Abstract
Alzheimer's disease (AD) is caused by the cerebral deposition of beta-amyloid (Abeta), a 38-43-amino acid peptide derived by proteolytic cleavage of the amyloid precursor protein (APP). Initial studies indicated that final cleavage of APP by the gamma-secretase (a complex containing presenilin and nicastrin) to produce Abeta occurred in the endosomal/lysosomal system. However, other studies showing a predominant endoplasmic reticulum localization of the gamma-secretase proteins and a neutral pH optimum of in vitro gamma-secretase assays have challenged this conclusion. We have recently identified nicastrin as a major lysosomal membrane protein. In the present work, we use Western blotting and immunogold electron microscopy to demonstrate that significant amounts of mature nicastrin, presenilin-1, and APP are co-localized with lysosomal associated membrane protein-1 (cAMP-1) in the outer membranes of lysosomes. Furthermore, we demonstrate that these membranes contain an acidic gamma-secretase activity, which is immunoprecipitable with an antibody to nicastrin. These experiments establish APP, nicastrin, and presenilin-1 as resident lysosomal membrane proteins and indicate that gamma-secretase is a lysosomal protease. These data reassert the importance of the lysosomal/endosomal system in the generation of Abeta and suggest a role for lysosomes in the pathophysiology of AD.
Highlights
PS-1, APP, and Nicastrin Are Resident Lysosomal Proteins— These studies demonstrate that APP, nicastrin, and PS-1 are bona fide LM proteins, and are not just present as substrates for degradation
APP and PS-1 both possess C-terminal endocytosis/lysosomal sorting sequences based on the consensus sequence Tyr-X-Xbulky-hydrophobic amino acid (Y-X-X-Ø) (Table III) [55]
The Y-X-X-Ø on the cytoplasmic tail of lysosomal acid phosphatase directs its rapid endocytosis into endosomes
Summary
Antibodies—Antibodies used were: APP C-terminal (Sigma), nicastrin (Affinity Bioreagents), A, PS-1 (N-terminal), calnexin, LAMP-1 (Santa Cruz Biotechnology), and rab (CytoSignal). 6E10 was purchased as part of the “-Amyloid Multipeptide ProteinChip Kit” (Ciphergen). The membrane-associated proteins were solubilized by resuspending the pellet in 0.1 M NaCO3, pH 11.0, incubated on ice for 30 min, with membranes removed by centrifugation at 355,000 ϫ g for 30 min. Routine Transmission Electron Microscopy Preparation—Tyloxapolfilled lysosomes in sucrose were brought to 2% paraformaldehyde, 0.5% glutaraldehyde, 0.05 M phosphate buffer, pH 7.2, incubated 1 h on ice, and pelleted in a benchtop microcentrifuge for 15 min. The grids were rinsed thoroughly in PBS/BSA prior to incubation in goat anti-rabbit IgG 5-nm gold complexes for 1 h (room temperature). In Vitro ␥-Secretase Assay—Membranes were prepared by freeze/ thaw lysis of whole lysosomes (or other fractions) in PBS containing 0.5 M NaCl. After 30 min on ice, membranes were recovered by centrifugation (355,000 ϫ g, 15 min). The matrix, ␣-cyano-4-hydroxycinnamic acid was applied to each spot on the array and mass analysis was performed by SELDI-TOF-MS, using the ProteinChipTM Biology System II
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