Abstract
Gamma-secretase cleaves type I transmembrane proteins, including beta-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 gamma-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-1.nicastrin subcomplex with presenilin that mediates trafficking of the presenilin.Aph-1.nicastrin 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-1.nicastrin subcomplex with presenilin is the prerequisite for the trafficking as well as the enzymatic activity of the gamma-secretase complex and that Aph-1 functions as a stabilizing scaffold in the assembly of this complex.
Highlights
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
Genetic studies in Caenorhabditis elegans and Drosophila melanogaster have revealed two additional polytopic membrane proteins, Aph-1 and Pen-2, that are required for the ␥-secretase activity mic reticulum; FL, full-length or holoprotein; GFP, green fluorescent protein; HMW, high molecular weight; mt, mutant; Nct, nicastrin; RNAi, double-stranded RNA-mediated interference; SC100, the carboxyl-terminal 99 amino acid fragment of APP fused to a signal peptide of rat preproenkephalin cDNA; TGN, trans-Golgi network; TMD, transmembrane domain; UTR, untranslated region; wt, wild-type
UTR-targeted RNAi in S2 cells expressing SC100, the carboxyl-terminal 99amino acid fragment of APP fused to a signal peptide of rat preproenkephalin cDNA, decreased the levels of Psn fragments in a similar manner to that observed in cells treated with coding sequence (CDS)-targeted RNAi (Fig. 1)
Summary
Mutations in presenilin (PS) genes account for the majority of early onset familial Alzheimer’s disease cases, causing an overproduction of those amyloid  peptides (A) ending at. Solubilized ␥-secretase activity is recovered in HMW fractions associated with PS fragments, and transition state analogue ␥-secretase inhibitors covalently label PS fragments but not PS holoproteins [11,12,13] These results suggest that the stabilized HMW PS complex harbors ␥-secretase activity and that the paired intramembrane aspartates in PS fragments serve as the active center of the ␥-secretase, which functions as a novel type of membrane-bound aspartic protease. Genetic studies in Caenorhabditis elegans and Drosophila melanogaster have revealed two additional polytopic membrane proteins, Aph-1 and Pen-2, that are required for the ␥-secretase activity mic reticulum; FL, full-length or holoprotein; GFP, green fluorescent protein; HMW, high molecular weight; mt, mutant; Nct, nicastrin; RNAi, double-stranded RNA-mediated interference; SC100, the carboxyl-terminal 99 amino acid fragment of APP fused to a signal peptide of rat preproenkephalin cDNA; TGN, trans-Golgi network; TMD, transmembrane domain; UTR, untranslated region; wt, wild-type. It was reported that Aph-1 preferentially interacts with immature Nct to form an intermediate subcomplex during the early biosynthetic process of ␥-secretase in mammalian cells [25,26,27], suggesting that the Aph-11⁄7Nct subcomplex functions as a stabilization factor for the ␥-secretase complex
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