The Transmembrane Domain of the Alzheimer's β-Secretase (BACE1) Determines Its Late Golgi Localization and Access to β-Amyloid Precursor Protein (APP) Substrate

Abstract

Release of A␤ peptides from ␤-amyloid precursor protein (APP) requires sequential cleavage by two endopeptidases, ␤-and ␥-secretases. ␤-Secretase was recently identified as a novel membrane-bound aspartyl protease, named BACE1, Asp2, or memapsin 2. Employing confocal microscopy and subcellular fractionation, we have found that BACE1 is largely situated in the distal Golgi membrane with a minor presence in the endoplasmic reticulum, endosomes, and plasma membrane in human neuroblastoma SHEP cells and in mouse Neuro-2a cell lines expressing either endogenous mouse BACE1 or additional exogenous human BACE1. The major cellular ␤-secretase activity is located in the late Golgi apparatus, consistent with its cellular localization. Furthermore, we demonstrate that the single transmembrane domain of BACE1 alone determines the retention of BACE1 to the Golgi compartments, through examination of recombinant proteins of various BACE1 fragments fused to a reporter green fluorescence protein. In addition, we show that the transmembrane domain of BACE1 is required for the access of BACE1 enzymatic activity to the cellular APP substrate and hence for the optimal generation of the C-terminal fragment of APP (CTF99). The results suggest a molecular and cell biological mechanism for the regulation of ␤-secretase activity in vivo.The pathological hallmarks of Alzheimer's disease are neuritic plaques and neurofibrillary tangles (see reviews in Refs. 1-3). The neuritic plaques, also known as senile plaques, are predominantly composed of A␤, a cluster of aggregated and heterogeneous peptides of 39 -43 amino acids (4). The most pathogenic A␤ peptide is the less soluble 42-amino acid peptide (A␤ 42 ), although the concentration of A␤ 40 is generally much higher than A␤ 42 . Excision of A␤ peptides from their amyloid precursor protein requires sequential proteolytic cleavages by the ␤-and ␥-secretases, respectively. ␤-secretase cleaves APP 1 to produce two major components: secreted ectodomain sAPP ␤ and the C-terminal fragment CTF99. The latter can be further cleaved by ␥-secretase to release A␤. Genetic linkage analysis suggests that increased total A␤ levels or an increase in the ratio of A␤ 42 /A␤ 40 is associated with the severity of AD dementia (reviewed in Refs.

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

“…The polyclonal antibody 7523 did not show any background staining in BACE1 Ϫ/Ϫ MEF transduced with an empty retroviral vector. In agreement with previous reports (16,58), analysis of BACE1 Ϫ/Ϫ MEF stably expressing wtBACE1 revealed predominant co-localization of wtBACE1 with ␥-adaptin and transferrin receptor, which are markers of the TGN and endosomes, respectively (Fig. 2, A and B).…”

Cleavage of Neuregulin-1 by BACE1 or ADAM10 Protein Produces Differential Effects on Myelination

“…Consistent with our expectations, the ADAM10-cleaved Nrg1 C-terminal fragment (Nrg1-ctf␣) migrated slower on the gels because Nrg1-ctf␣ is slightly longer than BACE1-cleaved Nrg1-ctf␤. Type I Nrg1 was chosen for this extensive characterization due to the structural similarity to APP, and constructs expressing either wild-type Nrg1 or type I Nrg1 variants with mutations surrounding the BACE1 cleavage site were generated for analyses in HM cells, which were generated by stably expressing HA-tagged BACE1 in HEK-293 cells (17). We observed that the C-terminal fragments of wild-type Nrg1, detected by an antibody recognizing the Nrg1 C terminus, could be separated on Western blots under our optimized conditions (Fig.…”

“…A cell line stably expressing BACE1 (HM cell line) was previously generated by expressing HA-tagged BACE1 in HEK-293 cells (17). Anti-myelin basic protein (MBP) antibody was purchased from Sternberger Monoclonal Inc. (Lutherville, MD).…”

Mint3/X11γ Is an ADP-Ribosylation Factor-dependent Adaptor that Regulates the Traffic of the Alzheimer's Precursor Protein from theTrans-Golgi Network

“…APP is a ubiquitous, single-pass transmembrane protein of unknown function that is highly expressed in brain. Processing involves the sequential actions of ␣-or ␤-plus ␥-secretases, each of which are found in multiple cellular locations (Kuentzel et al, 1993;Selkoe, 1998;Yan et al, 2001). Although processing may occur at any step, the trans-Golgi network (TGN) is the earliest site at which APP processing is thought to commence, and APP is internalized from the cell surface to endosomal compartments where ␥-secretase also acts (Selkoe et al, 1996;Greenfield et al, 1999;Lah and Levey, 2000;Bagshaw et al, 2003).…”