Functional gamma-secretase complex assembly in Golgi/trans-Golgi network: interactions among presenilin, nicastrin, Aph1, Pen-2, and gamma-secretase substrates.
Gamma-secretase is a proteolytic complex whose substrates include Notch, beta-amyloid precursor protein (APP), and several other type I transmembrane proteins. Presenilin (PS) and nicastrin are known components of this high-molecular-weight complex, and recent genetic screens in invertebrates have identified two additional gene products, Aph1 and Pen-2, as key factors in gamma-secretase activity. Here, we examined the interaction of the components of the gamma-secretase complex in Chinese hamster ovary cells stably expressing human forms of APP, PS1, Aph1, and Pen-2. Subcellular fractionation of membrane vesicles and subsequent coimmunoprecipitation of individual gamma-secretase components revealed that interactions among all proteins occurred in the Golgi/trans-Golgi network (TGN) compartments. Furthermore, incubation of the Golgi/TGN-enriched vesicles resulted in de novo generation of amyloid beta-protein and APP intracellular domain. Immunofluorescent staining of the individual gamma-secretase components supported our biochemical evidence that the gamma-secretase components assemble into the proteolytically active gamma-secretase complex in the Golgi/TGN compartment.
Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
Stephanie
S
Baulac
Matthew J
MJ
LaVoie
W Taylor
WT
Kimberly
Jennifer
J
Strahle
Michael S
MS
Wolfe
Dennis J
DJ
Selkoe
Weiming
W
Xia
D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D016466:CHO Cells; D006224:Cricetinae; D010450:Endopeptidases / Q000096:biosynthesis / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000378:metabolism* / Q000502:physiology; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism* / Q000502:physiology; D010447:Peptide Hydrolases; D053764:Presenilin-1; D021601:trans-Golgi Network / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology
D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C508853:PSEN1 protein, human; C469349:PSENEN protein, human; D053764:Presenilin-1; C413604:nicastrin protein; C475154:APH1A protein, human; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D010447:Peptide Hydrolases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human