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. 2004 Oct 22;279(43):44945-54.
doi: 10.1074/jbc.M407986200. Epub 2004 Aug 17.

Association of gamma-secretase with lipid rafts in post-Golgi and endosome membranes

Kulandaivelu S Vetrivel  1 , Haipeng Cheng, William Lin, Takashi Sakurai, Tong Li, Nobuyuki Nukina, Philip C Wong, Huaxi Xu, Gopal Thinakaran
Affiliations

Association of gamma-secretase with lipid rafts in post-Golgi and endosome membranes

Kulandaivelu S Vetrivel et al. J Biol Chem. .

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.

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Figures

Fig. 1
Fig. 1. Analysis of the detergent solubility characteristics of PS1 and nicastrin
N2aWt.11 cells were solubilized either in 0.5% Brij 96 (A), 1% CHAPSO (B), 0.5% Lubrol WX (C), or 1% Triton X-100 (D) at 4 °C for 30 min. The lysates were then subject to flotation centrifugation on discontinuous sucrose gradients as described under “Experimental Procedures.” The gradients were harvested from the top, and the distribution of PS1 and nicastrin were determined by fractionating 60-μl aliquots of gradient fractions (1–12; top to bottom) on SDS gels followed by Western blot analysis. Flotillin-2 and calnexin were analyzed in parallel as a marker to indicate DRM and detergent-soluble fractions, respectively.
Fig. 2
Fig. 2. DIM association of γ-secretase components is sensitive to cholesterol depletion
N2aWt.11 cells were treated or not with 5 mm MβCD for 2 h at 37 °C, lysed in 0.5% Lubrol WX, and analyzed by flotation on sucrose gradients. DIM localization of γ-secretase components, and raft-associated proteins PrP and flotillin-2, were assessed by Western blotting.
Fig. 3
Fig. 3. DIM localization of endogenous γ-secretase components in neuronal and non-neuronal cells
A, mouse embryonic fibroblasts were lysed in 0.5% Lubrol WX and subjected to flotation sucrose density gradient centrifugation. Equal volumes of each fraction was analyzed by Western blotting with antibodies against PS1, nicastrin and PEN-2. Caveolin, flotillin-1, and flotillin-2 mark low buoyant density lipid raft fractions. Non-raft proteins residing in the ER (BiP and calnexin), Golgi (P115), and the TGN (γ-adaptin) (TGN) are recovered in heavier fractions. B, mouse primary neurons were subject to sucrose density gradient fractionation and analyzed as above using anti-PS1 loop, mAb anti-nicastrin, and PrP antibodies. Lipid raft fractions were identified by the presence of GPI-anchored PrP protein as the marker.
Fig. 4
Fig. 4. γ-Secretase components are recruited in Lubrol WX DIM following their assembly
A, PS1−/−/PS2−/− fibroblasts were solubilized in 0.5% Lubrol WX and subject to sucrose gradient fractionation. In the absence of PS1 and PS2, immature nicastrin fails to become Lubrol WX-resistant and were recovered in non-raft fractions. B, wild-type and NCT−/− fibroblasts stably transfected with an empty vector (Vec) or human wild-type nicastrin cDNA were lysed and fractionated on a sucrose gradients. Raft (fractions 4 and 5) and non-raft fractions 8–12 were pooled, methanol/chloroform-precipitated, and analyzed by Western blotting. In the absence of nicastrin, APH-1, and PEN-2 remain in the non-raft fractions. Introduction of nicastrin in NCT−/− fibroblasts restores raft association of γ-secretase components.
Fig. 5
Fig. 5. Subcellular localization of endogenous PS1 by confocal microscopy
HeLa cells were grown on coverslips were fixed and analyzed by double immunofluorescence staining with PS1 N-terminal antiserum Ab14, and antibodies raised against resident proteins of the ER (anti-KDEL), cis-Golgi (GM130), TGN (γ-adaptin), TGN/TGN-derived vesicles (syntaxin 6), plasma membrane (Na+/K+-ATPase), or early endosomes (EEA1). Note the colocalization between PS1 (green) and Golgi/TGN markers (red). Scale bar, 10 μm.
Fig. 6
Fig. 6. Subcellular fractionation of γ-secretase components in sucrose gradients
