Localization of presenilin-nicastrin complexes and gamma-secretase activity to the trans-Golgi network.
Abundant biochemical and genetic evidence suggests that presenilins are catalytic components of gamma-secretase, the protease responsible for generating the Alzheimer amyloid beta-protein. However, the differential localization of presenilins to early secretory compartments and gamma-secretase substrates to late secretory compartments and the plasma membrane (the "spatial paradox") argues against this view. We investigated this issue by studying the localization of nicastrin, another putative gamma-secretase component, and its association with presenilin-1 into proteolytically active complexes. Glycosidase digests revealed that nicastrin exists in multiple glycoforms and is terminally sialylated, a modification often associated with the trans-Golgi network. Trafficking of nicastrin to the trans-Golgi network was confirmed by density gradient fractionation and immunofluorescence microscopy. In presenilin-deficient cells, however, nicastrin trafficking and maturation were abnormal, as the protein was restricted to early secretory compartments and failed to be sialylated. Mature sialylated nicastrin in trans-Golgi network fractions was complexed quantitatively with N- and C-terminal fragments of presenilin-1, whereas immature nicastrin present in early secretory compartments was not. Additionally, trans-Golgi network fractions contained the gamma-secretase substrate beta-amyloid precursor protein C83 and were enriched in presenilin-dependent gamma-secretase proteolytic activity. The results resolve the apparent spatial paradox by demonstrating that presenilin-nicastrin complexes and presenilin-dependent gamma-secretase activity are co-localized to a late secretory compartment. The findings provide further evidence that presenilin-containing complexes are the gamma-secretase, and indicate that presenilins also regulate gamma-secretase assembly.
Siman
Robert
R
Department of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, JMB162, Philadelphia, PA 19104-6084, USA. siman@pharm.med.upenn.edu
Velji
Jamel
J
D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D002451:Cell Compartmentation / Q000502:physiology; D002478:Cells, Cultured; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D005347:Fibroblasts / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D006031:Glycosylation; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000172:deficiency / Q000378:metabolism; D051379:Mice; D053764:Presenilin-1; D053766:Presenilin-2; D021381:Protein Transport / Q000502:physiology; D013234:Stem Cells / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry; D021601:trans-Golgi Network / Q000737:chemistry* / Q000378:metabolism
D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D053764:Presenilin-1; D053766:Presenilin-2; C413604:nicastrin protein; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509282:Bace1 protein, mouse