Phosphorylation, Subcellular Localization, and Membrane Orientation of the Alzheimer's Disease-associated Presenilins

doi:10.1074/jbc.272.6.3590

Phosphorylation, Subcellular Localization, and Membrane Orientation of the Alzheimer's Disease-associated Presenilins*

From the^‡ Experimental Genetics Group and
^∥ Molecular Oncology Group, Center for Human Genetics, and the
^¶ Laboratory of Biochemistry, Katholieke Universiteit, 3000 Leuven, Belgium the
** Center for Research in Neurodegenerative Diseases, Department of Medicine (Neurology) and Medical Biophysics, University of Toronto, Toronto, Ontario M5S 1A8, Canada, and
^‡‡ Scios Inc., Sunnyvale, California 94043

^§ Onderzoeksleider of the National Fund for Scientific Research, Belgium (NFWO). To whom correspondence should be addressed:
Campus Gasthuisberg, O&N 06, 3000 Leuven, Belgium
. Fax: 32-16-345871; E-mail: bart.destrooper{at}med.kuleuven.ac.be

Abstract

Presenilins 1 and 2 are unglycosylated proteins with apparent molecular mass of 45 and 50 kDa, respectively, in transfected COS-1 and Chinese hamster ovary cells. They colocalize with proteins from the endoplasmic reticulum and the Golgi apparatus in transfected and untransfected cells. In COS-1 cells low amounts of intact endogeneous presenilin 1 migrating at 45 kDa are detected together with relative larger amounts of presenilin 1 fragments migrating between 18 and 30 kDa. The presenilins have a strong tendency to form aggregates (mass of 100-250 kDa) in SDS-polyacrylamide gel electrophoresis, which can be partially resolved when denatured by SDS at 37°C instead of 95°C. Sulfation, glycosaminoglycan modification, or acylation of the presenilins was not observed, but both proteins are posttranslationally phosphorylated on serine residues. The mutations Ala-246 → Glu or Cys-410 → Tyr that cause Alzheimer's disease do not interfere with the biosynthesis or phosphorylation of presenilin 1. Finally, using low concentrations of digitonin to selectively permeabilize the cell membrane but not the endoplasmic reticulum membrane, it is demonstrated that the two major hydrophilic domains of presenilin 1 are oriented to the cytoplasm. The current investigation documents the posttranslational modifications and subcellular localization of the presenilins and indicates that postulated interactions with amyloid precursor protein metabolism should occur in the early compartments of the biosynthetic pathway.

Footnotes

↵* This investigation was supported by grants from the Fonds voor Geneeskundig Wetenschappelijk Onderzoek (FGWO-NFWO), the Human Frontiers of Science Program (HFSP), the Action Program for Biotechnology of the Flemish Government (VLAB), the Flemish Institute for Biotechnology (VIB), the Katholieke Universiteit Leuven, the Alzheimer's Society of Ontario, and the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PS2

presenilin 2

PS1

presenilin 1

APP

amyloid precursor protein

mAb

monoclonal antibody

PBS

phosphate-buffered saline

TBS

Tris-buffered saline

FITC

fluorescein isothiocyanate

TRITC

tetramethylrhodamine isothiocyanate

PAGE

polyacrylamide gel electrophoresis

Pipes

1,4-piperazinediethanesulfonic acid

NSP

neuroendocrine-specific protein.
↵² P. Fraser, unpublished results.
↵³ B. De Strooper and K. Craessaerts, unpublished results.
↵⁴ D. Moechars, B. De Strooper, and F. Van Leuven, unpublished results.
- Received May 30, 1996.
- Revision received October 1, 1996.