Amyloid precursor protein and Notch intracellular domains are generated after transport of their precursors to the cell surface.
Alzheimer's disease is characterized by brain deposition of extracellular amyloid beta-peptide (Abeta)-containing plaques. The cellular site of gamma-secretase activity, which releases Abeta and the corresponding amyloid precursor protein intracellular domain (AICD), remains controversial. Proposed cleavage sites range from the endoplasmic reticulum (ER), the Golgi apparatus, and the cell surface to endosomal compartments. We now used C99-green fluorescent protein (GFP), a fluorescent reporter substrate for gamma-secretase activity and monitored AICD production in living cells. C99-GFP is efficiently cleaved by gamma-secretase, and AICD-GFP is released into the cytosol. Inhibiting gamma-secretase results in accumulation of C99-GFP in early endosomes. By blocking selective transport steps along the secretory pathway, we demonstrate that gamma-secretase does not cleave its substrates in the ER, the Golgi/trans-Golgi network, or in secretory vesicles. In contrast, inhibition of endocytosis did not inhibit cleavage of C99-GFP. Similar results were obtained for another gamma-secretase substrate, NotchDeltaE. Our results suggest that intracellular domains are generated by gamma-secretase at the plasma membrane and/or early endosomes.
Department of Biochemistry, Adolf-Butenandt-Institute, Laboratory for Alzheimer's and Parkinson's Disease Research, Ludwig-Maximilians-University, 80336 München, Germany.
Christoph
C
Kaether
Stephanie
S
Schmitt
Michael
M
Willem
Christian
C
Haass
D000544:Alzheimer Disease; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017434:Protein Structure, Tertiary; D051880:Receptors, Notch / Q000737:chemistry / Q000378:metabolism*
D016564:Amyloid beta-Protein Precursor; D004791:Enzyme Inhibitors; D051880:Receptors, Notch; D049452:Green Fluorescent Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human