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SEC24 isoform specificity regulates the assembly of γ‐secretase from dimeric subcomplexes
Author(s) -
Wouters Rosanne,
Michiels Christine,
Kleizen Bertrand,
Dillen Katleen,
Vermeire Wendy,
Demedts David,
Sannerud Ragna,
Schekman Randy,
Annaert Wim
Publication year - 2020
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1002/alz.042844
Subject(s) - nicastrin , golgi apparatus , presenilin , endoplasmic reticulum , protein subunit , microbiology and biotechnology , biology , cell fractionation , secretory pathway , amyloid precursor protein , hek 293 cells , copii , transmembrane protein , chemistry , biochemistry , enzyme , gene , alzheimer's disease , medicine , receptor , disease , pathology
Background γ‐Secretase is a diaspartyl protease with presenilin (PSEN) as the catalytic subunit. It targets a plethora of type I transmembrane proteins and, as such, affects a broad range of physiological processes. Because of this, aberrant functioning of γ‐secretase is linked to several diseases including cancer and Alzheimer’s disease (AD). Herein, mutations in the PSEN genes are associated with rare cases of familial AD and shift the production to longer, more aggregation prone amyloid β peptides. γ‐Secretase consists, besides the catalytic component PSEN, of nicastrin, APH1 and PEN‐2 that are co‐translationally translocated in the endoplasmic reticulum (ER). As γ‐secretase has a stoichiometry of 1:1:1:1, the assembly process is spatially and temporally regulated. Although it is generally agreed that this occurs in early biosynthetic compartments, it remains far from understood whether full assembly occurs already in the ER and to what extent Golgi‐to‐ER recycling is involved. Method We used advanced cell fractionation protocols and an in vitro reconstituted ER budding assay to explore in detail the stepwise assembly process of γ‐secretase in early biosynthetic compartments. Result Cell fractionation combined with blue native electrophoresis shows that subcomplexes are mainly present in ER‐enriched fractions while full complexes appear in the ER‐Golgi intermediate compartment, indicating that final assembly occurs beyond the ER. In vitro reconstitution of ER export reveals that none of the γ‐secretase subunits is required for ER‐exit of the others. However, knock‐out of any subunit induces the accumulation of preceding subcomplexes in de novo formed COPII vesicles. Mutating a motif in the cytosolic loop domain of PSEN1, which is lost in the familial AD‐associated PSEN1∆E9 mutant, abrogates ER‐exit of PSEN1 as well as PEN‐2, but not of nicastrin. This is explained molecularly by the selectivity of Sec24A/B and Sec24C/D in packaging PSEN1 and nicastrin, respectively, and further argues against full complex assembly prior to ER‐exit. Conclusion In conclusion, our data support a model wherein individual subunits and dimeric subcomplexes preferentially exit the ER. Full assembly likely occurs in intermediate compartment/ cis ‐Golgi allowing complexes to escape active Golgi‐to‐ER recycling as part of a secondary quality control mechanism in controlling cellular γ‐secretase levels and activity.