Association of γ-Secretase with Lipid Rafts in Post-Golgi and Endosome Membranes

Kulandaivelu S Vetrivel,Haipeng Cheng,William Lin,Takashi Sakurai,Tong Li,Nobuyuki Nukina,Philip C Wong,Huaxi Xu,Gopal Thinakaran

doi:10.1074/jbc.m407986200

Abstract

Alzheimer's disease-associated beta-amyloid peptides (Abeta) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases. There is growing evidence that cholesterol- and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major beta-secretase in neurons is a palmitoylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the gamma-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1(-/-)/PS2(-/-) and NCT(-/-) fibroblasts, gamma-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires gamma-secretase complex assembly. Biochemical evidence shows that subunits of the gamma-secretase complex and three TGN/endosome-resident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1- or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of gamma-secretase in lipid raft microdomains of post-Golgi and endosomes, organelles previously implicated in amyloidogenic processing of APP.

Highlights

Alzheimer’s disease-associated ␤-amyloid peptides (A␤) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by ␤- and ␥-secretases
APH-1 is presumed to have a role in the initial assembly and maturation of PS-nicastrin complexes [14], while PEN-2 is required for endoproteolytic processing of protein; PS, presenilin(s); NTF, N-terminal fragment; C-terminal fragments (CTF), C-terminal fragment; ER, endoplasmic reticulum; TGN, trans-Golgi network; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; detergent insoluble membrane (DIM), detergent-insoluble membrane; GTEL, Golgi/ TGN/endosomes/lysosome-enriched membranes; MES, 4-morpholineethanesulfonic acid; SNARE, soluble NSF attachment protein receptors; VAMP, vesicle-associated membrane protein; M␤CD, methyl-␤cyclodextrin; GPI, glycosylphosphatidylinositol
By subcellular fractionation, confocal microscopy, and immunoisolation methods, we show that endogenous presenilin 1 (PS1), nicastrin, APH-1, and PEN-2 associate with raft microdomains in post-Golgi and endosome membranes enriched in syntaxin 6, syntaxin 13, and VAMP4

Summary

The abbreviations used are

A␤ is generated by the sequential processing of amyloid precursor protein (APP) by ␤- and ␥-secretases [1, 2]. A recent study reported that ␥-secretase cleavage occurs in lipid rafts, but ␥-secretase catalytic activity is independent of the presence of cholesterol [38] It appears that APP processing within cholesterol-rich lipid rafts by secretases, and not the cholesterol levels per se, determines the levels of A␤ production. By subcellular fractionation, confocal microscopy, and immunoisolation methods, we show that endogenous PS1, nicastrin, APH-1, and PEN-2 associate with raft microdomains in post-Golgi and endosome membranes enriched in syntaxin 6, syntaxin 13, and VAMP4. These findings provide novel insights into the subcellular localization of ␥-secretase, which has important implications in understanding the regulation of amyloidogenic processing of APP

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION