Assembly of γ-secretase occurs through stable dimers after exit from the endoplasmic reticulum

Rosanne Wouters; Christine Michiels; Ragna Sannerud; Bertrand Kleizen; Katleen Dillen; Wendy Vermeire; Abril Escamilla Ayala; David Demedts; Randy Schekman; Wim Annaert

doi:10.1083/jcb.201911104

Assembly of γ-secretase occurs through stable dimers after exit from the endoplasmic reticulum

Rosanne Wouters, Christine Michiels, Ragna Sannerud, Bertrand Kleizen, Katleen Dillen, Wendy Vermeire, Abril Escamilla Ayala, David Demedts, Randy Schekman, Wim Annaert^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

γ-Secretase affects many physiological processes through targeting >100 substrates; malfunctioning links γ-secretase to cancer and Alzheimer’s disease. The spatiotemporal regulation of its stoichiometric assembly remains unresolved. Fractionation, biochemical assays, and imaging support prior formation of stable dimers in the ER, which, after ER exit, assemble into full complexes. In vitro ER budding shows that none of the subunits is required for the exit of others. However, knockout of any subunit leads to the accumulation of incomplete subcomplexes in COPII vesicles. Mutating a DPE motif in presenilin 1 (PSEN1) abrogates ER exit of PSEN1 and PEN-2 but not nicastrin. We explain this by the preferential sorting of PSEN1 and nicastrin through Sec24A and Sec24C/D, respectively, arguing against full assembly before ER exit. Thus, dimeric subcomplexes aided by Sec24 paralog selectivity support a stepwise assembly of γ-secretase, controlling final levels in postGolgi compartments.

Original language	English
Article number	e201911104
Pages (from-to)	1-26
Journal	Journal of Cell Biology
Volume	220
Issue number	9
DOIs	https://doi.org/10.1083/jcb.201911104
Publication status	Published - 22 Jul 2021

Bibliographical note

Funding Information:
Research Foundation (S#20030). R. Wouters held a Research Foundation–Flanders aspirant fellowship, and B. Kleizen held a long-term European Molecular Biology Organization fellowship. R. Schekman is supported as an investigator of the Howard Hughes Medical Institute and the Adolph C. and Mary Sprague Miller Institute for Basic Research in Science, University of California, Berkeley. The authors declare no competing financial interests.

Funding Information:
We thank P. Wong (Baltimore, MD) for NCT KO MEFs and B. De Strooper (Katholieke Universiteit Leuven, Leuven, Belgium) for other subunit KO MEFs. This work was financed through Vlaams Instituut voor Biotechnologie, Katholieke Universiteit Leuven (C16/15/073, C14/21/095), Research Foundation?Flanders (S006617N, G078117N, G056017N, AKUL13/39), and Stichting AlzheimerOnderzoek?Alzheimer Research Foundation (S#20030). R. Wouters held a Research Foundation?Flanders aspirant fellowship, and B. Kleizen held a long-term European Molecular Biology Organization fellowship. R. Schekman is supported as an investigator of the Howard Hughes Medical Institute and the Adolph C. and Mary Sprague Miller Institute for Basic Research in Science, University of California, Berkeley. The authors declare no competing financial interests..

Funding Information:
This work was financed through Vlaams Instituut voor Bio-technologie, Katholieke Universiteit Leuven (C16/15/073, C14/21/095), Research Foundation–Flanders (S006617N, G078117N, G056017N, AKUL13/39), and Stichting Alzheimer Onderzoek–Alzheimer

Publisher Copyright:
© 2021 Wouters et al.

Funding

Research Foundation (S#20030). R. Wouters held a Research Foundation–Flanders aspirant fellowship, and B. Kleizen held a long-term European Molecular Biology Organization fellowship. R. Schekman is supported as an investigator of the Howard Hughes Medical Institute and the Adolph C. and Mary Sprague Miller Institute for Basic Research in Science, University of California, Berkeley. The authors declare no competing financial interests. We thank P. Wong (Baltimore, MD) for NCT KO MEFs and B. De Strooper (Katholieke Universiteit Leuven, Leuven, Belgium) for other subunit KO MEFs. This work was financed through Vlaams Instituut voor Biotechnologie, Katholieke Universiteit Leuven (C16/15/073, C14/21/095), Research Foundation?Flanders (S006617N, G078117N, G056017N, AKUL13/39), and Stichting AlzheimerOnderzoek?Alzheimer Research Foundation (S#20030). R. Wouters held a Research Foundation?Flanders aspirant fellowship, and B. Kleizen held a long-term European Molecular Biology Organization fellowship. R. Schekman is supported as an investigator of the Howard Hughes Medical Institute and the Adolph C. and Mary Sprague Miller Institute for Basic Research in Science, University of California, Berkeley. The authors declare no competing financial interests.. This work was financed through Vlaams Instituut voor Bio-technologie, Katholieke Universiteit Leuven (C16/15/073, C14/21/095), Research Foundation–Flanders (S006617N, G078117N, G056017N, AKUL13/39), and Stichting Alzheimer Onderzoek–Alzheimer

