Alzheimer Disease Aβ Production in the Absence of S-Palmitoylation-dependent Targeting of BACE1 to Lipid Rafts

doi:10.1074/jbc.M808920200

Alzheimer Disease Aβ Production in the Absence of S-Palmitoylation-dependent Targeting of BACE1 to Lipid Rafts^*

^‡Departments of Neurobiology, Neurology, and Pathology, The University of Chicago, Chicago, Illinois 60637, ^§TorreyPines Therapeutics, Inc., La Jolla, California 92037, the ^¶Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada, the ^∥Department of Cell and Molecular Biology, The Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, the ^**Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and the ^‡‡Division of Membrane Physiology, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki 444-8787, Japan

2 To whom correspondence should be addressed: Knapp R212, 924 E. 57th St., Chicago, IL 60637. Tel.: 773-834-3752; Fax: 773-834-3808; E-mail: gopal{at}uchicago.edu.

Abstract

Alzheimer disease β-amyloid (Aβ) peptides are generated via sequential proteolysis of amyloid precursor protein (APP) by BACE1 and γ-secretase. A subset of BACE1 localizes to cholesterol-rich membrane microdomains, termed lipid rafts. BACE1 processing in raft microdomains of cultured cells and neurons was characterized in previous studies by disrupting the integrity of lipid rafts by cholesterol depletion. These studies found either inhibition or elevation of Aβ production depending on the extent of cholesterol depletion, generating controversy. The intricate interplay between cholesterol levels, APP trafficking, and BACE1 processing is not clearly understood because cholesterol depletion has pleiotropic effects on Golgi morphology, vesicular trafficking, and membrane bulk fluidity. In this study, we used an alternate strategy to explore the function of BACE1 in membrane microdomains without altering the cellular cholesterol level. We demonstrate that BACE1 undergoes S-palmitoylation at four Cys residues at the junction of transmembrane and cytosolic domains, and Ala substitution at these four residues is sufficient to displace BACE1 from lipid rafts. Analysis of wild type and mutant BACE1 expressed in BACE1 null fibroblasts and neuroblastoma cells revealed that S-palmitoylation neither contributes to protein stability nor subcellular localization of BACE1. Surprisingly, non-raft localization of palmitoylation-deficient BACE1 did not have discernible influence on BACE1 processing of APP or secretion of Aβ. These results indicate that post-translational S-palmitoylation of BACE1 is not required for APP processing, and that BACE1 can efficiently cleave APP in both raft and non-raft microdomains.

Footnotes

↵3 The abbreviations used are: Aβ, β-amyloid; APP, amyloid precursor protein; BACE1, β-site APP cleaving enzyme 1; CTF, C-terminal fragment; DHHC, Asp-His-His-Cys; DRM, detergent-resistant membrane; ELISA, enzyme-linked immunosorbent assay; FL, full-length; GPI, glycosylphosphatidylinositol; mAb, monoclonal antibody; MEF, mouse embryonic fibroblasts; PAT, protein acyltransferase; PLAP, placental alkaline phosphatase; PS, presenilin(s); WT, wild-type; IRES, internal ribosome entry site; TGN, trans-Golgi network; 3C/A, C478A/C482A/C485A; 4C/A, C474A/C478A/C482A/C485A.
↵* This work was supported, in whole or in part, by National Institutes of Health Grants AG021495 and AG019070 (to G. T.). This work was also supported by Alzheimer's Association Grants IIRG (to G. T.) and NIRG (to K. S. V.), Canadian Institutes of Health Research Grant MOP 44363 (to N. G. S.), and an American Federation for Aging Research Fellowship (to X. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and additional data.
↵1 Both authors contributed equally to this study.
- Received November 25, 2008.
- Revision received December 12, 2008.
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