Abstract
Proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases generates beta-amyloid (Abeta) peptides, which accumulate in the brains of individuals affected by Alzheimer disease. Detergent-resistant membrane microdomains (DRM) rich in cholesterol and sphingolipid, termed lipid rafts, have been implicated in Abeta production. Previously, we and others reported that the four integral subunits of the gamma-secretase associate with DRM. In this study we investigated the mechanisms underlying DRM association of gamma-secretase subunits. We report that in cultured cells and in brain the gamma-secretase subunits nicastrin and APH-1 undergo S-palmitoylation, the post-translational covalent attachment of the long chain fatty acid palmitate common in lipid raft-associated proteins. By mutagenesis we show that nicastrin is S-palmitoylated at Cys(689), and APH-1 is S-palmitoylated at Cys(182) and Cys(245). S-Palmitoylation-defective nicastrin and APH-1 form stable gamma-secretase complexes when expressed in knock-out fibroblasts lacking wild type subunits, suggesting that S-palmitoylation is not essential for gamma-secretase assembly. Nevertheless, fractionation studies show that S-palmitoylation contributes to DRM association of nicastrin and APH-1. Moreover, pulse-chase analyses reveal that S-palmitoylation is important for nascent polypeptide stability of both proteins. Co-expression of S-palmitoylation-deficient nicastrin and APH-1 in cultured cells neither affects Abeta40, Abeta42, and AICD production, nor intramembrane processing of Notch and N-cadherin. Our findings suggest that S-palmitoylation plays a role in stability and raft localization of nicastrin and APH-1, but does not directly modulate gamma-secretase processing of APP and other substrates.
Highlights
A growing number of type I integral membrane proteins has been identified as ␥-secretase substrates within the last few years, including Notch1 homologues, Notch ligands, Delta and JANUARY 16, 2009 VOLUME 284 NUMBER 3
By direct metabolic labeling and a highly sensitive acyl thiol exchange labeling method [39], we demonstrate that nicastrin and APH-1 undergo S-palmitoylation in cultured cells and in the brain
By expression of S-palmitoylation-deficient nicastrin and APH-1 in NCTϪ/Ϫ and APH-1acϪ/Ϫ mouse embryonic fibroblasts (MEF), respectively, we show that this modification is not essential for ␥-secretase complex formation
Summary
Plasmids—cDNAs encoding nicastrin mutant C689S, and APH-1 mutants C182S, C245S, and C182S/C245S were generated by overlap PCR, and all PCR products were verified by sequencing. Rabbit polyclonal antibody A1tag was raised against a synthetic peptide corresponding to the C-terminal 15 amino acids of APH-1aL followed by residues SSRGPSSAEVLLLPVS. For biotin-BMCC labeling of nicastrin, cells were lysed in methylthiolation buffer (20 mM Tris-HCl, pH 7.7, 2.5% SDS, 20 mM S-methylmethanethiosulfonic acid, 1 mM EDTA, and 0.1 mM neocuproine). For [3H]palmitic acid labeling, transfected HEK293 cells were starved for 30 min in Dulbecco’s modified Eagle’s medium containing 1% dialyzed fetal bovine serum and incubated with 100 Ci/ml of [10, 11-3H]palmitic acid (American Radiolabeled Chemicals) for 6 h. NCTϪ/Ϫ cells overexpressing WT or mutant nicastrin and APH-1 in 60-mm dishes were infected with retrovirus encoding APPSwe and harvested 48 h later in buffer A (50 mM HEPES, 150 mM NaCl, 5 mM 1,10-phenanthroline monohydrate, pH 7.4). The samples were centrifuged at 10,000 ϫ g for 15 min and the supernatant containing soluble proteins was collected and analyzed by Western blotting
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
