alternative interpretation of the amyloid Aβ hypothesis with regard to the pathogenesis of Alzheimer's disease

“…Recent reports highlight the functional importance of GxxxG motifs in the TM segments of APh1 (26,27), and these findings raise the possibility that A␤ peptides might also affect the proteolytic processing of APP molecules by interfering with the assembly of the presenilin1͞␥-secretase complex. The sequences of the two isoforms of Aph-1 that contain the GxxxG motifs are shown in Fig.…”

“…The role of Aph1 as a necessary component of the PS1͞␥-secretase activity was first discovered in Caenorhabditis elegans (26), and a mutant form, which had a loss-of-function phenotype, was found to have an amino acid substitution (aspartic) for a critical glycine residue in the fourth transmembrane segment of the molecule. Follow-up studies confirmed that mutated glycines in GxxxG domains of mammalian cells block the ability of APH-1͞Nicastrin subcomplexes to stabilize the PS1͞␥-secretase complex (27). One has to ask whether excessive amounts of A␤ peptides, which contain similar GxxxG domains, can modify in some way the capacity of GxxxG-containing segments of Aph1 to assemble functional multiprotein complexes.…”

Nicastrin Is Dispensable for γ-Secretase Protease Activity in the Presence of Specific Presenilin Mutations

“…On the other hand, Pen2 triggers the endoproteolysis of PS into amino-and carboxyl-terminal fragments (called NTF and CTF, respectively) as a part of the maturation of the protease complex (9). Aph-1 is thought to be a scaffold for the assembly and contributes to the stability of the entire complex and its trafficking to the Golgi apparatus (10). The interactions between the subunits and the arrangement of PS1 transmembrane domains were partly understood from biochemical analyses.…”