Keywords

Amyloid Precursor Protein Secretases/chemistry
Animals
Biological Transport
COP-Coated Vesicles/chemistry
Cell Line
Cell Line, Tumor
Cerebral Cortex/cytology
Endopeptidases/chemistry
Endoplasmic Reticulum/metabolism
Fibroblasts/cytology
Gene Expression Regulation
Golgi Apparatus/metabolism
Humans
Membrane Proteins/chemistry
Mice
Models, Molecular
Neurons/cytology
Presenilin-1/chemistry
Primary Cell Culture
Protein Binding
Protein Conformation
Protein Isoforms/chemistry
Protein Multimerization
Rats
Rats, Wistar
Signal Transduction
Vesicular Transport Proteins/genetics

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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jcb_201911104Final published version, 7.05 MBLicence: CC BY-NC-SA

Cite this

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title = "Assembly of γ-secretase occurs through stable dimers after exit from the endoplasmic reticulum",

abstract = "γ-Secretase affects many physiological processes through targeting >100 substrates; malfunctioning links γ-secretase to cancer and Alzheimer{\textquoteright}s disease. The spatiotemporal regulation of its stoichiometric assembly remains unresolved. Fractionation, biochemical assays, and imaging support prior formation of stable dimers in the ER, which, after ER exit, assemble into full complexes. In vitro ER budding shows that none of the subunits is required for the exit of others. However, knockout of any subunit leads to the accumulation of incomplete subcomplexes in COPII vesicles. Mutating a DPE motif in presenilin 1 (PSEN1) abrogates ER exit of PSEN1 and PEN-2 but not nicastrin. We explain this by the preferential sorting of PSEN1 and nicastrin through Sec24A and Sec24C/D, respectively, arguing against full assembly before ER exit. Thus, dimeric subcomplexes aided by Sec24 paralog selectivity support a stepwise assembly of γ-secretase, controlling final levels in postGolgi compartments.",

keywords = "Amyloid Precursor Protein Secretases/chemistry, Animals, Biological Transport, COP-Coated Vesicles/chemistry, Cell Line, Cell Line, Tumor, Cerebral Cortex/cytology, Endopeptidases/chemistry, Endoplasmic Reticulum/metabolism, Fibroblasts/cytology, Gene Expression Regulation, Golgi Apparatus/metabolism, Humans, Membrane Proteins/chemistry, Mice, Models, Molecular, Neurons/cytology, Presenilin-1/chemistry, Primary Cell Culture, Protein Binding, Protein Conformation, Protein Isoforms/chemistry, Protein Multimerization, Rats, Rats, Wistar, Signal Transduction, Vesicular Transport Proteins/genetics",

author = "Rosanne Wouters and Christine Michiels and Ragna Sannerud and Bertrand Kleizen and Katleen Dillen and Wendy Vermeire and Ayala, {Abril Escamilla} and David Demedts and Randy Schekman and Wim Annaert",

note = "Funding Information: Research Foundation (S#20030). R. Wouters held a Research Foundation–Flanders aspirant fellowship, and B. Kleizen held a long-term European Molecular Biology Organization fellowship. R. Schekman is supported as an investigator of the Howard Hughes Medical Institute and the Adolph C. and Mary Sprague Miller Institute for Basic Research in Science, University of California, Berkeley. The authors declare no competing financial interests. Funding Information: We thank P. Wong (Baltimore, MD) for NCT KO MEFs and B. De Strooper (Katholieke Universiteit Leuven, Leuven, Belgium) for other subunit KO MEFs. This work was financed through Vlaams Instituut voor Biotechnologie, Katholieke Universiteit Leuven (C16/15/073, C14/21/095), Research Foundation?Flanders (S006617N, G078117N, G056017N, AKUL13/39), and Stichting AlzheimerOnderzoek?Alzheimer Research Foundation (S#20030). R. Wouters held a Research Foundation?Flanders aspirant fellowship, and B. Kleizen held a long-term European Molecular Biology Organization fellowship. R. Schekman is supported as an investigator of the Howard Hughes Medical Institute and the Adolph C. and Mary Sprague Miller Institute for Basic Research in Science, University of California, Berkeley. The authors declare no competing financial interests.. Funding Information: This work was financed through Vlaams Instituut voor Bio-technologie, Katholieke Universiteit Leuven (C16/15/073, C14/21/095), Research Foundation–Flanders (S006617N, G078117N, G056017N, AKUL13/39), and Stichting Alzheimer Onderzoek–Alzheimer Publisher Copyright: {\textcopyright} 2021 Wouters et al.",

year = "2021",

month = jul,

day = "22",

doi = "10.1083/jcb.201911104",

language = "English",

volume = "220",

pages = "1--26",

journal = "Journal of Cell Biology",

issn = "0021-9525",

publisher = "Rockefeller University Press",

number = "9",

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TY - JOUR

T1 - Assembly of γ-secretase occurs through stable dimers after exit from the endoplasmic reticulum