N2aWt.11 cells were grown to confluency, and cells were lysed using a ball-bearing homogenizer and fractionated by velocity sedimentation. A, equal volume aliquots of fractions were analyzed by immunoblotting using antibodies against the following organelle-specific markers: calnexin (ER), GM130 (Golgi), syntaxin 6, and VAMP4 (TGN/TGN-derived vesicles), Na+/K+-ATPase (plasma membrane), syntaxin 13 (late endosome), and LAMP-1 (lysosome). Ganglioside GM1 distribution in the gradient fractions was detected by binding of cholera toxin followed by incubation with anti-cholera toxin antiserum. Note that significant amounts of PS1 NTF, nicastrin, and PEN-2 are present in fraction 6, which is enriched in Golgi, TGN, endosome, and lysosome markers. An asterisk indicates nonspecific protein (migrating faster than immature nicastrin polypeptide) in fractions 1 and 2 that is reactive with nicastrin antibody. B, signal intensities of PS1 NTF, mature nicastrin and PEN-2 were quantified, and the relative distribution in each of the sucrose density fractions is plotted as % of total intensity for each protein. Note that the second peak for PEN-2 (fraction 11) likely represents nascent PEN-2 that is in the process of assembly with full-length PS1 and immature nicastrin (also abundant in fraction 11) within the ER.
Fig. 7
Fig. 7. Raft association of γ-secretase components in GTEL-enriched membranes
N2aWt.11 cells grown to confluency were lysed and fractionated on sucrose gradients as described under legend to Fig. 5. Membranes in fraction 6 were pooled from four gradients. Lipid rafts from these GTEL-enriched membranes were isolated by solubilization in 0.5% Lubrol WX and subject to sucrose gradient fractionation. Aliquot of each fraction were subject to SDS-PAGE and Western blot analyses. Raft fractions were identified by the presence of lipid raft markers flotillin-2 and GM1. Mature components of γ-secretase complex, syntaxin 6, syntaxin 13, and VAMP4 partition into DIM fractions, whereas GM130, γ-adaptin, and LAMP-1 remain Lubrol WX-soluble.
Fig. 8
Fig. 8. Syntaxin 6, syntaxin 13, and VAMP4 display detergent solubility characteristics similar to γ-secretase components
N2aWt.11 cells were lysed either in 0.5% Brij 96, 1% CHAPSO, 0.5% Lubrol WX, or 1% Triton X-100 at 4 °C for 30 min and fractionated on discontinuous sucrose gradients. Aliquots of fractions (1–12; from top to bottom) were resolved on SDS-PAGE and analyzed by Western blotting.
Fig. 9
Fig. 9. Co-residence of γ-secretase components in syntaxin 6, syntaxin 13 and VAMP4 containing raft domains revealed by immunoisolation
A and B, N2aWt.11 cells were solubilized in 0.5% Lubrol WX and fractionated for raft isolation on sucrose gradients. Raft fractions (4 and 5) were pooled and an aliquot was incubated with magnetic beads coated with monoclonal syntaxin 6 antibody, OKT8 (negative control), affinity-purified polyclonal PS1 antibodies, or STC2 antibody (negative control). Bound DIM were analyzed by Western blotting using indicated antibodies. An aliquot of the input (1/30th volume) was also separated in the same gel for comparison. C, N2a cells were lysed by a non-detergent method (0.5 m sodium carbonate/sonication) and analyzed by flotation on sucrose gradients. DIM localization of PS1 NTF/CTF and nicastrin was assessed by Western blotting. Raft and non-raft fractions were identified by blotting with flotillin-2 and GM130 antibodies, respectively. D, non-detergent raft fractions were incubated with magnetic beads coated with syntaxin 6 or OKT8 antibody and immunoisolated membranes were analyzed by Western blotting using indicated antibodies.
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References

    1. Selkoe DJ. Physiol Rev. 2001;81:741–766. - PubMed
    1. Sisodia SS, St George-Hyslop PH. Nat Rev Neurosci. 2002;3:281–290. - PubMed
    1. Vassar R. J Mol Neurosci. 2004;23:105–114. - PubMed
    1. Iwatsubo T. Curr Opin Neurobiol. 2004;14:379–383. - PubMed
    1. Price DL, Tanzi RE, Borchelt DR, Sisodia SS. Annu Rev Genet. 1998;32:461–493. - PubMed

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