Active gamma-secretase complexes contain only one of each component.
Gamma-secretase is an intramembrane aspartyl protease complex that cleaves type I integral membrane proteins, including the amyloid beta-protein precursor and the Notch receptor, and is composed of presenilin, Pen-2, nicastrin, and Aph-1. Although all four of these membrane proteins are essential for assembly and activity, the stoichiometry of the complex is unknown, with the number of presenilin molecules present being especially controversial. Here we analyze functional gamma-secretase complexes, isolated by immunoprecipitation from solubilized membrane fractions and able to produce amyloid beta-peptides and amyloid beta-protein precursor intracellular domain. We show that the active isolated protease contains only one presenilin per complex, which excludes certain models of the active site that require aspartate dyads formed between two presenilin molecules. We also quantified components in the isolated complexes by Western blot using protein standards and found that the amounts of Pen-2 and nicastrin were the same as that of presenilin. Moreover, we found that one Aph-1 was not co-immunoprecipitated with another in active complexes, evidence that Aph-1 is likewise present as a monomer. Taken together, these results demonstrate that the stoichiometry of gamma-components presenilin:Pen-2:nicastrin:Aph-1 is 1:1:1:1.
Sato
Toru
T
Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
Diehl
Thekla S
TS
Narayanan
Saravanakumar
S
Funamoto
Satoru
S
Ihara
Yasuo
Y
De Strooper
Bart
B
Steiner
Harald
H
Haass
Christian
C
Wolfe
Michael S
MS
D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D010450:Endopeptidases; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D010447:Peptide Hydrolases; D053763:Presenilins / Q000235:genetics / Q000378:metabolism; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism
D016564:Amyloid beta-Protein Precursor; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D046912:Multiprotein Complexes; C469349:PSENEN protein, human; D053763:Presenilins; D051880:Receptors, Notch; C413604:nicastrin protein; C475154:APH1A protein, human; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D010447:Peptide Hydrolases