AU - Wouters, Rosanne

AU - Michiels, Christine

AU - Sannerud, Ragna

AU - Kleizen, Bertrand

AU - Dillen, Katleen

AU - Vermeire, Wendy

AU - Ayala, Abril Escamilla

AU - Demedts, David

AU - Schekman, Randy

AU - Annaert, Wim

N1 - Funding Information: Research Foundation (S#20030). R. Wouters held a Research Foundation–Flanders aspirant fellowship, and B. Kleizen held a long-term European Molecular Biology Organization fellowship. R. Schekman is supported as an investigator of the Howard Hughes Medical Institute and the Adolph C. and Mary Sprague Miller Institute for Basic Research in Science, University of California, Berkeley. The authors declare no competing financial interests. Funding Information: We thank P. Wong (Baltimore, MD) for NCT KO MEFs and B. De Strooper (Katholieke Universiteit Leuven, Leuven, Belgium) for other subunit KO MEFs. This work was financed through Vlaams Instituut voor Biotechnologie, Katholieke Universiteit Leuven (C16/15/073, C14/21/095), Research Foundation?Flanders (S006617N, G078117N, G056017N, AKUL13/39), and Stichting AlzheimerOnderzoek?Alzheimer Research Foundation (S#20030). R. Wouters held a Research Foundation?Flanders aspirant fellowship, and B. Kleizen held a long-term European Molecular Biology Organization fellowship. R. Schekman is supported as an investigator of the Howard Hughes Medical Institute and the Adolph C. and Mary Sprague Miller Institute for Basic Research in Science, University of California, Berkeley. The authors declare no competing financial interests.. Funding Information: This work was financed through Vlaams Instituut voor Bio-technologie, Katholieke Universiteit Leuven (C16/15/073, C14/21/095), Research Foundation–Flanders (S006617N, G078117N, G056017N, AKUL13/39), and Stichting Alzheimer Onderzoek–Alzheimer Publisher Copyright: © 2021 Wouters et al.

PY - 2021/7/22

Y1 - 2021/7/22

N2 - γ-Secretase affects many physiological processes through targeting >100 substrates; malfunctioning links γ-secretase to cancer and Alzheimer’s disease. The spatiotemporal regulation of its stoichiometric assembly remains unresolved. Fractionation, biochemical assays, and imaging support prior formation of stable dimers in the ER, which, after ER exit, assemble into full complexes. In vitro ER budding shows that none of the subunits is required for the exit of others. However, knockout of any subunit leads to the accumulation of incomplete subcomplexes in COPII vesicles. Mutating a DPE motif in presenilin 1 (PSEN1) abrogates ER exit of PSEN1 and PEN-2 but not nicastrin. We explain this by the preferential sorting of PSEN1 and nicastrin through Sec24A and Sec24C/D, respectively, arguing against full assembly before ER exit. Thus, dimeric subcomplexes aided by Sec24 paralog selectivity support a stepwise assembly of γ-secretase, controlling final levels in postGolgi compartments.

AB - γ-Secretase affects many physiological processes through targeting >100 substrates; malfunctioning links γ-secretase to cancer and Alzheimer’s disease. The spatiotemporal regulation of its stoichiometric assembly remains unresolved. Fractionation, biochemical assays, and imaging support prior formation of stable dimers in the ER, which, after ER exit, assemble into full complexes. In vitro ER budding shows that none of the subunits is required for the exit of others. However, knockout of any subunit leads to the accumulation of incomplete subcomplexes in COPII vesicles. Mutating a DPE motif in presenilin 1 (PSEN1) abrogates ER exit of PSEN1 and PEN-2 but not nicastrin. We explain this by the preferential sorting of PSEN1 and nicastrin through Sec24A and Sec24C/D, respectively, arguing against full assembly before ER exit. Thus, dimeric subcomplexes aided by Sec24 paralog selectivity support a stepwise assembly of γ-secretase, controlling final levels in postGolgi compartments.

KW - Amyloid Precursor Protein Secretases/chemistry

KW - Animals

KW - Biological Transport

KW - COP-Coated Vesicles/chemistry

KW - Cell Line

KW - Cell Line, Tumor

KW - Cerebral Cortex/cytology

KW - Endopeptidases/chemistry

KW - Endoplasmic Reticulum/metabolism

KW - Fibroblasts/cytology

KW - Gene Expression Regulation

KW - Golgi Apparatus/metabolism

KW - Humans

KW - Membrane Proteins/chemistry

KW - Mice

KW - Models, Molecular

KW - Neurons/cytology

KW - Presenilin-1/chemistry

KW - Primary Cell Culture

KW - Protein Binding

KW - Protein Conformation

KW - Protein Isoforms/chemistry

KW - Protein Multimerization

KW - Rats

KW - Rats, Wistar

KW - Signal Transduction

KW - Vesicular Transport Proteins/genetics

U2 - 10.1083/jcb.201911104

DO - 10.1083/jcb.201911104

M3 - Article

C2 - 34292306

AN - SCOPUS:85112127551

SN - 0021-9525

VL - 220

SP - 1

EP - 26

JO - Journal of Cell Biology

JF - Journal of Cell Biology

IS - 9

M1 - e201911104

ER -

Assembly of γ-secretase occurs through stable dimers after exit from the endoplasmic reticulum

Abstract

Bibliographical note

Funding

Keywords

UN SDGs

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